rapid hyaluronan uptake assoclated with
TRANSCRIPT
RAPID HYALURONAN UPTAKE IS ASSOCLATED WITH ENHANCED MOTILITY AND TRANSFORMATION:
IMPLICATIONS FOR AN INTRACELLULAR MODE OF ACTION
Lisa Janine Collis
A thesis submitted in conformity with the requirements
for the Degree Master of Science,
Graduate Department of Anatorny and Ce11 Biology
University of Toronto
O Copyright by Lisa Janine Collis, 1999
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Rapid Hyaluronan Uptake is Associated with Enhanced Motility and
Transformation: Implications for an Intracellular Mode of Action
Lisa Janine Collis
For the Degree Master of Science, 1999
Department of Ariatomy and Cd1 Biobgy
University of Toronto
ABSTRACT
Transformed fibroblasts rapidly intemalize Texas Red labeled hyaluronan (TR-
HA), and target it to ce11 nuclei. processes and the pennuclear region. TR-HA uptake is
blocked by: excess polymeric HA, HA-dodecascuicharide, colchicine, BrefeldinA, and by
digestion of TR-HA with hyaluronidase. Uptake is dose dependent, saturable and strongly
enhanced upon ceIl attachment, monolayer wounding and transformation. TR-HA nuclear
targeting requires a minimum of 30 HA-saccharides, suggesting the existence of novel
hyaladherins. Enhanced uptake correlates with increased CD44 and RHAMM expression
and both CD44 antibodies and RHAMM peptides alter uptake. Interestingly, MEK
inhibition also affects uptake. Connexin43 overexpression, regulated by RHAMM
expression, enhances HA uptake and targeting to cell processes. In vitro, HA enhances
DNasel-DNA interaction, suggesting nuclear HA'S potential influence on gene expression.
Roles for intracellular hyaluronan in cell function are suggested by a correlation of ce11
process HA targeting with increased motility, after oncogene transformation, monolayer
u-ounding, or phorbol ester treatment.
I would like to express the deepest admiration and thanks to my parents who are a conrinual
source of love and support and to Chad for his love, patience and constant encouragement,
and it is to thent that I dedicate this thesis.
This thesis would not have been completed without help and support from
numerous CO-workers and friends.
1 would first like to thank my supervisor Dr. Eva Turley, for providing me with the
opportunity to do this work, and for her guidance and support throughout this study. 1 am
likewise indebted to our collaborators Dr. Glen Prestwich and Michael Ziebell of the
University of Utah for their assistance in developing and analyzing the TR-HA conjugate,
Dr. Markku Tammi and Dr. Raija Tammi of the University of Kuopio, Finland for
providing me with purified and labelled HA oligosaccharides, Dr. Jim Nagy of the
University of Manitoba for providing the connexin43 transfected glioma cells and Bruce
Lynn of the University of Manitoba for his patience during our attempts at live uptake, and
his instruction on image analysis. 1 further wish to express my sincere gratitude to my
cornmittee members Dr. Michael Opas and Dr. Sean Egan for their time and knowledge.
1 am also indebted to ail the members of Dr. Turley's research group, past and
present. My thanks extend especially to Frouz Paiwand and Rene Harrison who have
been such great benchmates, confidants and friends during this formative experirnce. 1
would also like to express my thanks to Jingbo A, Shiwen Zhang, Arian Khandani, Jie
Lu, FuSheng Wang, Joycelyn Entwistle and Judy Edwards for their support as both
friends and colleagues.
This study was performed at The Hospital for Sick Children in the Department of
Cardiovascular Research under the aegis of the Department of Anatomy and Cell Biology
of the University of Toronto during the yean 1997 - 1999 with financial support from an
MRC studentship and Hyal Pharrnaceutical Corporation.
TABLE OF CONTENTS
ABSTRACT ...............................................................................................................
DEDICATION ...........................................................................................................
ACKNO WLEDGMENTS .........................................................................................
TABLE OF CONTENTS ..........................................................................................
LIST OF FIGURES ...................................................................................................
LIST OF ABBREVIATIONS ...................................................................................
1 . INTRODUCTION ..............................................................................................
.................................................................. . 1.1 Biochemical Properties of HA
1.1 . 1. Structure .....................................................................................
................................................... 1.1.2. Family of glycosaminoglycans
1.1.3. Synthesis of HA ........................................................................
1.1.4. HA degradation ...........................................................................
1.2. Functions of HA ..................... .... ............................................................
.............................................. 1.2.1. Physicochemical funciions of HA
..................................... 1.2.2. HA binding proteins - the hyaladherins
1.2.2.1. CD44 ...........................................................................
..................................... 1.2.2.2. RHAMM and related proteins
L2.3. Hyaluronan and tumorigenesis ...................................................
.................................................. L2.3. Hyaluronan and wound healing
1.3. Rationale of this study ...............................................................................
II . MATERIALS AND METHODS .......................................................................
II.2. Conjugation of HA with Texas Red ........................................................
II.3. Treatment of cells with TR-HA rind analysis of resultant intracellular
fluorescence ...........................................................................................
Digestion of TR-HA with hyaluronidase ................................
Cornpetition with unlabeled HA and HA oligosaccharides ....
Treatment of celis with Texas Red probe alone ......................
Treatment of cells with Syto16 ...............................................
.................................................. TR-HA concentration curves
............................ TR-HA treatment of cells over timecourses
Prebinding of peptides to TR-HA ...........................................
................. Anti-CD44 and Anti-RHAMM antibody blocking
.......... AMAC labeled HA-oligosaccharide treatment of cells
II.4. Wounding assays ......................................................................................
................................................................................ fI.5. Immunofluorescence
U.6. Flow cytometry (FACS analysis) .............................................................
...................................................................................... IT.7. DNaseI digestions
III . RESULTS ............................................................................................................
El . 1 . Texas Red labeled hyaiuronan (TR-HA) is rapidly taken up into ras-
transforrned cells ..................................................................................
III.2. TR-HA uptake is dose and time dependent and increases with cellular
transformation ......................................................................................
............................ OI.3. Specific HA oligosaccharides inhibit TR-HA uptake
m.4. Thiny HA-saccharide minimum length is required for effective
targeting of HA to the nucleus .............................................................
m.5. Anti-CD44 antibodies affect TR-HA uptake .........................................
iiI.6. Subcellular distribution of RHAMM is dramatically altered upon HA
............................................................................................... treatmen t
m.7. RHAMM peptides block TR-HA targeting to the nucleus .....................
iII.8. TR-HA uptake is unaffected by blocking with RHAMMexon9
antibody ................................................................................................
IU.9. HA uptake varies depending upon length of ce11 plating time and
......................... positively correlates with functional RHAMM levels
III . 10 . Connexin43 transfected cells show incmased TR-HA uptake and
.................................................... targeting to the tips of ce11 processes
iII . 1 1 . TR-HA uptake is decreased upon MEK inhibition ...............................
III . 12 . HA is targeted to the ce11 processes upon phorbol ester treatment of
................................................................... parental iOT112 fibroblasts
III . 13 . TR-HA shows enhanced uptake and tatgeting to the tips of the ce11
processes at the wound edge in parental IOT112 fibroblasts ................
IV . DISCUSSION ......................................................................................................
IV . 1 . The phenornenon of rapid HA uptake ....................................................
IV.2. Mechanisms of HA uptake .....................................................................
N.2.1. The potentiai role of ce11 surface receptors in TR-HA uptake ...
N.3. Potential roles for intracellular targeting of HA .....................................
TV.3.1. Potentiai role of HA in the nucleus ..........................................
vii
......................... N.3.2. Potential roles of HA uptake in transformation
................... N.3.3. Potential roles of intracellular HA in ce11 motility
iV.4. Conclusions ............................................................................................
V . REFERENCES ....................................................................................................
VI . APPENDIX 1 - ADDITIONAL RESULTS SECTION ....................................
VI . 1 . Resul ts ......................................................................................................
........ . VI 1.1. Colchicine treatment prevents nuclear uptake of TR-HA
VI . 1.2. Brefeldin A pretreatrnent blocks uptake of TR-HA by
....................................................... 10T 1 /2 parenta1 fi broblasts
.............. . VI 1.3. HA alter'; the ability of DNaseI to interact with DNA
VI.2. Discussion ................................................................................................
..................................... Vf.2.1. Cytosolic factors in the uptake process
........ VI.2.2. Potential influence of HA on DNA - protein interaction
................... ................... VIL APPENDIX 2 - HYALURONAN DETECTION ..
......... ...................................... . VI 1.1. Existing probes for hyaluronan ..
.................................... . VI 1.2. The Texas Red - hyaluronan conjugate
viii
LIST OF FIGURES
1 . INTRODUCTION
..................................................................... Figure 1.1. Structure of hyaluronan 4
Figure 1.2. Extrusion mode1 for HA synthesis ................................................... 6
Figure 1.3. Hyaluronan binding proteins - the hyaladherins ............................... 14
II . METHODS
. . Figure II 1 Conjugation of HA to Texas Red hydrazide ..................................... 30
III . RESULTS
Figure UI . 1 . TR-HA is targeted to the nucleus. perinuclear area. and ruffles of
.................................................................. ras transformed ce1 1s
Figure III.2. TR-HA and Syto l6 are found in the nucleus in the same focal
............................................................................................. plane
Figure m.3. TR-HA uptake is HA specific ............................................................
Figure m.4. TR-HA uptake is dose and time dependent. and increased in
................................................................. transformed ceIl lines
................................... Figure m.5. Oligosaccharides of HA block TR-HA uptake
Figure m.6. AMAC labeled Oligosaccharides of HA can be targeted to the
perinuctear and nuclear compartments of cells in a size
dependent manner ......................................................................
Figure iIi.7. CD44 mAbs alter TR-HA uptake .......................................................
Figure III.8. Subcellular distribution of RHAMM is dramatically aitered upon
HA treatment ..............................................................................
Figure III.9. RHAMM peptides block TR-HA targeting to the nucleus .................
Figure III. 10 HA uptake is unaffected by blocking with RHAMMexon9
an tibody . . . . . . . . .. . . . . .. . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .
Figure m. 1 1 HA uptake varies depending upon length of ceil plating time and
positively correlates with functional RHAMM levels ...............
Figure III. 12 Connexin43 transfected cells show increased TR-HA uptake and
targeting to the tips of ceIl processes ..........................................
Figure III. 13 TR-HA uptake is decreased upon MEK inhibition ............................
Figure III. 17 HA is targeted to the ce11 processes upon phorbol ester treatment
of parental 1 OT 1 /2 fibroblasts . . . .. . . . . . . . . . .. .. . . . .. . . . .. . .. . . . . . . . . .. . . . . . . ... . .
Figure m. 18 TR-HA shows enhanced uptake and targeting to the tips of the ce11
processes at the wound edge in parental lOT112 fibroblasts .......
VI. APPENDIX 1
Figure VI. 1 Colchicine treatment prevents nuclear uptake of TR-HA .................
Figure V1.2 Brefeldin A pretreatment blocks uptake of TR-HA by 10T 112
parental fibroblasts ..... . .. .. . . . . . . .. . . . . ... .. . . . .. . . . .. . . . .. . . . . .. . . . .. . .. .. . . . . . . .. .
Figure VL3 HA alters the ability of DNaseI to interact with DNA .......................
Figure V1.4 Mode1 of potential uptake mechanisms and intraceliular functions
of HA .........................................................................................
VII. APPENDIX 2
Figure VU. 1 .HPLC analysis of TR-NA conjugate ................................................
aa
BSA
CD44
CD44v
cDNA
CPC
CS
Da
DMEM
DMSO
ECM
EDTA
ERK
FACS
FCS
FITC
GAG
GPI
HA
HA#
HABP
HARLEC
HAS
Amino acid(s)
Bovine serum albumen
Standard CD44 isoform
Variant CD44 isoform
Complimentary DNA
Cetylpyridinium chloride
Chondroitin sulphate
Dalton
Dulbecco's modified eagle's medium
Dimethyl sulfoxide
Extracellular matrix
Ethylenediaminetetraücetate
Extracellular signal regulated protein kinase
Fluorescence activated ce11 sorter (used in flow cytometry)
Fetal calf semm
Fluorescein isothiocyanate
Gl ycosaminogl ycan
Glycosylphosphatidyl inositol
Hyaluronan, hyaiuronic acid, hyaluronate
Hyaluronic acid oligosaccharide of "#" monosaccharide units
Hyaluronan binding protein(s)
Hyaluronan receptor for liver endothelial cells
Hyaiuronan synthase
HBSS
HMW
HS
IgG
kb
kDa
LMW
m Ab
MAP
MAPK
MEK
MW
MWCO
PA"
PBS
PDGF
PKC
PMA
rER
RHAMM
RHAMMA 1 -5
RHAMMexon9
RITC
TR
TR-HA
Hank's batanced salt solution
High molecular weight
heparin sulphate
Immunoglobulin G
Kilobase(s) or lOOObp
Kilodalton or 1 OOOda
Low molecular weight
Monoclonal anti body
Mitogen associated protein
Mitogen associated protein kinase
MAPWERK kinase
Molecular weight
Molecular weight cut-off
Polyclonal antibody
Phosphate-buffered saline
Platelet derived growth factor
Protein kinase C
Phorbol rnyristate acetate
Rough endoplasmic reticulum
Receptor for hyaluronan mediated motility
RHAMM exons 1-5 deleted (old nomenclature WAMMv4)
old nomenclature = exon4
Rhodarnine isothiocyanate
Texas red
Texas red - hyaluronan conjugate
CHAPTER 1
INTRODUCTION
Hyaluronan (HA) was first descnbed as a polysaccharide isolated from the
vitreous humour in 1934 (Meyer et al. 1934). It contained uronic acid and Meyer
named the polysaccharide hyaluronic acid from hyulos (glassy, vitreous) and uronic
acid. However at physiological pH al1 carboxyl groups on the uronic acid residues
are dissociated and the polysaccharide has therefore been named sodium hyaluronate
when sodium is the counter ion. It is often difficult to specify the counter ion, for
example in a tissue, and Balzacs et al. (1989) therefore suggested that the name
hyaluronan (HA) should be used.
Hyaluronan is a molecule of paradoxes and contrasts. On the one hand, it is
a homopolymer of simple disaccharide units endlessly repeated. present in some
tissues (such as the cockscomb and vitreous humor) in large amounts and
performing very simple mechanical jobs as a space filler or lubricant (in synovial
joints). On the other hand, HA takes part in ce11 surface phenornena of great
specificity when used at very low dilution in the presence of specific receptors.
Interest in HA has increased dramatically since 1980 when major clinical
applications in optharnology and in the treatment of joint disease were introduced.
Since then, HA has found multiple applications in drug delivery and surgery. HA
has been found to enhance absorption of drugs and proteins through mucus tissues
(Morimoto et al. 199 1). When combined with HA, the efficacy of many agents
(such as anti-inflammatory drugs) are strengthened (Goa and Benfield 1996; Miller
et al. 1997). HA has found important applications in the field of viscosurgery,
viscosupplementation and wound healing (Davidson et al. 1991; Juhlin et al. 1997).
in reproductive medicine, HA is used to improve retention of the mobility of
cryopreserved and thawed spermatozoa (S bracia et al. 1997).
HA is a ubiquitous compound and there is no known genetic disease in
which HA is not synthesized. HA may therefore be of fundamental importance in
the animal organism and mutations causing defects in HA synthesis may be lethal.
HA is found in virtually every species in the animal kingdom, in every tissue in the
human body as well as the capsule of certain microorganisms. Moreover the HA
repeating disaccharide is identical in al1 tissues and species and consequently HA is
never recognized as immunologicdly foreign (Fraser and Laurent 1997).
1.1. Biochemical Properties of HA
1.1.1. Structure
The polysaccharide HA is a negatively charged linear pol ymer with the
structure 143-4 D-glucuronic acid ( 1 -[P-3) N-acetyl-D-glucosamine ( 1 -1,-P-4)
(Figure 1.1.) that can have a molecular mass of up to several million daitons. The
relative rnolecular weight of HA varies dramatically depending upon its source. For
example, the HA isolated from human synovial fluid is about 7000kDa, from
human blood serum it is approximately 200kDa, and from human urine it rnay be as
small as 4- 12kDa (Fraser and Laurent 1996).
Studies on conformation of HA by X-ray diffraction and spectroscopy
indicate that the molecule c m take up helical conformations stabilized by hydrogen
bonds that run in parallel with the chah axis (Scott and Tigwell 1978; Scott 1989).
The polyrner consequently takes up a stiffened helical configuration which gives the
molecuie an overail expanded coi1 structure in solution. High molecular weight
(HMW) forms of HA can have a diameter in the order of 500nm (Laurent 1989).
repeating disaccharide
glucuronic acid Ai-acetylglucosamine
Figure 1.1. Structure of hyaluronan (rnodified from Alberts et al. 1994).
Along the HA molecule there are clusters of adjacent CH groups forming
patches of a highly hydrophobic character. These hydrophobic patches are repeated
at regular intervals on altemate sides of the molecule (Scott 1989). The altemation
of clustered hydrophobic and hydrophilic segments in HA facilitates its interactions
with biological membranes, hydrophobic proteins (e.g.: link protein) and also in HA
self-aggregation (Scott 1989; Scott et al. 1992).
Intramolecular interactions of HA in solution dampen interactions with other
molecules, and promote its neutral space filling and molecule passing roles (Scott
1998). HA networks are more easily formed by HMW HA. Welsh et al. (1980)
showed that an admixture of LMW HA dramatically alter the rheologic properties
of HMW HA which these authors tentatively suggest is an aggregation breaking
effect.
1.1.2. Family of glysosaminoglycans
HA is one of a family of connective tissue heteropolysaccharides containing
hexosamine, collectively called glycosaminoglycans (GAG). Other prominent
GAGS include heparin sulphate (HS), heparin, chondroitin sulphate (CS), dermatan
sulphate (DS), and keratin sulphate (reviewed in Sarnes 1994; Soldani and
Romagnol i 199 1 ).
HA is the simplest GAG, and is structurally unique in at least three respects.
Firstly it is the only GAG which does not exist covalently linked to a core protein
(Le. as proteoglycan). Secondly it has no sulphate content. Thirdly, its linear
polymers can reach a much greater relative molecular m m . For example, HA in
joint fluid can be up to several million daltons in size, while proteoglycans are
commonly less than 30kDa (Fraser 1994).
1.1.3. Synthesis of HA
Eukaryotic HA synthases are localized to the inner leaflet of the plasma
membrane where they extrude HA directly into the extracellular space (Prehm 1989;
Weigel et al. 1997). HA chains are elongated at the reducing end by alternate
transfer of sugar residues from the substrates UDP-glucuronic acid and UDP-N-
acetylglucosarnine (Prehm 1983a,b). This occurs inside the plasma membrane, and
the chain (with the non-reducing end ahead) is extruded into the pencellular space,
whiIe the growing HA chah remains bound to the enzyme (Prehm 1984; Heldin et
al. 1993). The mechanisrn for release of HA extracellularly is not known. HA
chah release typically occurs after s20,000 monosaccharides s4 x 1 0 6 ~ a ) have
been assembled. HA extrusion to the extracellular milieu is presently viewed as a
process requiring the enzyme to form a pore or channel-like pocket to guide the
hydrophilic HA chain through the lipid layer (see Figure 1.2). Extrusion of the
growing chain into the extracellular space allows unconstrained growth of the HA
polymer. This mechanism of synthesis and release is in contrast to the smaller
GAGS which are synthesized in the Golgi cornpartment and released by exocytosis
(Hascall et al. 1998).
4 p lasma membrane & .a
UDP- h yaluronan
Figure 1.2. Extrusion mode1 for HA synthesis (modified from Prehm et al. 1983)
There are three HA synthase (HAS) genes in eukaryotes (Itano and Kimata
1996; Spicer et al. 1996; Fulop et al. 1997; Spicer et al. 1997a). In contrat with
the low levels of identity between HAS within a species (HAS 1/HAS2, 55%
identity; HAS l/HAS3,57% identity; HAS2/HAS3, 7 1 5% identity). the sequence
between individual human and mousc HAS orthologues is highIy conserved
(identity 96%) (Spicer et al. 1997b; Weigel et al. 1997). These sequence
characteristics suggest a conservation of the functionally important residues of HAS
during evoiution and the differences of action of the three HAS proteins (Spicer et
al. 1997b; Weigel et al. 1997). Localization of the three HAS gcnes on different
chromosomes and the appearance of HA throughout the vertebrate class suggest that
this gene family is ancient and the isozymes appeared by duplication early in the
evolution of vertebrates (Spicer et al. 1997b). Recently, Spicer and Nguyen ( 1999)
demonstrated that HAS-2 deficient embryos were deficient in HA and had severe
developmental defects, including a failure to form the endocardial cushions of the
hem (Spicer and Nguyen 1999). HAS- 1 and HAS-3 mutant mice have yet to be
reported.
The HAS protein has four trammembrane domains and one or more
amphipathic helices that may be partially embedded in the membrane (Watanbe et
al. 1996; Spicer et al. 1997a). Translocation of an HA chah across the bilayer by
such a small protein is difficult to envision. It has recenily been shown that
streptococcal HA synthases do not oligomerize, but require cardiolipin molecules
for maximal activity and stability (Tlapak-Simmons et al. 1998). Thus it has been
proposed that a collection of cardiolipin molecules may function with HAS proteins
to create or maintain a pore-like structure (Tlapak-Simmons et al. 1998).
It seems likely that most cells can synthesize HA at some point in their
natural life cycle and most of al1 during periods of rapid growth (Fraser and Laurent
1989; Fraser et al. 1994). The activity of hyaluronan synthase correlates with a
variety of cellular parameters inciuding ce11 growth, transformation and
differentiation (Prehm et ul. 1980), metastasis (Toole et al. 1979), ceIl migration
(Chen et al. 1989; Schor et al. 1989: Ellis et al. 1992; Ellis et al. 1996) and mitosis
(Moscatelli and Rubin 1975; Brecht et al. 1986). HA synthesis has been proposed
to increase dunng active division and decline at confluence. with fluctuations
during the ce11 cycle peaking at mitosis (Brecht et al. 1986; Matuoka et al. 1987;
Yoneda et al. 1988). HA extrusion ont0 the ce11 surface just prior to mitosis may
create a hydrated microenvironment which promotes partial detachment and
rounding of dividing cells (Brecht et al. 1986; Hall et al. 1994; Koochekpour et ul.
1 995).
HA synthesis is responsive to environmental factors such as hormones
(glucocorticoids, th yroid and sex hormones), vitarnins (vitamin C and retinoic acid).
growth factors (EGF, PDGF, TGFP, FGF, IGF- 1) and cytokines (IL- 1, IL-6 and
TNF) which can directly activate HA synthesis (Heldin et al. 1989; Honda et al.
1989; Sampson et al. 1992; Tammi et al. 1994; Ellis and Schor 1995; Tirone et al.
1997). Many of these factors, especially the growth factors, cm act in autocrine or
paracnne modes, and are frequently related to matrix changes in neoplasia and
inflammation. Furthemore, cyclohexirnide and protein kinase inhibitors have been
shown to inhibit synthesis, indicating that transcription as well as phosphorylation is
required for activation (Klewes and Prehm 1994).
1.1.4. HA degradation
Circulating levels of HA in human semm are nomally -30-40ng/ml
(Laurent et al. 1995). This is due to a rapid turnover of HA in the bloodstream that
results in a mean half-life for HA of only 2.5 to 5.0 minutes in heaithy adults (Fraser
and Laurent 1989; Laurent et al. 1996). Of the 1 O- 100mg/day entering the
bloodstream the majority (about 90%) goes to the liver, while the remainder goes to
the kidney (Laurent et al. 1987) and spleen (Bentsen et al. 1986).
Studies with separated liver cells (Smedsrod et al. 1984) have shown that
sinusoidal liver endothelial cells are pnmarily responsible for HA uptake.
Metabolic degradation of HA is principally intracellular and relies on receptor
mediated uptake (Eriksson et al. 1983; Smedsrod et al. 1984; Fraser et al. 1985;
Laurent and Fraser 1986; Raja et al. 1988). Endocytic degradation in the liver is
thought to be mediated by HARLEC an HA receptor complex of liver endothelial
cells which has been isolated from rat sinusoidal liver endothelial celis as. This
complex contains three proteins of 100, 166, and 175kDa proteins (Gustafson 1996;
Yannariello-Brown et al. 19961. HARLEC also recognizes chondroitin sulphate
(CS) and dermatan suIphate (DS). Thus CS given intravenously into rats prior to
intravenous HA c m inhibit the uptake of HA by the liver (Gustafson and Bjorkman
1997).
Abnormal HA accumulation has been noted in a variety of fibrotic disorders
(Whiteside and Buckingham 1989). Examples include sclerodenna (Freitas et al.
1 W6), hypertrophic scarring (Savage and Swann 1985) and fibrosis of liver and
lung (Frebourg et al. 1986; Bjemer et al. 1989). Because HA accumulation in
fibrotic disorden. understanding how HA is removed from connective tissues has
long been of interest (Fraser and Laurent 1989; Roden et al. 1989). Elevation of
serum HA levels also occuts in rheumatoid arthritis, liver cirrhosis and various
malignancies (Engstrom-Laurent 1989: Coppes 1993; Laurent et al. 1996).
HA is turned over locally within cells and tissues, particularly during
ernbryogenesis, tumourigenesis and upon injury. This process occurs in embryonic
tissues during development and also in remodeling tissues (Fraser and Laurent
1989). Numerous ce11 types including human synovial cells (Truppe et ul. 1977),
SV40-trinsformed 3T3 fibroblasts (Culty et al., 1992), embryonic myocardial cells
(Bernanke and Orkin 1984), macrophages (Culty et al. 1992; Gustafson and
Forsberg 199 1) and human breast cancer ce11 Iines (Culty et al. 1994) have been
shown to degrade HA.
Studies both on cultured cells and on developing tissues suggest that the
mechanism of HA degradation may require intemalization/endocytoçis before
degradation. Degradation of HA is thought to occur in lysosomes because the
hyaluronidase is an acidic pH requiring enzyme (Orkin and Toole 1980; Orkin et al.
1982; Bernanke and Orkin 1984). Indeed, inhibitors of lysosomal acidification such
as ammonium chloride and chloroquine block HA degradation (Alston-Smith et al.
1992; Culty et al. 1992).
HA degradation in macrophages fibroblasts (Culty et al. l992),
chondrocytes (Hua et al. 1993) and several tumour ce11 lines (Culty et al. 1994).
requires CD44 Underhill and coworkers (Underhill et al. 1993; Pavasant et al.
1994) have shown that CD44 mediated removal of HA occurs at several stages of
developrnent. For instance, dunng lung developrnent. macrophages expressing
CD44 increase in number in parallel with a marked decrease in interstitial HA
concentration; injection of a hlocking antibody against CD44 into newbom mice
was shown to cause an increase in lung HA relative to controls (Underhill et al.
1993). In embryonic skin, newly forming hair follicles are associated with
condensing mesoderm; this process of mesodemal condensation is also associated
with lung HA degradation and elevation of CD44 (Underhill et al. 1993). During
bone development, lacunae surrounding hypertrophic chondrocytes are highly
enriched in HA (Pavasant et ni. 1994) and swelling pressure exerted by this HA
contributes to expansion of lacunae as bone growth occurs (Pavasant et al. 1996).
Subsequently, in the zone of erosion, the HA within these lacunae has been
proposed to be removed via CD44-rnediated endocytosis (Pavasant et al. 1994).
Upon injury, metabolism of HA has been observed to rnodify the outcome of a
wound (Burns er al. 1997). Likewise, in neoplasia HA deposition exceeds
degradation (Auvinen et al. 1997; Ropponen et al. 19%).
Under specific conditions HA degradation may also occur extracellularly.
For exarnple, HA within the cartilage ECM becomes partially depolyrnerized, most
likely due to the action of free radicais (Baker et al. 1989; Ng et al. 1992). Free
radicals can be generated by ionizing radiation and are also present at wound sites
(Forschi et al. I W O ) . Such initial depolyrnerization of HMW HA may an initial
step which facilitates HA catabolisrn. For instance, McGuire et al. ( 1987)
demonstrated that myocardial cells bind, intemalize and degrade partially
depolymerized HA more efficiently than HMW native HA. Thus partial
extracellular together with endocytosis results in the complete degradation of the
HA (Hua et al. 1993).
1.2. Functions of HA
Through its complex interactions with matrix components and cells, HA has
multiple roles in biology. These biological roles range from mechanical functions
in the extracellular matrix to regulation of cellular behavior. HA affects many
biologic properties including wound healing (Chen and Abatangelo 1999),
angiogenesis (Rooney et al. 1995), embryonic morphogenesis (Toole 1997), turnour
metastasis (Bourguignon et al. 1997), inflammation (McKee et al. 1996), cell
adhesion (Hall et al. 1994; Klein et al. 1996) ce11 migration (Chen et al. 1989;
Melrose et al. 1996) and proliferation (Wiig et al. 1996; Bertrand et al. 1997).
1.2.1. Physicochemical functions of HA
HA function has long been attrîbuted to its physical and chemical properties
and its interaction with oiher macromolecules. Because of its water attracting
properties, HA plays a role in the osmotic homeostasis of the extracellular space
(Comper and Laurent 1978; Comper and Zamparo 1990). HA meshworks also
exhibit macromoleular exclusion and thus can regulate the distribution and transport
of plasma proteins in the tissues such that small molecules move freely in the
network, while larger particles become immobilized (Scott et al. 1989). This steric
exclusion property of HA are thought to serve to protect cells by hindering the
approach of particles that would otherwise cause darnage (Presti and Scott 1989)
and serve as a barrier against the spread of infectious agents (Laurent and Fraser
1992). The rheological properties of HA solutions showing both viscoelasticity and
shear dependence have been related to its lubricating functions in joints and other
tissues (Balzacs and Delinger 1993).
1.2.2. HA binding proteins: the hyaladherins
Although HA is not covalently linked to proteins like other GAGS, several
extracellular intracellular and plasma membrane proteins bind to it with high
affinity (Figure 1.3.). These HA binding proteins, or hyaladherins, are widely
distributed in the body and have diverse functions (Toole 1997). Extracellular and
ceIl surface hyaladhenns are important in modifying matrix assembly, cell-matrix
and ceil-ce11 interactions. Besides their extracellular structural and orgünizational
functions, of particuiar interest is the discovery of hyaladherins that exist within
cells.
Cellular hyaladherins can be grouped into at least two classes of proteins.
CD44 is a prototype of the type 1 transmembrane HA receptor. This protein is
predominantly present at the ce11 surface but can be shed into the extracellular
space. CD44 binds to HA via a complex site known as the link module (Kohda et
al. 1996). RHAMM is a prototype of cell-associated hyaladherins that occur at
ECM Proteins versicad
CD40 Family hyaluronec tin link protein aggreçan neurocan brevican fibrinogen
Soluble Proteins Trypsin inhibitor 1
I
Figure 1.3. Hyaluronan binding proteins - the Hyaladherins (modified from Pniwand et al. 1999)
multiple cellular loci, including the cell surface, cytoplasm and nucleus. This
group, including RHAMM, cdc37, and p68 are characterized by a lack
transmembrane domain, signal sequence or link module. HA binds to this class of
hyaladherins through short, basic amino acid motifs (Yang er al. 1994; Kohda et al.
1 996).
1.2.2.1. CD44
CD44 was first described as a ce11 surface molecule of T-lymphocytes,
granulocytes, and cortical thymocytes (Dalchau et al. 1980). This protein was then
rediscovered as the phagocytic glycoprotein 1 (Pgp-1) (Mackay et al. 1988), and
GP90 Hennes (Goldstein et al. 1989). CD44 is now known to be a widely-
expressed receptor for HA (Underhill et al. 1987; Aniffo et al. 1 990).
CD44 consists of an external domain, highly conserved transmernbrane
domains and cytoplasmic domains (Naor et al. 1997). There are 20 known isofoms
of CD44 which are created through alternative splicing of 10 variant exons
(reviewed in Ponta et al. 1998). The smaliest isoform, known as CD44s, lacks al1
ten variant exons. CD44s is the most abundant isoform in vivo. Additional post-
translational modifications of CD44 include phosphorylation and palmitoylation in
the cytoplasmic domain, N- and 0- linked glycosylation and addition of GAGS in
the external domain (Lesley et al. 1993). Dimerization of CD44 has been
implicated in enhanced binding of HA can occur naturally with CD44v4v7
(Sleeman et al. 1997), caii be induced experimentally with certain divalent
antibodies to CD44 (Lesley et al. 1993), or can be induced by constructing CD44
with cysteine in the intramernbranous domain that forms disulfide cross 1 inks
(Perschl et al. 1995).
Interestingly, not al1 CD44-expressing cells are able to bind HA. This
property can be acquired or can occur transiently (Hyman et al. 199 1). The ability
of HA to bind to CD44 is regulated by both protein conformation, rather like
integrin activation, and by glycosylation patterns. Thus, CD44 can be stimulated to
bind HA by phorbol esters, anti-CD44 antibodies, or degIycosylation (Sherman et
al. 1994). Blocking anti-CD44 mAbs studies suggest that topography of the CD44
epitopes and their orientation toward the HA binding site determine the ability of
antibodies to interfere with HA binding (Lesley et al. 1993; Zheng et al. 1995).
Clustenng of CD44 proteins, which is dependent upon cytoskeletal proteins, also
seems important to its ability to bind HA (Lokeshwar et al. 1994). Certain cells,
including some B and T ce11 lines, appear constituitively able to bind HA.
However. further studies are required to define the molecular mechanisrns that
result in CD44/HA interactions as well as to assess the impact that these
interactions have on ce11 behaviour.
CD44 is a multifunctional receptor involved in cell-ceIl and ceII-ECM
adhesion, i.e. ce11 motility, trafficking, 1 ymph node homing, lymphocyte activation,
presentation of chemokines and growth factors to traveling cells, and transmission
of these growth signals (Lesley et al. 1993). As well, CD44 participates in the
endocytic uptake and intracellular degradation of HA (Culty et al. 1992; Hua et al.
1993), and transmission of signals mediating hematopoiesis and apoptosis (Ayroldi
et al. 1995; Henke et al. 1996) that are relevant to tumour progression and wound
repair. CD44 is constituitively expressed in regions of active ceIl growth (Mackay
et al. 1994).
CD44 expressed on turnour cells and host tissue stroma1 hyaluronan can
enhance growth and invasiveness of certain tumours. Disruption of CD44-HA
interaction by soluble recombinant CD44 has been recently shown to inhibit tumour
formation by lymphoma and melanoma cells transfected with CD44 (Zeng et al.
1998). CD44 is expressed on many types of cancer cells, such as malignant gliomas
(Koochekpour et al. 1995) human breast cancer cells (Culty et al. 1994), colon
cancer cells (Ropponen et al. 1998) ovarian cancer cells (Catterall et al. 1997) and
basal ce11 carcinomas (Baum et al. 1996). A diverse set of CD44 receptor isofoms
is found on breast cancer ce11 lines, not only among different ce11 lines but also
within individual ce11 lines (Culty et al. 1994). Al1 ce11 lines investigated can bind
HA. Interestingly HA binding and CD44 expression is positively correlated with
invasive potential of breast ovarian and colon cancer ceIl lines (Culty et al. 1994;
Catterall et al. 1997: Ropponen a al. 1998). Disruption of the CD44 function on
mammary carcinoma cells induces apoptosis (Yu et al. 1997). In basal ce11
carcinoma expression of CD44 is found to be down regulated, while the expression
of the CD4.4~6 isoform is increased (Baum et al. 1996). This variant of the receptor
seems to be linked to cellular functions, while the absence of the CD44s accounted
for the noninvasive character of this type of tumour.
The interaction of HA with the CD44 receptor is also involved in
inflammation (Lesley et al. 1997), and liver cirrhosis (Rockey et al. 1998).
Synovial fibroblasts isolated from patients with osteo- or rheumatoid arthritis were
shown to exhibit complex splicing variations in their CD44 expression (Croft et al.
1997). Noninflamed tissue samples showed no such splicing events. It was found
that rheumatoid tissue contained higher levels of expressed CD44, while
osteoarthritic tissue samples showed greater variety in their CD44 expression. The
interaction of HA fragments with the CD44 receptor on macrophages in inflamed
tissues can induce the expression of inflammatory genes of the chemokine family
(IL- 1 B. TNF-a and IGF- 1 ; Noble et al. 1993; McKee et al. 1996) and nitric-oxide
synthase through a nuclear factor kappa beta (NFuP) dependent mechanism (Noble
et al. 1996; McKee et al. 1997). A similar effect of HA fragments on nitric oxide
synthase was observed in liver endothelid cells and Kupffer cells (Rockey et al.
1998). It is proposed that the various interactions of CD44 with HA fragments
might sustain or increase the process of inflammation.
An extensive body of evidence exists suggesting that CD44 is involved in
angiogenesis (Goshen et al. 1996; Trochon et al. 1996; Deed et al. 1997; Griffioen
et al. 1997; Ozer et al. 1997). Endothelia1 cells of solid turnour vasculature have
been found to display an enhanced expression of CD44, an upregulaiion that could
be induced by angiogenic factors such as FGF and VEGF (Griffioen et al. 1997).
Further, it has been demonstrated that LMW oligosaccharides HA (HAS-50) can
induce angiogenesis (West et al. 1985), while HMW HA in the ECM has been
shown to inhibit angiogenesis (Dvorak et al. 1987; West and Kumar 1989). Both
should bind to ce11 surface recepton but how they have different elfects on cells in
cornparison to HA oligosaccharides is unclear.
CD44 interactions with angiogenic oligosaccharides of HA have also been
found to upregulate the expression of early-response genes such as c-Jos, c-jun,
junB. Krox-20 or Krox-24 in endothelial cells (Deed et al. 1997). The products of
these genes are known to act as transcription factors for genes encoding
extracellular proteinases, which are additional factors that promote endothelial ce11
migration and thus angiogenesis.
1.2.2.2. RHAMM and related proteins
RHAMM is a member of a group of cell-associated hyaladherins including
p68 and cdc37 that localize to several subcellular loci and that perform multiple
functions in regulating ce11 motility and ce11 cycle (Turley et al. 1982, 1994;
Grammatikakis et al. 1995; Deb and Datta 1996; Mohapatra et al. 1996; Toole et al.
1997; Wang et al. 1998). For instance, cell surface foms of RHAMM are
transiently expressed in most cells but are nevertheless key to regulating cell
motility as determined by antibody blocking experiments (Samuel et al. 1993; Hall
et al. 1994, 1995, Pilarski et al. 1994; Turley et al. 1994; Savani et al. 1995a,
1995b; Nagy et al. 1995; Masellis-Smith et al. 1996; Delpech et al. 1997; Zhang et
al. 1998). Intracellular forms of this class of hyaladherins, including RHAMM,
bind to and chaperone signaling molecules involved in regulating cell cycle and ce11
motility (Grammatikakis et al. 1995; Mohapatra et al. 1996; Kimura et al. 1997).
These types of hyaladherins may also perform functions within the nucleus. Such
hyaladherins typically lack a link module for binding HA but rather utilize short
sequences encoding basic amino acid motifs (Yang et al. 1994) which are required
for ce11 motility and proliferation (Samuel et al. 1993; Sherman et al. 1994). Even
though they are present on the cell surface (Samuel et al. 1993; Sherman et al.
1994; Grammatikakis et al. 1995; Kirnura et al. 1997; Crainie et al. 1999) this class
of hyaladherins is also characterized by an absence of both signal sequences and
transmembrane domains. Therefore, the molecular bais for their subcellular
distribution is not yet clear. Based upon their modular and dynamic subcellular
location and the different mechanisms by which they bind to HA, these proteins
likely regulate cell motility and ce11 cycle in a manner that is fundamentally distinct
from the more well characterized HA receptor, CD44
Surface RHAMM regulates signals generated by both HA (Turley 1982;
Entwistle et al. 1996; Toole et al. 1997) and growth factors such as PDGF (Zhang et
al. 1998) and TGFP (Samuel et al. 1993). The ability of HA to signal motility via
RHAMM implies that the HA binding domains are also necessary for signal
transduction. Interestingly, the HA binding domains of intracellular foms of
RHAMM are required for activation of erk kinase by mutant active ras and by
growth factors that activate ras, such as PDGF (Zhang et al. 1998).
Signais from HAkell surface RHAMM interactions are associated with
activation of src (Hall et al. 1996), focal adhesion turnover (Hall et al. 1994) and a
reduction in the tyrosine phosphorylation focal adhesion kinase (FAK) (Hall et al.
1995). RHAMM-mediated activation of src is required for HAIRHAMM-regulated
fibroblast motility and both locomotion of these cells and activation of src by HA
can be blocked with anti- RHAMM antibodies (Hall et al. 1996). However, celis
transfected with either a constituitively active f o m of src or v-src no longer require
ceIl surface RHAMM (as detected by antibody blocking) for signaling motility,
indicating that src is downstream of ce11 surface RHAMM. Nevenheless, v-src-
induced disassembIy of focal adhesions cannot occur in the absence of cellular
expression of RHAMM (Hall et al. 1996). This, although not confirmed
experimentally, is most easily interpreted by proposing that intracellular isoforms of
RHAMM are required for src-generated effects on the cytoskeleton and is consistent
with the ability of a RHAMM isofom to CO-immunoprecipitate with src (Hall et al.
1 996).
In addition to its effect on src signding, RHAMM isoforms encoding exon
9, appear under certain conditions (e.g. subconfluence) to control the erk kinase
cascade through ras (Zhang et al. 1998). Mutations of domains of intracellular
RHAMM isoforms block signaling through ras (Hall et al. 1995) and activation of
erk (Zhang et al. 1998). Specifically, RHAMMA 1-5 (73 kD) interacts specifically
with erk 1 kinase and its upstream activator MEK in transfonned cells (Zhang et al.
1998; Paiwand et al. in preparation) and its overexpression constituitively activates
this cascade. Thus, at least one form of RHAMM, in a manner possibly analogous
to cdc37 (Kimura et al. 1997, Silverstein et al. 1998), directly associates with
kinases that regulate transformation, proliferation and motility (Zhang et al. 1998).
Imponantly, a RHAMM isofortn of the same molecular weight predicted by the
RHAMMA 1-5 cDNA (e.g. 70-73 kDa) is uniquely upregulated after wounding of
smooth muscle ceII monolayea (Savani et al. 1995). Furthemore, manipulation of
RHAMMA 1-5 expression or funciion alters the ability of PDGF, a key growth
factor in response-to-injury processes, to activate signaling cascades (Zhang et al.
1998) and modify actin assembly (Hall et al. 1994, Chang et al. 1997). The role of
erk in these functions remains to be investigated.
A recently characterized intracellular HA binding protein encodes a 29.3kDa
chicken homologue protein homologous to cdc37, an essential ce11 cycle regulatory
factor previously characterized genetically in yeast and Drosophila (Grammatikakis
et ai. 1995). These findings suggest a role for HA in cell division control. Cdc37 is
associated with Cdk4 through HSPSO, further suggesting that cdc37 may regulate
the rnammalian ce11 cycle through direct effect on Cdk4 (and thus the GIS
boundary) (Dai et al. 1996). Further, raf and src are known to directly interact with
HSP90lp50-cdc37 complexes (Perdew et al. 1997).
A recently characterized 34kDa HABP is a new member of the cellular
hyaladhenns as demonstrated in a wide viuiety of ceIl lines. This protein has been
found to form a homodimer of 68kDa (p68) that binds specifically to HA (Deb and
Datta 1996). P68 has been shown to be highly phosphorylated in transformed
fibroblasts compared to normal fibroblasts, and phosphorylation was enhanced in
the presence of HA, PMA and calyculin-A (Rao et al. 1997). The phosphorylated
form of p68 is show on the ce11 surface and cm be detected in semm free medium
(Rao et al. 1997). P68 has a 100% homology with gClq, the complement protein
(Ghebrehiwet et al. 1994) and has been demonstrated to be the human counterpart
of munne YL2 (Luo et <il. 1994). Thus it has been proposed to modulate the
function of Rev which is expressed at the early stage of HIV 1 and act as viral
transactivator for the replication of HIV I (Das et al. 1997). Interestingly, the cDNA
sequcnce of p68 has complete homology with the cDNA sequence of a protein P-32.
CO-purified with the human pre-mRNA splicing factor SF2 (Krainer et al. 1991).
1.2.2.3. Hyaluronan and tumourigenesis
It has been clear for years that the HA content of tumours is often elevated
(Knudson et al. 1989), which is consistent with the raised concentrations of HA in
semm of cancer patients (Kumar et al. 1989). Although increased HA synthesis is
not a universal characteristic of tumours, there seems to be an overall tendency for
transfomed cells to exhibit higher levels of HA production (Kimata et al. 1983;
Turley and Tretiak 1985).
In cell culture many cell types have reduced capacities to synthesize HA
even when derived from tumours enriched in HA (reviewed in Knudson et al.
1989). These cm nevertheless stimulate HA synthesis by normal fibroblasts.
Production of tumour associated HA occurs via tumour-stroma1 cell interactions.
Also some human tumour cells also possess unoccupied high affinity ce11 surface
binding sites for HA which rnay allow tumour ceils to interact directly with HA
enriched ECM. This may allow them to use HA as a support for adhesion and
locomotion and guide them into surrounding stroma.
Takeuchi et al. ( 1976) reported that the HA con tent of malignant breast
lesions was higher than that of normal tissue. Bertrand et al. (1992) confirmed the
finding and extended it by showing that the increase was more marked in the
peripheral invasive areas of the tumour than in the central parts. Histochemical
staining localized HA over-expression in the malignant stroma, whereas the tumour
epithelium was HA negative (de la Torre et al. 1993). Further, Auvinen et al.
( 1997) have dernonstrated HA expression was restricted to the stroma1 connective
tissue in benign lesions, while in malignant breast tumours the intensity of stroma1
HA staining was significantly stronger and was also detected in the cell membranes
and cytoplasm of adenocarcinorna cells. Victor et al. ( 1999) recently showed that
the passage of human breast cancer cells from the primary state to the metastatic
state was characterized by a drarnatic increase of HA and hyaluronidase production
and expression of HA-binding sites at the invasion areas. Thus, both synthesis and
degradation of HA presumably support tumour invasion and growth.
It has recently been shown that invasive breast adenocarcinorna has a
significantly higher level of hyaluronidase activity, correlating with its invasive
potential (Madan et al. 1999). This hyaluronidase can disrupt basement membrane
integrity and produce an angiogenic response that has been implicated in tumour
invasiveness and metastasis (Madan et al. 1999). In contrat, HYALI, a widely
expressed hyaluronidase in somatic tissues (Frost et al. 1997) has been proposed as
a candidate tumour suppressor gene as it is found at the sight of a common tumour
suppressor locus (Csoka et al. 1998). This is a locus frequently deleted either as a
loss of heterozygocity or hornozygous deletion in a nurnber of human rnalignancies,
particularly tobmco-related lesions of the mouth, head and neck, and of the lung
(Killary et al. 1992; Buchhagen 1996). HYAL-I activity is absent or strongly
suppressed in squamous ceIl carcinorna cell lines compared to normal keratinocyte
controls (Frost et al. in prepnration).
In a recent study, HA intensity was shown to be stronger in high grade
colorectal tumours (Ropponen et al. 1998) and to be prognostic of poor outcorne.
The abnormal expression of HA in the neoplastic colon epithelial cells is suggested
to provide a distinct advantage for invasive growth and metastasis.
Itano et al. ( 1999) have recently implicated HA in turnourigenesis through
selection of mouse mammary carcinoma cell lines mutant deficient in HA. These
mutants show loss of HA production and loss of periceIlular HA coats, and more
important, decreased metastatic ability. Further, transfection with the HA synthase
HAS- 1 can rescue these mutants (Itano et al. 1999). In a related study, human
HTlOSO cells were transfected with HAS2, which caused the ceils to grow more
rapidly and produce tumours that were 2-4 times larger than control (Kosaki et al.
1999), providing more direct evidence for HA'S role in tumourigenicity.
1.2.2.4. Hyaluronan and wound healing
As wound healing progresses in vivo there are changes in the composition of
the extracel1ular matrix. Initially, a wound contains a fibrin-rich matrix, followed
by a HA-nch matrix (Bertolami and Donoff 1978). HA is then removed by
hyaluronidases and then there is a deposition of a fine soft ternporary reticular
collagen, called Type DI, which is in tum repiaced by fiben of Type 1 coilagen. The
latter is the hallmark of a mature scar. In fetal wounds there is no scarring. It is
tempting to speculate that the high concentrations of HA which persist in fetal
wounds (Longaker et al. 1989; 1990; 199 1 ; Mast et al. 1992) may in part underlie
scar free healing.
HA also functions in wound repüir as a regulator of ce11 motility. This may
be mediated through both its physicochemical properties as well as through direct
interactions with cells. in the former, HA appears to provide an open, hydrated
matrix that pemits cell migration (Toole et al. 1997), whereas in the latter, HA:cell
interactions via cell surface hyaiadherins actively promote migration. RHAMM in
particular forms links with several protein kinases associated with ce11 locomotion,
for e.g., ERK, p IZFak and pp6Oc-src (Hall et al. 1994; Hall et al. 1996; Wang et
al. 1998). Kobayashi and Terao (1997) have shown a dose-dependent increase of
the proinflammatory cytokines TNF-a, IL- 1 and IL-8 production by human uterine
fibroblasts at HA concentrations of IOug/ml to lmg/ml via a CD44 rnediated
mechanism, which may also contribute to ce11 motility as well as impact on the
progress of wound healing.
Consistent with the model that scarless wound healing is due in part to HA.
many reports have attested to the ability of exogenous HA to produce beneficial
wound healing outcome (reviewed in Chen and Abatangelo 1999). In animal
experiments. topically applied HA has been shown to accelerate skin wound healing
in rats (Abatangelo et al. 1983; Cabera et al. 1995) and hamsters (King et al. 199 1 ),
and to enhance repair of tympanic membranes (Hellstrom and Laurent 1987). and
comeal epitheliai wounds (Nakamura et al. 1992). In chronic wounds such as
venous Ieg ulcers, HA application has been shown to promote healing (Falanga et
al. 1996). These results and the observation that intrauterine treatment of fetal
wounds with hyaluronidase convened the healing frorn a fetal type to the scamng
rnodel observed ex utero (Mast et al. 1992; West et al. 1997) are consistent with a
persistence of HA as one necessary feature for scarless healing to take place.
1.3. Rationale of this study
In addition to its role in the ECM and at ceIl surfaces, evidence is growing
that HA is also present in the cytoplasm and nuclei of cells in a number of tissues in
vivo (Margolis et al. 1976; Furukawa and Terayama 1977; 1979; Londono and
Bendayan 1988; Ripellino et al. 1989; Kan 1990; Eggli and Graber 1995).
Furthermore, recent studies have shown that there are a number of intracellular
hyaladherins that may be important in regulation of the ce11 cycle or in gene
transcription (Grarnmatikakis et al. 1995; Deb and Datta 1996; Hofmann et al.
1998; Zhang et al. 1998). For example, intracellular foms of the hyaluronan
receptor, RHAMM, have been shown to regulate erk kinase activity (Hofmann et al.
1998; Zhang et al. 1998). and a vertebrate homologue of the ce11 cycle control
protein cdc37 was recently cloned and found to bind HA (Grammatikakis et al.
1995). Thus we postulate that intracellular HA may be targeted to sites relevant to
intracellular hyaladherin expression by a novel mechanism, there to exert effects
relevant to diverse cell processes. To investigate this we have created a fluorescent
conjugate of HA which can be used to determine the mechanisrn of transport and
potential functions of intracellular HA.
MATERIALS AND lMETHODS
11.1.. Ce11 Culture
Murine IOTU2 parental, ras-transformed (C3) (Egan et al. 1987) or
vRHAMMA 1 4-transfected cells (LR2 I ; Hall et al. 1995); parental and connexin43
transfected gliorna cells (provided by J. Nagy, Winnipeg, MB); and human breast
cancer MDA-MB-23 1 and MCF-7 cell lines purchased from American Type Culture
Coilection (Rockwell, MD) were al1 maintained at 37°C in 5% CO2 on lûûmm
plastic tissue culture dishes (Nunclon) in DMEM (GibcoBRL) supplemented with
10% (vlv) fetal calf semm (FCS) (Intergen) and 1 OmM HEPES (Sigma) (growth
medium), pH 7.3. Cells were routinely subcultured using 0.25% trypsinll mM EDTA
in HBSS (GIBCO BRL) from 80% confluent cultures and passaged at a 1 : 10
dilution. Transfected cells were routinely selected using 900pg/ml geneticin (Gibco
BRL). Al1 cells were used under passage 25. Frozen stocks of cells were prepared
by resuspending the cells in freezing medium (70% DMEM, 20% FCS, 10%
DMSO), and then stored under liquid nitrogen.
11.2. Conjugation of HA with Texas Red
HA (Hyal Pharmaceutical Corp., Mississauga, Ontario) suspended in 20mM
MES (Sigma), pH 4.5, with 30% ethanol, was mixed with three-fold excess EDCI
(Aldrich) as per the number of HA disaccharides. Texas red hydrazide (Molecular
Probes) dissolved in DMF (Aldrich) at a molar ratio of probe to disaccharide of 1 : 10
was added and the mixture was shaken overnight at room temperature.
Unconjugated material was then removed with didysis in 10 000 MWCO
membranes (Pierce) against 75mM NaCl and 40% ethanol for 6 days at 4°C in the
dark. The product (see Figure 11.1) was lyophilized for storage. Analysis of success
of conjugation was conducted with gel permeation chromatography using refractive
index and UV absorption to detemine purity and the molecular size range of the
bioconjugate (see Appendix 2). The molecular weight of the HA conjugated to the
probe was shown to be lOOkDa and the success of conjugation was shown to be
approximately one rnolecule of Texas Red per fifteen hyaluronan disaccharides (see
Appendix 2 - Figure VII.1.).
TEXAS RED HYDRAZIDE HYALURONAN
Figure II.1. Conjugation of hyaluronan to Texas Red hydrazide
11.3. Treatment of cells with TR-HA and analysis of resultant
intraceliular fluorescence
Cells were seeded in DMEM (Gibco BRL) supplemented with 10% FCS
(Intergen) at 60% confluence on 25mm sterile g las coverslips (VWR) in 35mm
tissue culture plates (Nuncion) [except cells plated for oligosaccharide treatments,
which were plated on 13mm glas covenlips (VWR) plated in 24 well plates
(Falcon)]. After 8 h at 37°C the culture medium was aspirated, cells were rinsed and
medium was replaced with serum-free DMEM containing 4 pg/ml transfemn
(Human, Gibco BRL) 4pglml insulin (Bovine; Gibco BRL) and lOmM Hepes
(Sigma) (defined medium), for 12 h at 37°C. Where indicated cells were pretrcated
with 5Opghl BrefeldinA (Molecular Probes) for 30 minutes; lOmM colchicine
(Sigma) for 30 minutes; lOOnM phorbol 12-myristate 13-acetate (PMA; Calbiochem)
for up tto 4 h; or 50pg/ml MEK inhibitor (PW98059; Oncogene Science; dissolved
in DMSO; Alessi et ni. 1995) for 2 hours; at 37°C and 5% CO2 in defined media.
The cells were then gently exposed to 100pg/ml (unless concentration otherwise
indicated) TR-HA conjugate in 1 ml of (37OC) defined medium, wrapped in foi1
incubated at 37OC with 5% CO? for 1 O minutes (unless incubation time otherwise
indicated). The cells were then rinsed twice in cold 5X PBS to prevent release of
bound hyaluronate during washing (Underhill and Toole 1980; McGuire et al. 1987),
and fixed in a solution of 2% paraformaidehyde and 1 % cetylpyridinium chloride
(CPC), which precipitaies glycosarninoglycans, in a O. 1 M Na-phosphate buffer pH
7.4 for 10 minutes at room temperature. Coverslips were then washed three times
for 5 minutes each in 1 XPBS (2.7mM KCI; 1.1 rnM KH2P04; l28mM NaCI; 8.1 mM
Na2HP04; pH 7.4), mounted with elvanol (PVA 15%, glycerin 30%), and viewed on
a Zeiss Axiophot 100 confocat microscope using the 63X objective (unless othenuise
specified). To ensure that confocal images represented internalized TR-HA, some
cultures were exposed ro TR-HA for 10 minutes, washed, then digested with
Streptomyces hyaluronidase ( 1 IU/ml at 37OC for 1 h to remove TR-HA remaining
on the ceIl surface that might interfere with confocal images), prior to viewing.
Images represent a z-axis slice through the center of a cell interior. Analysis was
done with UTHSCA Image Tool version 1.28 (University of Texas Health Sciences
Centre in San Antonio, Texas) using zero thresholding of equal central sections
taken from the perinuclear areas of cells. Staining of cells with different ce11
thicknesses were equilibrated by dividing mean fluorescent intensi ty by the intensity
of the FITC-Dextran counterstain ( 10,ûûûMW lysine fixable; Molecular Probes).
Laser settings were constant for al1 images compared.
11.3.1. Digestion of TR-HA with hyaluronidase
Digestion of 1 5 0 ~ of TR-labeled HA prior to addition to cells was carried
out in acetate buffer pH 5.0, using 15 TRU of Streptomyces hyaluronidase (Sigma)
for 24 h at 37°C in a proteinase inhibitor buffer (25rnglml ovomucoid; 1 .Omg/ml
pepstatin A; 18.6mg/ml iodoacetic acid; 37rng/ml EDTA; 17.1 mg/ml PMSF). The
mixture was heat inactivated at 56°C for 30 minutes to destroy enzyme activity.
Control incubations (minus the hyaluronidase) were carried out under the sarne
conditions. Digestion products were used to treat cells as in IT.3.
11.3.2. Cornpetition with unlabeled HA and HA oligosaccharides
To assess specificity of uptake. cultures were also exposed to 80pg/ml TR-
labeled HA cornbined with excess (4mg/ml; i.e. 5OX excess) unlabeled HA (Hyal
Pharmaceutical, Mississauga, Ontario) or HA oligosaccharide (HA4, HA8, HA 10,
HA12; provided by R. Tammi, Kuopio, Finland). Cells were treated and fixed as
described above in II.3.
11.3.3. Treatment of cells with Texas Red hydrazide probe alone
Cells were treated with 6.67pg/ml TR-hydrazide probe (equivalent aniount of
probe for 100pg/ml TR-HA given the determined ratio of 1 HA disaccharide per 15
Texas red molecule success of conjugation - see Figure VII.1.) followed by fixation
and analysis as above in II.3.
11.3.4. Treatment of cells with Sytol6
Cells were treated simultaneously with 5p.M Syto 16 live ceIl nucleic acid
stain (Molecular Probes) and with 100pg/rnl TR-HA for 10 minutes at 37°C and
fixed and analyzed as above in 11.3.
11.3.5. TR-HA concentration curves
vRHAMM t -5 transfected, ras transfected and parental 10T 112 rnurine cells,
and MDA-MB-23 1 and MCF-7 breast cancer cells were exposed to 0,O.O 1,0.05,
0.1,0.2,0.5,0.75, 1.25,2.O and 3.0mgM TR-HA, and 1 mg/ml FITC-dextran
( 10,000MW lysine fixable; Molecular Probes) for 15 minutes at 37OC followed by
fixation and analysis as above in 11.3. Confocal laser settings were kept constant
throughout andysis, on a low, medium and high setting (contrast = 153; 202; 264)
for Texas Red, and on a single laser for FITC-dextran, and different lasers were
equil i brated upon analysis.
11.3.6. TR-HA treatment of cells over tirnecourses
Cells were exposed to 150pg/ml TR-HA for 1 minutes. 15 minutes, 45
minutes, 2 hours, 6 hours and 20 hours at 37°C and 5% CO2. Al1 samples were
incubated with 1 mg/ml FITC-dextran ( 10,000MW lysine fixable; Mol Probes) 10
minutes prior to fixation as above in II.3.
11.3.7. Prebinding of peptides to TR-HA
150pg TR-HA was preincubated with 1 mg/ml of a peptide that mimics the
, Yang et al. 1994) or 1 mg/ml of a HA binding domain 1 of RHAMM (peptidem4''43'*
scrambled peptide of domain 1 which has been shown not to bind to HA (Yang et ai.
1994). for 2 h ai 37OC prior to cell treatment as in II.3.
11.3.8. Anti-CD44 and Anti-RHAMM antibody blocking
Live cells were preincubated for 30 minutes at 37°C and 5% CO? with
5Opg/ml with, anti-CD44 mAbs: KM201 (R&D Systems) or Hennes I (Endogen); or
~ ~ ~ ~ - R H A M M ~ ~ ~ " ~ pAb, or IgG (Sigma) diluted in defined media prior to addition of
SOpg/ml TR-HA for 5 minutes at 37°C and 5% CO?, followed by fixation and
analysis as in iI.3. (note: KM201 and RH^^^^^ antibodies were dialyzed
exhaustively to rernove sodium azide in 10,000MWCO membranes; Pierce).
11.3.9. AMAC labeled HA-oligosaccharide treatment of cells
Cells were treated with 1.67mg/ml AMAC Iabeled hyaluronan
oligosaccharides of discrete length (HA& HA12, HA26, HA30; provided by R.
Tammi, Kuopio, Finland), for 10 minutes at 37OC, and fixed and analyzed for
fluorescence as described in iI.3.
11.4. Wounding assays
100% confluent 10T 1/2 ce11 monolayers growing in DMEM (Gibco BRL)
supplemented with 10% FCS (Intergen) were scratch wounded, rinsed w i th fresh
DMEM/! 0%FCS and allowed to recover for up to 24 hours before TR-HA uptake
and analysis was performed as in II.3. Trypan blue (Gibco BRL) addition was used
to exclude areas of cells which sustained unrepaired injury.
11.5. Immunofluorescence
10T1/2 cells were seeded at 70% confluence, ont0 25mm glas coversiips and
allowed to attach for 24 hours in growth medium, washed and placed in defined
medium for 24 hours. Cells were rinsed 5x in lXPBS (pH 7.3) and fixed with
freshly prepared 3 8 paraformaldehyde (Sigma) in PBS, for 10 minutes at RT,
washed 5x for 5 minutes with LXPBS. Cells were permeabilized for 2 minutes with
0.2% Triton X-100 (VWK) in PBS, and washed 5 x for 5 minutes with 1XPBS. Cells
were then blocked for 1 hour at RT in 3% BSA (Sigma) in PBS washed and
incubated with 1 OAbs: anti-RHAMM R 10.1 pAb or anti-caveolin mAb
(Transduction Laboratories) at 150 in 1 % BSA (Sigma) in PBS, at 37°C for 1 hour.
Cells were then washed 5x for 5 minutes in lXPBS and incubated with 2"
antibodies: goat anti-mouse-RITC (Jackson) and goat anti-rabbit-FITC (Jackson) for
1 hour at 37OC in the dark, then washed 5x in IXPBS. Coverslips were then washed
in xylene, dried on paper towels, and mounted using "anti-fade" media (l0mM p-
phenylenediamine, l 18mM Tris-HCl, 90% glycerol, pH 7.4) and sealed. Cells were
visualized with a Nikon confocal laser scanning microscope.
11.6. Flow cytometry (FACS analysis)
70% confluent IOT1/2 cells, which had been in defined medium for 24 hours
previously, were incubated with 2.0m.M EDTA (ethylene-diaminetetraacetic acid) in
HBSS (Hank's balanced salt solution, Gibco), with 1OmM Hepes, for 5-6 minutes,
gently harvested with FACS buffer (10% FCS in HBSS with 20mM Hepes), and
pelleted by centrifugation at 1500rpm for IO minutes. Cells were then resuspended in
FACS buffer. An aliquot of the ce11 suspension was stained with trypan blue (0.4%
Sigma) and ceilular viability was determined to be above 90% using a
hemacytometer and trypan blue exclusion. Samples of 3x 10' cells were treated in
suspension with O. 1Wml HA (Healon) over a time course of 0,0.5, 1,3,5, 10, 15,
20, 25,30,60 minutes, followed by wash and centrifugation at 4OC, and
resuspension in 0.5ml of wash solution (HBSS; 10% FCS; lOmM Hepes) at 4°C.
Cells were then incubated with 1 : 100 dilution of 1 O Ab: anti-RHAMM R 10.1 pAb,
or IgG alone, for 45 minutes at 4OC, washed by centrifugation 2x, and resuspended
in wash solution. Cells were then incubated with 2' Ab: Goat anti-RHAMM-cy3 for
45 minutes at 4"C, washed 2x, and resuspended in 1 % paraformaldehyde (Sigma).
Cells were scanned using a coulter electronics EPICS 754 flow cytomeier for surface
RHAMM expression. Al1 tests were duplicated 3x.
11.7. DNaseI digestions
Genomic DNA was isolated from ras-transformed ceIl monolayers (as shown
in Current Protocols). Briefly cells were suspended with trypsin (Sigma), washed 2x
in HBSS, and resuspended in lysis buffer with proteinase K and incubated with
shaking at 50°C for 18 hours, then extracted. Precipitate was recovered with g las
rod and resuspended in lOmM Tris pH 8.0. Extraction was repeated with RNase
treatment.
Each sample consisted of: 0.24pg genomic DNA, 5p1 of 2x DNA binding
buffer and either 2 . 5 ~ 1 ( IOpg or 50pg) of HA or chondroitin sulfate or water.
Samples were incubated together at RT for 2 hours with shaking. Then DNaseI (0.0,
0.6, 1.8. 3.0,4.2, 5.4,6.6, or 7.8 x 104units respectively) was added to each sarnple
and incubated for a further 30 minutes. DNaseI was inactivated by heating at 65°C
for 15 minutes with 5mM EDTA. Sarnples were loaded onto a 1% agarose gel.
CHAPTER III
111.1. Texas Red labeled hyaluronan (TR-HA) is rapidly taken up
into ras-transformed cells and targeted to cell processes and the
nucleus.
Optical sectioning of cells, from which cell surface-bound TR-HA has been
removed with hyaluronidase, shows an intracellular location of the TR-HA within 10
minutes after treatment of subconfluent monolayers of ras-transfomed cells (Fipre
111.1.). TR-HA rapidly accumulates within ce11 lamellae and nuclei (Figure III.1.).
Some general cytoplasmic and striking perinuclear accumulation is also observed
(Figure 111.1.). TR-HA within the nucleus is seen in the same confocal plane as a
live cell nucleic acid stain. confirming its location in the nucleus (Figure 111.2.)
Streptomyces hyaluronidase-digested TR-HA (Figure I113a.) or TR-HA rnixed with
excess unlabeled HA (Figure I11.3b.) block of the TR-HA uptake by cells,
indicating that unlabeled HA cornpetes effectively for the uptake of TR-HA and that
the uptake requires larger than hexa- saccharide units of HA respectively. Cells
treated with Texas-red hydrazide aione (Figure Il1.3c.) show no staining.
111.2. TR-HA uptake is dose and time dependent and increases with
cellular transformation.
TR-HA concentration curves indicate that uptake of TR-HA is dose
dependent until an apparent saturation point is reached (Figure III.4.A.). Of the
five ce11 lines examined, the highest levels of intracellular HA accumulation occur
in ras and RHAMM transformed and as compared to their parental IOT112 cells
(Figure 1IIA.A.). Further, overd1 HA uptake is enhanced in invasive human breast
epi thelial ce11 line MDA-MB-23 1 cells as compared to the non-invasive MCF-7
breast cancer cell lines (Figure II1.4.A.) (note, MDA-23 I contains mutant active
ras, while MCF-7 does not). When these ce11 lines are examined over a 20 hour
tirnecourse (Figure 111.4.B.) it is seen that TR-HA uptake achieves its highest levels
within the first hour of incubation and decreases thereafter. Confocal images
illustrate differences in both TR-HA intensity and intracellular distribution amongst
the five cell lines (Figure III.4.C.). RHAMM and ras transformed, as well as
MDA-23 1 invasive breast cancer cells accumulate significant amounts of
intracellular TR-HA, funher, HA appears to be targeted strongly to the tips of ceil
processes and lamellae of these cells (Figure III.4.C.). Interestingly, non-
transformed IOT1/2 and non-invasive MCF-7 (which do not express mutant active
ras) cell lines display weaker TR-HA accumulation and more diffuse intracellular
distributions.
111.3. Specific HA oligosaccharides inhibit TR-HA uptake
Simultaneous treatment of cells with TR-HA and excess amounts of different
sizes of HA-oligosaccharides were used to determine the minimum effective length
of oligosaccharide which would interfere with cellular uptake of the TR-HA probe.
Cells treated with HA oligosaccharides composed of ten monosaccharides (HA IO) or
fewer appear to have no significant effect upon TR-HA uptake (Figure In.5.).
However, cells treated wi th excess HA oligosaccharide of 1 2 monosaccharides
(HA 1 2) show a significant overall decrease in TR-HA intracellular intensity (Figure
III-S.), indicating that the HA 12 is the minimum effective length of oligosaccharide
that is able to effectively compete with full length TR-HA for uptake. However the
degree of blocking with HA12 does not achieve the level of inhibition shown for
cornpetition with excess unlabeled HA (Figure 1II.S.A.g).
111.4. Thirty HA-saccharide minimum length is required for
effective targeting of HA to the nucleus.
In order to determine the minimum size of HA necessary to achieve cellular
uptake and intracellular targeting to different cellular compartments. different sizes
of HA oligosaccharides were fluorescently labeled such that their intracellular
accumulation could be monitored. It appears that HA8 and HA12 are internalized by
celis at extremely low levels, if at d l , while HA26 is intemalized and intracellularly
distributed into discrete perinuclear vesicles. Interestingly, HA30 is internalized and
distributed into both perinuclear structures and to the nucleus (Figure 111.6). Thus it
would appear that a minimum of 30 HA monosaccharides is necessary for nuclear
targeting.
Ili.5. Anti-CD44 antibodies affect TR-HA uptake
The KM-201 mAb blocks the binding of HA to CD44 in murine (Culty et al.
1992) hamster (Miyake et al. 1990) and human cells (Thomas et al. 1992). TR-HA
uptake into cells significantly decreases in both RHAMM transfected, and 10T 1/2
cells following preincubation with KM201 mAb (Figure III.7.A.B.).
111.6. Subcellular distribution of RHAMM is dramatically altered
upon HA treatment.
Immunofluorescent staining of semm starved 10T 1/2 cells shows that
RHAMM and caveolin CO-localize (Figure III.8a.). Within 5 minutes after HA
addition RHAMM leaves the caveolin compartment and the two no longer colocalize
(Figure 111.8.b.). Further. flow cytometry shows that HA induces a rapid fluctuation
in display of ce11 surface RHAMM expression during the first 15 minutes of HA
treatment.
111.7. RHAMM peptides block TR-HA targeting to the nucleus.
Preincubation of TR-HA with peptides that mimic the HA binding domains
of RHAMM (Yang et al. 1994) causes a dramatic decrease in the degree of nuclear
accumulation of TR-HA, while targeting of TR-HA to the cytosol, perinuclear area
and ceIl processes appears unaffected (Figure III.9.a.). This effect appears to
require the HA binding capability of the peptide, since pretreatment of TR-HA with
scrambied peptides made of these domains which lack the ability to bind HA (Yang
et al. 1994) will target HA to $1 intracellular compartments including the nucleus
(Figure II1.9.b.).
111.8. TR-HA uptake is unaffected by blocking with RHAMMexon9
antibody . In an attempt to determine the roie of RHAMM in TR-HA upiake we
preincubated cells with anti RHAMMexon9 pAb pnor to treatment with TR-HA.
Intracellular accumulation and subcellular targeting within RHAMMAI-5 and
parental IOT112 cell cell lines was unaffected by the presence of the antibody
(Figure 111.10.).
111.9. HA uptake varies depending upon length of ce11 plating time
and posi tively correlates wi th functional cellular RHAMM levels.
Image analysis of relative cellular intensities indicates that when cells are
subcultured and allowed to attach for only 3 hours, RHAMM transfected and
parental 1 OT 1 /2 cells appear to take up TR-HA equally. but by 1 2 hours after
attachment the capability of RHAMM transfected cells to take up HA significantly
exceeds their parental 1 OT 1 /2 counterparts (Figure 111.11.). 24 houn after
subculture RHAMM transfected cells are still maintaining their high levels of TR-
HA uptake, while parental IOT1/2 cells have dropped to IR their original intensity -
the same basal level at which another set of WAMM transfectants, those with
rnutated HA binding domains, have been displaying intracellular TR-HA uptake at
al1 time points (Figure 111.11.).
III.10. Connexin43 transfected cells show increased TR-HA uptake
and targeting to the tips of the ce11 processes.
Treatment of connexin43 transfected glioma cells with TR-HA results in
cellular uptake to the cytoplasm, nucleus and notably to the tips of the ce11 processes
(Figure III.lZ.a.), whereas parental glioma cells do not target HA to the tips of ce11
processes (Fipre II1.12.b.). When these same cells are plated at confluence and
treated with TR-HA it is seen that intracellular TR-HA intensity is much higher in
connexin43 transfected glioma cells (Figure III.12.c.), than in their parental glioma
counierparts (Figure III.12.d.).
111.11. TR-HA uptake is decreased upon MEK inhibition
Treatment of breast cancer cells with a specific inhibitor for MEK
(PD098059; dissolved in DMSO; Alessi et al. 1995) prior to TR-HA treatment show
that TR-HA uptake is dramatically reduced upon MEK inhibition (Figure III.13.A.
a-c). as compared to cells treated with DMSO alone (Figure II1.13.A. d-f'). Image
analysis indicates that reduction in fluorescent intensities is significant (Figure
III.13.B.).
III.12. HA is targeted to the ce11 processes upon phorbol ester
treatment of parental 10T1/2 cells.
1 OT 1 /2 cells treated wiih TR-HA for 10 minutes show diffuse cytoplasmic
and nuclear staining (Figure 111.14.a.). Following 45 minutes with phorbol ester
1 OT1/2 cells exhibit an intracellular accumulation pattern of TR-HA in the cytoplasm
that resembles ras-transfomed cells in that TR-HA accumulation occurs at the tips
of ce11 processes (Figure I11.14.b.). Furthemore, treatment of cells with PMA for 4
h appears to enhance TR-HA uptake in a dramatic cytoplasmic and lamellar pattern
(Figure III.14.c.).
IIIml3m TR-HA shows enhanced uptake and targeting to the tips of
the ce11 processes at the wound edge in parental 10T112 cells.
Scratch wounding of 10T1/2 cell monolayers shows that TR-HA
accumulation is enhanced at the wound edge and that celis at the wound edge show
HA targeting to the tips of ce11 processes (Figure III.15.A.B.). Cornparison of cells
30 minutes and 24 hours post wounding indicates that TR-HA uptake enhancement
is most pronounced immediately after wounding and declines with time (Figure
111.15.C.).
FigureIII.1. Confocal optical sectioning of ras-transformed cells exposed to
100pg/rnl TR-HA. Consecutive optical sections (beginning from the basal surface of
the ce11 and proceeding ai 0Spm increments in the z-axis towards the apical surface
of the cell) show TR-HA in the cell processes (indicated by arrow), perinuclear area
and nucleus of a ras-transformed 10T1/2 fibroblast 10 minutes after its addition to
the culture medium. Bar = IO Pm.
Figure 111.2. Cells treated simultaneously with 100pg/ml TR-HA and 5p.M Syto 16
live cell nucleic acid stain for 10 minutes at 37°C. (a) Dual fluorescence image of
Syto16 and TR-HA shows staining of the two probes in the same z-axis plane of
confocal imaging, though there is no signal colocalization. (b) TR-HA image of a.
shows fluorescence in the nucleus and perinuclearly. ( c ) Syto-16 image of a. shows
fluorescence in the nucleus. (d) Phase contrast image of the cell. Bar = 5pm.
Figure 111.3. (a) 100pg/ml TR-HA digested with Streptomyces hyaluronidase prior
to its addition to cells, (b) 100pg/ml TR-HA incubated simultaneously with 4mg/ml
unlabeled HA. Both treatments abolish cellular uptake of TR-HA. (c) Cells treated
with an equivalent amount of TR-hydrazide probe show no staining. Ali incubations
were for 15 minutes at 37°C Bar =lOpm.
Figure 111.4. A. The amount of uptake of TR-HA by cells is proporticnal to the
concentration of TR-HA applied, until an apparent saturation point is reached (at
-2mg/ml), at which the amount of uptake does not increase any funher. Five cell
lines are shown here, and it appears that ras and RHAMM transformed ce11 lines
intemalize HA to a greater extent than their parental IOT 112 counterparts. As well.
HA uptake is enhanced in invasive MDA-MB-23 1 breast cancer cells compared to
their non-invasive MCF-7 counterparts. n=SO. B. After an initial peak in intensity at
- 1 hr of TR-HA incubation, levels of internalized HA decline over a -20hr
timecourse. RHAMMA 1-5 transfected fibroblasts display the greatest HA uptake
over the timecourse. n=50. C. Confocal images of the five celi lines used in A and B
show (a) RHAMMA 1-5 transfected 10T 1/2 fibroblasts (b) ras-transfected 10T 112
fibroblasts (c) parental IOT112 fibroblasts (d) MCF-7 human breast cancer cells (e)
MDA-23 1 human breast cancer cells; treated with 10pg/ml TR-HA for 15 minutes at
37°C. (a) Ras and (b) RHAMM transfected fibroblasts and (e) MDA-23 1 human
breast cancer cells show TR-HA accumulation in ce11 processes, nucleus and a
str i king amount perinuclearly. (c) Parental 10T 112 fibroblasts show TR-HA
accumulation diffusely in the nucleus and in the cytoplasm. (d) MCF-7 cells show
weak TR-HA signal across the entire cell. Bar = 1Op.rn.
,, 4 ras-transfected
E l-~ RHAMM-transfected -14-- 10T1/2 parental
m w?- MCF-7 (non-inmsiw) ..-.--.--*._.._ -a- - . MDA-MB-231 ( insiw) *-•
m c
0.0 0.5 1 .O 1.5 2.0 2.5 3.0
TRHA treatment concentration (mglml)
-i-- parental 1 OT1/2 4 ras-transfected +, RHAMM-transfected
- ,I - - MCF-7 (non-inasiw) - -e - . MDA-231 (inasim)
O 2 4 6 8 10 12 14 16 18 20
time of TRHA incubation (hourr)
Figure 111.5. A. Confocal images of RHAMM transfected cells treated with 4rng/ml
excess HA-oligosaccharide: (a) none, (b) HA4, (c) HA6, (d) HA8, (e) HA 10, (0
HA 12, or (g) 100kDa HA; at the time of TR-HA addition show an overall decrease
in TR-HA intracellular intensity in treatments which included oligosaccharides
composed of (f) 12 saccharide units, though inhibition does not achieve the level of
inhibition obtained using (g) full length (1ûûkD) unlabeled HA. Bar = IOpm. B.
Analysis of cellular intensities in A. n=30. Bars represent SEM.
Figure 111.6. A. RHAMM transfected cells treated for 10 minutes at 37°C with
1.67mg/ml AMAC labeled oligosaccharides of (a) HA8, (b) HA12, (c) HA26, and
(d) HA30 (monosaccharide units of HA). HA8 and HA12 do not appear to be taken
up into cells to any significant degree. HA26 is intracellularly distributed into
discrete perinuclear vesicular structures, whereas HA30 appears to be the minimum
effective size of HA that shows targeting to the nucleus. (a'-d') show phase-contrat
images of the cells. Bar = 1Op-n. B. Image analysis of (n=4O) cells shows mean
fluorescent intensity of n=40 cells of each sample. Bars indicate SEM. Line indicates
degree of autofluorescence in this experiment.
Figure 111.7. A. Preincubation of (a) RHAMM transfected fibroblasts and (b)
parental 10T112 fibroblasts with anti-murineCD44 mAb KM201 (50pg/ml), which
has been previously shown to block HA binding to CD44 (Culty et al. 1992; Hua et
ui. i993), appears to significantly decrease TR-HA intemalization in both ce11 lines
as compared to preincubation of (c) RHAMM transfected fibroblasts and (d) parental
IOT 112 fibroblasts with IgG. B. Image analysis of experiments in A. n=60.
RHAMM transfected parental 1 OT1/2
Figure 111.8. (a) 10T112 cells serum starved for 24 hours show colocalizirtion
( yellow ) between caveolin (green fluorescence) and RHAMM (red fluorescence).
(b) Addition of HA (O. lpg/ml) for 5 minutes caused a redistribution of RHAMM,
and a reduction of its colocalization with RHAMM over the ce11 body. Bars = 1 Opm.
(c) Flow cytometry of 10T112 cells serum starved 24 hours prior to treatment with
HA (O. lpg/ml) for up to I hour, and stained for RHAMM show that ce11 surface
RHAMM intensity fluctuates rapidly within the fint 15 minutes after HA addition.
Blocking with RHAMM fusion peptide could ablate fluctuation (data not shown)
Error bars = SEM.
O 10 a0 30 43 90 €D
10ndml HA incubation (minutes)
Figure 111.9. RHAMMAI -5 transfected cells treated for 10 minutes with 100pg/ml
TR-HA that has been preincubated for 2 hours with: (a) 1 mglml of a peptide
mimicking an HA binding domain of RHAMM will traffic TR-HA to the ce11
processes and perinuclear area but no TR-HA signal is evident within the nucleus:
(b) I mglm1 of a scrambled peptide that is unable to bind HA show distribution of
HA to al1 intracellular compartments. Arrows indicate ce11 processes. Bar = 10pm. n
= 50.
Figure 111.10. A. Confocal images of parental (a, c, and e) and RHAMM
transformed (b, d, and f) 10T1/2 fibroblasts preincubated for 30 minutes with: (a+b)
Opg/ml, (c+d) I O~glml and (e+D 50ug/mI, of anti-RHAMM an tibody pnor to TR-
HA incubation. Intracellular accumulation of TR-HA appears unaffected by
antibody treatments. Bars =25pm. B. Image analysis of cellular intensities of A.
(n=60). Error bars indicate SEM.
Figure IILl1. RHAMMA 1-5 transfected, mutant RHAMMA 1-5 (unable to bind
HA) transfected, and parental 1 OT 112 fïbroblasts were subcultured and allowed to
attach for 2, 12 or 24 hours prior to TR-HA treatment (150pglml; 10 minutes). Cells
were analyzed for fluorescent uptake of TR-HA. While RHAMMAI -5 transfected
and parental IOT112 levels of uptake are initially equal. by 12 hours after plating
RHAMMA 1-5 transfectants clearly have more intracellular TR-HA. 24 houn after
plating, parental 1OT 112 levels of uptake decrease until they are equal to HA uptake
of the transfectants mutated in their HA binding domains, which have had
significantly lower TR-HA intracellular staining at al1 three plating times. n=60.
Error bars represent SEM.
RHAMMA 1-5
O parental 1 OT112
RHAMMAI-5 HABD- T
2 hours 12 hours 24 hours
Figure 111.12. TR-HA ( 1 ûûpg/ml; 10 minutes) treated (a) connexin43 transfected
glioma cells display nuclear and cytoplasmic HA accumulation as well as high levels
of TR-HA targeted to the tips of the ce11 processes, while (b) parental glioma cells do
not show HA targeted to the tips of cell processes. (c) These same connexin43
transfected cells plated at confluence show greater overall TR-HA uptake than their
(d) parental glioma counterparts plated at confluence. Bars = IOpm.
O DMSO
PD98059
Figure 111.13. A. Human breast cancer cell lines: (a,d) MDA-MB-23 1 , (b,e) MCF-7
and (c,f) MCF- IO neo 2T treated (a-c) with 50p.M MEK inhibitor PD098059 or
DMSO alone (d-f) for 2 hours prior to treatment with TR-HA ( lûûyg/ml; 10
minutes). Bar=lOpm. B. Analysis of mean fluorescent ce11 intensities indicates that
al1 three ceIl lines display reduced TR-HA uptake in the presence of MEK inhibitor.
Bars represent SEM. n=30.
Figure 111.14. Confocal analysis of (a) 1 OT ln parental fibroblasts treated with TR-
HA (100pg/ml; 10 minutes) shows TR-HA distributed to the nucleus and diffusely
present in the cytoplasm. (b) 10T112 fibroblasts treated with 1 OOnM PMA for 45
minutes prior to TR-HA treatment show TR-HA localized at the edge of cell
processes and increased in the perinuclear area, although nuclear stüining remains
unchanged. (c) lOT112 fibroblasts treated with lOOnM PMA for 4 h pnor to TR-HA
addition show dramatically enhanced uptake of TR-HA into ce11 processes. Bar =
10pm.
Figure 111.15. A. 1 OT 1 /2 ce11 monolayer scratch wounded and treated with TR-HA
30 minutes post wounding shows TR-HA strongly in the nucleus, perinuclear area
and ce11 processes of cells at the wound edge. Those cells that are further from the
wound edge display much weaker TR-HA staining. Bright outline indicates cells at
the wound edge. Arrows indicate ce11 processes. Image captured with 63x objective.
B. Analysis of ce11 intensities in different layers of the 30 minute post-wounded
culture indicates that TR-HA uptake significantly increases with proximity to the
wound edge. n=30. Bars indicate SEM. C. Confocal images captured with the 40x
objective show the wound edge of 10T 112 parental fibroblast cultures. While
enhanced uptake at the wound edge can be seen clearly at (a) 30 minutes post
wounding, the contrast is less vivid (b) at 24 hours post wounding, though cells at
the wound edge do clearly demonstrate increased intracellular TR-HA.
DISCUSSION
Whereas intracellular HA degradation in the lysosomes has been extensively
reviewed in many studies (Truppe et al. 1977; Orkin and Toole 1980; Orkin et al.
1982; Bemanke and Orkin 1984; Alston-Smith et al. 1992; Culty et al. 1992; Hua et
al. 1993; Culty et al. 1994). few studies have focussed upon the modes by which
hyaluronan may target to altemate intracellular sites (Collis et al. 1998; Evanko and
Wight in press). This is interesting, since hyaluronan has been localized to many
such altemate subcellular sites including the nucleus (Margolis et al. 1976;
Furukawa and Terayama 1977; 1979; Castejon and Castejon 1987; Ripellino et al.
1988; Kan 1990: Eggli and Graber 1995: Evanko and Wight in press), rER and golgi
(Kan 1 WO), caveolae (Eggli and Graber 1993, mitochondria (Kan 1990; Eggli and
Graber 1995) and membrane mffles (Evanko and Wight in press). Since the HA
synthases are present only at the ce11 membrane and are thought to extrude newly
synthesized HA directly into the extracellular space (Prehm et al. 1989; Weigel et al.
1997). presumably mechanisms must exist to deliver HA back into these intracellular
compartments. These intracellular sites have also been shown to contain
intracellular hyaluronan binding proteins which have a number of significant roles in
ce11 signaling. We begin to delineate some aspects of a novel HA intemalization
process, and its cellular mechanisms in this study.
IV.1. The phenornenon of rapid HA uptake
Fluorochrome-tagged HA can be rapidly taken up by the transfomied ce11 and
accumulate wi thin subcellular compartrnents, such as ce11 processes (Figure 111.1)
and the nucleus (Figure III.2), which have been shown to contain intracellula-
hyaladherins (Frost er al. 1990; Grammatikakis et al. 1995; Deb and Datta 1996;
Entwistle et al. 1996: Zhang et al. 1998). The TR-HA uptake process is HA specific
since it is blocked by predigestion of the TR-HA probe with hyaluronidases (Figure
II1.3.a.). is effectively competed by excess unlabeled HA (Figure 111.3.b.), and since
TR hydrazide alone is not taken up by cells (Figure II1.3.c.).
The presence of nuclear HA is consistent with histological studies in which
endogenous HA has been localized to nucleoli, nuclear clefts, heterochromatin and to
areas of condensed chromatin in the nuclear periphery of oocytes, cumulus cells, and
smooth muscle cells using nanogold labeled hyaladherins (Kan 1990; Eggli and
Graber 1995; Evanko and Wight in press) and in biochemical studies of rat liver and
brain nuclei purified by cell fractionaiion (Margolis et al. 1976; Purukawa and
Terayarna 1977; 1979). Our observations that exogenous HA can rapidly target to
the nuclear cornpartment are supported by laser scanning confocal microscopy,
which resolves the fluorescent signal in adjacent 1 .Opm thick optical sections
(Figure III.l.), and demonstrates that TR-HA staining is present in the sarne focal
plane as a nucleic acid specific stain, confirming its nuclear localization (Figure
111.2.).
The process appears to be dose dependent and saturable in at least five cell
Iines as demonstrated by concentration curves (Figure III.4.A.). This indicates that
it is dependent upon some limiting factor, such as receptor sites. Examination of
TR-HA uptake over a 20 hour tirnecourse demonstrates the rapidness of this process,
as the greatest arnounts of intracellular HA are seen within 1 hour of TR-HA
incubation (Figure 111.4.B.).
IV.2. Mechanisms of HA uptake
IV.2.1. The potential role of cell surface receptors in TR-HA uptake
TR-HA uptake was decreased in the presence of excess HA oligosaccharides
of 12 monosaccharides in length (HA 12) (Figure 111.5.). It remains unclear whether
HA 12 is able to enter the ce11 itself (Figure III.6.), and thus HA 12 may be
interfering with TR-HA uptake through direct cornpetition through cellular
internalization (and consequent involvement of intracellular sites) as full length
unlabeled HA does, or by statically occupying binding sites in the HA uptake
machinery at the cell surface. One candidate for HA 12 binding is CD44, since it has
been showed previously to require a minimum binding site of HA6 (Culty et al.
1992; Hua et al. 1993). Tammi et al. (1998) have more recently showed that
displacement of HA pericellular matrices frorn CD44 in vivo requires HA l O and they
postulate that tight HA binding necessitates dimerization of CD44 or its cooperative
interaction with other cell surface molecules. Our result of HA12 being required to
inhibit intemalization of HA (Figure 1115.) is consistent with a cornplex process
potentially mediated by aggregates of CD44, or the involvement multiple forms of
ce11 surface receptors. The inhibition of TR-HA achieved using HA 12 was
incomplete, as evidenced by the rnuch greater degree of blocking achieved using
unlabelled HA (Figure 111.5.). Thus, a significant amount of HA uptake is still
occurring in the presence of HA1 2. It is likely that larger oligos would be more
effective at blocking HA internalization, further suggesting the complexity of the ceIl
surface/ intracellular protein binding interactions at work here.
Interestingly, it appears that nuclear targeting of HA requires a minimum of
30 monosaccharides (Figure 111.6.). Since no single protein has been shown to
require such a large number of HA saccharides for binding it would appear that if
protein binding is important for nuclear entry (as implicated by Figure II1.9.), then
either a complex of proteins, or a novel hyaladhenn is required for nuclear
trafficking of HA.
HA uptake to the lysosomal degradation pathway has been shown to be
mediated by a clathrin dependent process (Alston-Smith et al. 1992) which involves
CD44 (Culty et al. 1992; Hua et al. 1993), as determined by antibody blocking. In
this study we treated cells with two different anti-CD44 antibodies prior to exposure
to exogenous TR-HA. It would appear that CD44 plays a role in the rapid uptake of
HA into 10T1/2 cells, since the KM201 antibody, which has previously been shown
to block CD44-HA binding (Culty et al. 1992; Thompson et al. 1992; Hua et al.
1993), was able to significantly inhibit TR-HA uptake in both transformed and non-
transformed cells (Figure 111.7.). It should be noted that the block was not a
complete one, which is consistent with the involvement of other molecules. It would
appear that CD44 is involved in targeting HA to multiple subcellular compartments
independent of the previously reported degradative pathway.
A role for the another HA receptor, RHAMM, is also suggested by drarnatic
alterations of RHAMM cytoplasmic and cell surface distributions upon HA
treatment (Figure 111.8.) and by the ability of RHAMM peptides to block HA uptake
to the nucleus (Figure 111.9). The redistribution of RHAMM to the ce11 periphery,
and indeed to the ce11 surface (Figure 111.8.~) as shown by flow cytometry, upon
exogenous HA administration however, is interesting, because it occurs rapidly, as
does HA uptake. Ul trastructural analyses have demonstrated that HA is associated
with caveolae (Eggli and Graber 1995), and these results would collectively
implicate pinocytosis as a potential mode of TR-HA uptake wonhy of further study.
CD44 is potentiafly found associated with the caveolae as well, since CD44 has been
demonstrated to have a strong association with GPI domains (llangumaran et al.
1997). Further, RHAMM and CD44 have been shown to colocalize and
coimmunoprecipitate in human breast cancer cells (Paiwand et ai., in preparation),
and could act cooperatively to mediate HA uptake.
Although RHAMM HA-binding peptides blocked nuclear uptake of TR-HA
(Figure III.9.), pretreatment of cells with anti-RHAMM antibodies proved
inconclusive (Figure II1.10.). These results suggest that the HA binding property
and not other domains shown to be necessary for cell motility, which these
antibodies target, are required for uptake. RHAMM therefore may perform multiple
functions at the ce11 surface to mediate cell locomotion.
Cellular RHAMM levels have been shown to vary depending upon time in
culture. This prompted an examination of the relationship between cellular
RHAMM levels, and relative TR-HA uptake. Fipre 111.11. shows that TR-HA
uptake is at its highest in RHAMMAI-5 transfected cells at 12 hours, this correlates
nicely with previous results using flow cytometry which indicate that RHAMM
expression on the cell surface is highest upon 12 hours of plating (Chang et al. in
preparation). Further, among the three ce11 lines used in these experiments, TR-HA
uptake is shown to positively correlate with functional (Le. RHAMM which is
capable of binding HA) cellular RHAMM levels at al1 tirnepoints (Figure 111.11.).
RHAMM has been proposed to regulate gap junctional intercellular
communication through connexin43 (Nagy et al. 1995), and a potential role for gap
junctional transport in TR-HA trafficking was investigated using connexin43
transfected gliorna cells. At confluence it can be seen that connexin43 transfected
cells show a greater amount of intracellular TR-HA than their parental glioma cells
and individual connexin43 transfectants appear to target TR-HA to the tips of cell
processes, while their parental glioma cells do not (Figure 111.12.). These results are
consistent with a role for gap junctions in TR-HA targeting, but it is not possible to
determine if this is a specific effect resulting from functionally competent gap
junctions between cells or hernigap junctions (Ebihara et al. 1995) at the plasma
membrane, or one reflecting other factors which are upregulated upon connexin43
transfection. Future experiments are needed to determine the role of connexin43 in
TR-HA uptake.
In a recent study HA treatment of rat embryonic cell Iines, under similar
conditions of confluence, plating time, and HA concentrations/times as those used in
this study, has been shown to rapidly activate tyrosine phosphorylation of cellular
proteins and MAP kinase in a time and dose dependent manner, and subsequently
stimulate cell growth (Serbulea et al. 1 999). This HA dependent activation of MAP
kinase was strongly suppressed by dominant negative ras expression (Serbulea et al.
1999). These results suggest that the ras-MAP kinase pathway is activated by HA
and may play an important role in HA dependent signaling (Serbulea et al. 1999).
Our lab has demonstrated that the HA binding domains of intracellular RHAMM are
required for interaction in MAP kinase signaling (Zhang et al. 1998). and these
results collectively suggest a role for intracellular HA in the MAP kinase cascade.
Conversely MEK inhibition with PD098059, which prevents the activation of MEK
by Raf in this cascade (Alessi et (11. 1995), appears to inhibit TR-HA internalization
(Figure II1.13.), and would suggest that HA internalization is somehow dependent
upon MAP kinase signaling. This effect may be due to multiple effects of the MEK
inhibitor, including hyperpol ymerizaton of microtubules (Harrison and Turley in
preparation) with consequent interference with vesicle rnediated transport of HA
dong microtubules (see Appendix 1 - Figure VI. 1 .). MEK inhibition also causes
decreased cell motility (Paiwand et al., in preparation), which may have far reaching
implications in decreasing levels of overall cellular activity and HA uptake.
IV.2. Potential roles for intracellular HA targeting
IV.2.1. Potential role of HA in the nucleus
Precedent for regulatory roles of nuclear GAGs corne from studies showing
that transient nuclear heparin sulfate is invol ved in growth control and transcriptional
regulation (Fedarko and Conrad 1986; Fedarko et al. 1989; Ishihara and Conrad
1989; Busch et al. 1992). Exogenous addition of heparin to cells has been show11 to
arrest the ce11 cycle in G 1 (Fedarko et al. 1989). stimulatelinhibit DNA synthesis in
nomal/tumor cells (Furukawa and Bhavanandan l982), compete for binding with
histones (Fumkawa and Bhavanandan 1983; Scheerer et al. 1989). suppress the
expression and DNA binding actions of AP- 1 (Au et al. 1994) and accumulate at the
nuclear membrane (Fedarko and Conrad 1986). Though multiple GAGs including
HA, CS, HS and heparin have d l been reported to be associated with chromatin in
nuclei (Furukawa and Terayama 1977), heparin has so far received more attention for
possible direct regulatory roles.
We show here a novel import of exogenous HA into the nucleus, and its
distribution appears ordered (Figures 111.1.-111.2.). Other studies have reported the
presence of endogenous HA in the nucleus (Margolis et al. 1976; Fumkawa and
Terayama 1977; 1979; Castejon and Castejon 1987; Ripellino et al. 1988; Kan 1990;
Eggli and Graber 1995; Evanko and Wight Ni press). Within the nucleus HA has
been found associated with nucleoli (Kan 1990; Evanko and Wight in press),
heterochromatin (Eggli and Graber 1995), fine filaments within the nuclei (Evanko
and Wight in press) and in nuclear clefts and surrounding condensed chromatin
during rnitosis (Evanko and Wight in press). GAGs have been found associated with
DNA in loops, many in aggregates or cycles resembling the pore complexes, which
suggests evidence for a structural role for GAGs in the nucleus (Engelhardt et al.
1982). As well, nuclear GAGs appear increased during liver regeneration (Funikawa
and Terayama 1977). The functional importance of these findings is not known.
The recent report of a cytoplasmic HA binding protein p68 which has been
CO-purified with the splicing factor SF2 (Deb and Datta 1996), as well as the
existance of other hyaladherins in the nucleus including chick ce11 cycle homologue
cdc37 (Gramrnatikakis et al. 19951, and RHAMM, al1 indicate the potential for a
multifaceted control of cell function by HA from within the nucleus.
Aside from its protein interaction, HA has the potential to exert influence in
the nucleus through basic biochemical and structural modalities. HA is a calcium
chelator and could potentially be involved in regulating nuclear calcium levels,
which control a variety of nuclear functions, including gene transcription, DNA
synthesis, DNA repair and nuclear envelope breakdown (Santella and Carafoli 1997).
The nucleus is a dynamic structure, with chromosome movements, and cycles of
condensation and decondensation (Larnond and Earnshaw 1998). The hydrating and
basic physicochemical properties of HA suggest that HA may be involved in
facilitating transport of small molecules within the nuclear interior just as it does in
the ECM.
How or whether HA might interact with nuclear pores remains unclear. but a
growing body of research on nuclear import of glycoconjugates suggests that when
proteins are substituted with 25 GlcNAc residues (linked in p 1-4 fashion), they are
pennitted to be transported from the cytotosol to the nucleus (Duverger et ni. 1993;
1995; 1996). This form of nuclear transport has been shown to use the nuclear pore
in a novel fashion which does not compete for cofactors associated with traditional
NLS dependent protein import (Duverger et al. 1995). It is possible that this form
of transport could be responsible for mediating the novel nuclear translocation of HA
seen in this study.
The size of HA preseni in both the nucleus and cytoplasm remains open for
speculation. Recently identified GPI linked ce11 surface hyaluronidases (Chan et al.
1999), and potential hyaluronidases within the cell may act to cleave HA as it is
being transported into the cell. Localization of HA in the nucleus using a
biotinylated aggrecan probe (Eggli and Graber 1995; Evanko and Wight in press)
demonstrates that the HA within the nucleus must be at least HAIO. since aggrecan
HABP requires at least a decasaccharide of HA to recognize and bind HA (Hascall
and Hinegard 1974).
Surprisingly, preliminary evidence has identified the nucleus as the
predominant cellular locdization of the newly characterized hyaluronidase HYAL 1
in normal epithelium both in vitro and in vivo (Frost et al. in preparafion). HYALl
is a candidate tumor suppressor gene (Frost et al. 1997), as it is found at the site of a
common tumor suppressor locus (Killary et al. 19%; Buchhagen 1996). The
putative localization of HYALl to the nucleus in normal epithelia has given insight
into possible mechanisms of how HYAL 1 might function as a tumor suppressor
gene. In addition, HYAL- 1 is unable to remove cell surface HA but may rather play
a functional role in the nucleus to remove nuclear GAGS that may regulate cellular
proliferation, differentiation, and or apoptosis (Frost et al. in preparation).
IV.3.2. Potential roles of HA uptake in transformation
HA production and metabolism are often altered during turnorigenesis
(Knudson and Knudson 1993) and a causal role for this modification has recently
been suggested by a study showing that HA accumulation around and within
colorectal tumor cells is prognostic of poor outcome (Ropponen et al. 1998).
Consistent with the notion that HA directly regulates cell behaviour, study of HA
binding proteins tenned hyaladherins has indicated that HA contributes to the control
of ceIl cycle and cell motility (Entwistle et al. 1996; Mohapatra et al. 1996; Wang et
al. 1998; Zhang et al. 1998). in spite of the importance of these observations, the
molecular mechanism by which HA directs these processes are not clear.
HA uptake was observed in five cell lines and levels of cellular
intemalization were shown to be highest in cells expressing the highest levels of
RHAMM, ras, CD44 and erk, as shown in both cells and invasive breast cancer cells
compared to their non transformed counterparts (Figure 111.4.). This trend
correlates with both increased motility and proliferation in these ce11 lines.
HA oligosaccharides have been shown to significantly reduce prolifention
and migration in response to PDGF (Evanko et al. 19991, and to inhibit tumor
formation in subcutaneous rnelanoma cells (Zeng et al. 1998). Thcse effects may be
mediated ihrough blocking accumulation of HA in intracellular compartments
relevant to tumorigenesis.
The potential uses of covalent! y modified HA for selectivel y targeting and
accumulating in aggressive cancer cells are manifold in both diagnostics and
treatment. One recent study used HA linked to sodium butyrate to target HA to
breast cancer cells and found that cells could internalize both HA and the covalently
linked compound, which resulted in inhibition of cellular growth rates (Coradini et
al. 1999).
IV.3.3. Potential roles of intracellular HA in cell motility
The ability of HA microinjected into cells that do not aggressively take up
HA or target it into the cell processes, to stimulate ceil motility provides the first
direct evidence of an effect of intracellular HA on ce11 behaviour (Collis et al. 1998).
Results in this study funher suggest that the uptake and intracellular targeting of HA
to cell processes is controlled by ras and PKC signaling pathways. lOT1/2 cells
which are not transformed or treated with PKC do not take up HA aggressively, and
do not target HA to the tips of ce11 processes (Figure III.14.a.). These same cells
will not respond to exogenous administration of HA with increased motility - as ras
and RHAMM transformed cells do (Collis et al. 1998). However, when 10T112 cells
are treated with phorbol ester. exogenous HA is targeted to the tips of ce11 processes
(Figure III.14.b.). concomitant with manifold increased motility (Collis et al. 1998).
The ability of HA to be directed into 10T1/2 ce11 processes after acute
phorbol ester treatments suggest an involvement of protein phosphorylation by
protein kinase C in this HA uptake. This protein serine/threonine kinase has
previously been implicated in cell motility (Liu et al. 1997) and its kinase activity
has also previously been linked to release of HA from the ce11 (Klewes and Prehm
1994; Thiebot et al. 1999), which may involve analogous mechanisms to uptake
noted here.
The dramatic alterations in TR-HA subcellular targeting upon phorbol ester
treatrnent (Figure 111.14) may be mediated through PKC and ras dependent
activation of microtubule-dependent particle motility (Alexandrova et al. 1993).
PMA treatment has been shown to target transfemn receptors to cellular
compartments which do not participate in normal routes of receptor traffic, or to
otherwise inaccessible compartments along new membrane traffic pathways
(Schonhorn et al. 1995). It would likewise be interesting to examine the effects of
PKC inhibitors upon this process.
Further evidence for intracellular HA upregulation upon onset of motility is
shown in wounded monolayers, where TR-HA is shown to accumulate intracellularly
at the wound edge (Figure III.15.A.B.). This effect is most dramatically enhanced
within 30 minutes post-wounding (Figure III.IS.C.), concomitant with increased
RHAMM expression during early wound responses (Savani et al. 1995b). Further,
wounding is shown to promote subcellular targeting of HA to the tips of cell
processes concomitant with their increasing motility (Figure III.1S.A.).
Previous reports have demonstrated HA accumulation at wound sites, both in culture
and in vivo (Gerdin and Hallgren 1997; Chen and Abatangelo 1999). but the
possibility that HA'S therapeutic activity at such sites is being mediated
intracellularly is a novel one.
IV.4. Conclusions
Wr note here that the rapid uptake of fluorochrome-tagged HA into cells
appears to be independent of the traditional receptor-mediated endocytic pathway,
since uptake is acute, is incompletely blocked by CD44 antibodies, and results in the
accumulation within ce1 1 processes, the nucleus, and the pennuclear area. The
localization of HA in the nucleus requires an interaction with subcellular proteins,
since exposing cells to peptides that mimic HA binding motifs of RHAMM block
this targeting and results in accumulation of HA only within the cytoplasm (Figure
111.9.). HA binding motifs (Yang et al. 1994) are found in RHAMM, cdc37. and
p68, which are d l potential candidates involved in the uptake of HA. Our results
also suggest the targeting of HA to ce11 processes involves a mechanism distinct
from nuclear targeting.
Collectively, Our results suggest that both HNcell surface receptor and
intracellular HNprotein interactions may be involved in regdation of ce11 behaviour.
A multifaceted role of intracel1uIa.r HA is suggested by its localization in the nucleus,
previous reports of HA binding to nuclear proteins (Furukawa and Terayama 1977;
1979) and the possibility that a hyaluronan binding protein may interact with RNA
splicing machinery (Deb and Datta 1996). Our results provide the first preliminary
evidence of the ability of exogenously added HA to accumulate within multiple
subcellular compartments, and to directly affect ce11 motility. Further analysis is
required to determine the precise role(s) of HA within the cell, to define the
mechanisms involved in this uptake and accumulation process and to determine the
signaling processes that intracellular HA might regulate. Clearly, study in this field
is only just beginning, and more work is necessary to elucidate the mechanisms
mediating HA traffic, and the role(s) of HA inside the cell.
CHAPTER V
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CHAPTER VI
APPENDIX 1 - ADDITIONAL RESULTS SECTION
VI.1. Results
VI.1.1. Colchicine treatment prevents nuclear uptake of TR-HA
To examine the potential role of the microtubules in subcellular trafficking of
HA. cells were pretreated with colchicine to disnipt their microtubule networks, then
exposed to TR-HA. Cells pretreated with colchicine display TR-HA staining
diffusely throughout the nucleus and cell processes, while nuclear targeting of TR-
HA is ablated and perinuclear accumulation is decreased (Figure V1.1.).
VI.1.2. Brefeldin A pretreatment blocks uptake of TR-HA by lOTll2 parental
cells.
In order to mess the potentid role of vesicle trafficking in TR-HA transport,
treatrnent with Brefeldin A were used. Pretreatment of ras transformed and parental
IOTU2 cells wiih Brefeldin A prior to addition of TR-HA caused a dramatic
decrease in the intracellular accumulation of TR-HA in al1 cellular compartments
(Figure VI.2.).
VI.1.3. HA alters the ability of DNaseI to interact with DNA
DNase digestion of genomic DNA is enhanced in the presence of HA as
evidenced by increased smear length (corresponding to fragmented DNA) across an
agarose gel (Figure VI.3.). Another glycosaminoglycan, chondroitin sulphate has no
effect on DNA degradation, indicating that the process is HA specific (Figure VI.3.).
This phenornenon would appear to suggest that HA may promote interactions
between protein and DNA on a wider basis, and suggests a fundamental role for HA
in the nucleus.
Figure VI.1. RHAMMA 1-5 transfected cells treated with 1OmM colchicine for 30
minutes prior to addition of TR-HA (100pg/ml) and funher incubation for (a) IO
minutes or (b) 30 minutes show no TR-HA trafficking to the nucleus, in contrast to
non-colchicine treated cells at (c) 10 minutes and (d) 30 minutes TR-HA. Bars =
1 Opm.
Figure VI.2. (a,b) Parental and (c,d) ras-transfonned fibroblasts pretreated with
(b+d) 50pg/rnl Brefeldin A for 30 minutes prior to TR-HA treatment (lOOClg/rnl; 10
minutes) show greatly decreased levels of TR-HA uptake compared to cells not
pretreated (a+c). Bar = 1 Opm.
Fipre V1.3. Lanes 1-8 are loaded with genomic DNA degraded by increasing
concentrations (0.0 - 7.8 x 104 uniis) of DNaseI. Prebinding of DNA with 10pg
(lanes 9- 16) or 5Opg (lanes 17-24) HA results in increased DNA degradation by
DNaseI (as visuaiized by increased smear length). 10pg chondroitin sulfate (lanes
25-28) had no effect on DNA degradation by DNaseI. Samples loaded on a 1%
agarose gel.
VI.2. Discussion
VI.2.1. Cytosolic factors in the uptake process
Cells which have had their microtubule networks disrupted by colchicine
have dramatically reduced transport of TR-HA to the nuclear and perinuclear
compartments, while entry of TR-HA into the cytoplasm and ce11 processes appears
unaffected (Figure VI.1.). This predicts a form of microtubule dependent transport
of HA across the cytoplasm, which is involved in targeting HA to sites relevant for
nuclear entry. Microtubules have been previously shown to direct a multistep
process of caveolae cycling between the Golgi and plasma membrane, and this
transport is intemipted by microtubule disruption using nocodazole (Conrad et al.
1995). Therefore we propose that at least part of the TR-HA transport could be
O C C U ~ ~ ~ via this rnicrotubule mediated transport of caveolae.
In previous studies, colchicine has been demonstrated to decrease levels of
cellular HA, potentially impinging upon HA synthesis (Brecht et al. 1986). Perhaps,
without an intact microtubule network, levels of HA accumulate at cellular and
intracellular sites which act to downregulate HA synthesis. Interestingly, addition of
HA has recentiy been shown to cause an increase in intracellular concentrations of
tubulin in human cells (Greco et al. 1 W8), and these results have implications for the
role of HA in ce11 division.
Vesicular transport within the ceIl is facilitated by recruitment of cytosolic
coat proteins that promote the budding of transport vesicles (Kreis and Pepperkok
1994). Brefeldin A inhibits recruitment of these coat proteins and formation of
transport vesicles. Brefeldin A has been shown to interfere with vesicular transport
causing disassembly and collapse of Golgi components back to the ER (Hidalgo et
al. 1992). Disruption of vesicular transport with Brefeldin A causes a decrease in
overall intracellular accumulation of TR-HA (Figure VI.2.), implicating vesicular
mediated transport at both the plasma membrane, and in cytoplasmic trafficking of
HA. These effects are also consistent with a microtubule mediated vesicular mode
of TR-HA transport (perhaps associated with caveolae), to the perinuclear/nuclear
compartments (see Figure VI.1.).
VI.2.2. Potential influence of HA on DNA - protein interactions
HA has been found associated with the chromatin fraction of the nucleus
(Furukawa and Terayama 1977; 1979; Eggli and Graber 1995), and previous work in
Our lab has demonstrated that HA is capable of binding to genomic DNA (Zhao et al.
in preparation). We show that HA is able to increase the effectiveness of DNaseI
interactions with DNA, resulting in increased degradation of DNA (Figure VI.3.).
At this time it is not possible to speculate whether this specific ability of HA to alter
protein-DNA interactions is efficacious within ce11 nuclei, but it does present us with
an interesting mode1 for HA activity. Likewise, in the cytoplasm, it is possible that
HA influences protein-protein interactions. The HA binding domains of RHAMM
are also required for RHAMM-erk binding to occur (Zhang et al. 1998). It is
possible that HA cm compete with erk for these binding sites, and thereby influence
downstream events. Peptides which mimic HA'S ability to bind these domain have
been developed and are being used to investigate this possibility (Ziebell et al. in
preparation) .
Figure V1.4. Model of potential uptake mechanisms and intracellular functions
of HA (see next page): HA uptake is potentially mediated by multiple processes:
( 1 ) Clathnn mediated HA uptake and targeting to lysosomes involves CD44
(Alston-Smith et al. 1992). (2) Caveolae rnediated pinocytic HA uptake rnay involve
RHAMM, CD44 and p68 proteins. This form of transport utilizes microtubules to
traffic vesicles to and from the golgi/ER pennuclear compartment. (3) The HA
synthase complex may at times direct HA cytoplasmically. (4) HA may interact with
the phospholipids of the cell membrane to form pores or channels (Pasquali-
Ronchetti et al. 1997), and thus enter the cell cytoplasm in a non energy dependent
fashion. Once internalized HA occurs notably at tips of ce11 processes upon onset of
motility as produced by ceIl transformation and PKC treatments. IntracelIular HA of
larger than HA30 also accumulates in ce11 nuclei. The presence of cytoplasmic and
intranuclear hyaladherins provide additionai functional targets for iniracellular HA.
1. Degradative . Endocytic
CHAPTER VI1
APPENDIX II. - HYALURONAN DETECTION
VII.1. Existing probes for hyaluronan
The andytical techniques for HA detection have been continuously refined.
For many yean it was necessary to purify the polysaccharide so that its sugar
constituents, glucuronic acid and glucosamine could be measured by colour
reactions. A ciassical technique was the carbazole method for uronic acids. These
analyses required at least 100pg of HA. With the discovery of specific rnicrobial
enzymes that degrade HA into unsaturated disaccharides, it became possible to
design techniques in which a few micrograms of poiymer were sufficient. The third
generation came in 1980. These make use of proteins with specific affinity for HA
and can be used as antibodies in ELISA4 ke assays to detect as little as 1 ng of
polysaccharide.
More recently a technique for the histolocalization of HA (Ripellino et al.
1985) that utilizes these highly specific HA binding peptides has become widely
used. However, even this HABP procedure may senously underestimate the HA
content of tissues fixed in a routine manner (Lin et al. 1997). Other GAGS are fixed
as a result of their covalent binding to profeoglycan core proteins, however the free
form of HA apparently diffuses slowly into the fixation solution during the process.
Although CPC improves the preservation of HA in such samples, total HA cannot be
accounted for (Lin et al. 1997).
VII.2. The Texas Red - hyaluronan conjugate
Four groups on the HA molecule are available for chernical modification: the
carboxylate, the acetarnido, the reducing end-group and the hydroxy groups.
Modification of HA with ethyl (N,N-dimethy1aminopropyl)carbodiimide (EDCI) has
been done since 1986 (Kuo et al. 1990). The productions of glucuronamides
requires the activation of the carboxylic group which can be accomplished using a
water soluble carbodiimide such as EDCI as the condensing agent.
Advantages include 1 ) preparation of HA derivatives under mild, aqueous
conditions which preserves the structural integrity and molecular size range of HA,
also 2) homogenous modified HA materials can be easily obtained by removal of al1
side products and unreacted reagents dunng dialsysis and 3) al1 new HA derivatives
based on this bishydrazide technology are chemicall y well-characterized (Prestwich
et al. 1998).
TR-HA can be prepared directly from HA and Texas Red sulphoylhydrazide
(TR-HA used in this study has been analyzed to have 1 in 15 disaccharides
successfully labeled withTR - Figure VII.1.). This gives maximal sensitivity while
preserving the minimum decasaccharide recognition unit of HA for HABDs. These
probes offer advantages over reducing end-modified HA derivatives (Raja et al.
1988; Yannarielio-Brown et al. 1996), in chat multiple probe moieties can be
included per HA rnolecule, and the probes are positioned in the intemal milieu of
HA.
Future directions in the chernical modification of HA include development of
covalent attachments of therapeutic dmgs to HA (Band et al. 1998). Attachment of
HA to proteins, short peptides, mti-cancer agents, anti inflammatory or anti infective
drugs or cell adhesion and growth pmmoting ligands provides the potential for drug
delivery through exploitation of HA recognition and receptor-rnediated endocytosis
for specific uptake by and delivery to target cells (Vercruysse and Prestwich 1998).
a i+r.r6 I l t r l r r i
C W 1 i m.. L...,..,.
e..&. ?"*"i
Y, II. Y.. PID.".bU -*Yi -
Figure VII.1. HPLC analysis of TR-HA conjugate. Analysis of success of
conjugation of TR-HA: the (a) refractive and (b) absorptive (at 590nm) indices
overlap at 19 minutes elution time in a single peak indicating the sample is pure.
(c) Molecular weight of the HA is shown to be approximately lOOkDa through
cornparison to other HAs of known molecular weights. To measure success of
conjugation (d) comparative absorbance of the TR-HA on a TR standard curve
shows that approximately 1 in 15 HA dissaccharides has been successfully
labeled.