Becky Wright: historyBecky Wright: history

2002-3 Postdoc (15months) Cardiovascular Genetics, 2002-3 Postdoc (15months) Cardiovascular Genetics, Rayne Bldg, UCLRayne Bldg, UCLAssociation between variation in the Alpha-1-Antitrypsin (AAT) gene and risk of cardiovascular disease (CVD).

1998-20031998-2003 Ph.D. Ph.D. St. George's Hosp, Uni of LondonSt. George's Hosp, Uni of London

Insulin-like Growth Factor Binding Proteins (IGFBPs) as Co-coordinators of Human Ovarian Function.

1996-98 RA -Molecular Immunology, NIMR, LondonT cell selection and the invariant chain

1995-96 RA -Interface Analysis Centre, Uni. of BristolXPS and EM analysis of top 10-30Å materials surface

Cardiovascular genetics Cardiovascular genetics Aims of project:Aims of project:to determine the association of AAT genotypes with the risk of CVD

(particularly atherosclerosis)

Hypothesis:Hypothesis:Polymorphisms are related to changes in the level or activity of AAT Polymorphisms are related to changes in the level or activity of AAT

protein. Low levels/activity of AAT will promote atherosclerosis.protein. Low levels/activity of AAT will promote atherosclerosis.

Background:Background: Produced by liver (and monocytes) as part of the acute phase Produced by liver (and monocytes) as part of the acute phase

response (APR)response (APR) Serine protease inhibitor (SERPIN)Serine protease inhibitor (SERPIN) In particular, AAT prevents the degradation of elastin by serine In particular, AAT prevents the degradation of elastin by serine

proteases (ELASTASES) esp in lungs and vasculature.proteases (ELASTASES) esp in lungs and vasculature.Cf metalloproteases and TIMPCf metalloproteases and TIMP

90% of anti-elastase activity in lung is due to AAT90% of anti-elastase activity in lung is due to AAT

Possible mechanisms by which Possible mechanisms by which AAT could affect atherogenesisAAT could affect atherogenesis

AAT

C-36

Activation of monocytesFoam cell formation

Oxidised AAT+LDL

?

Clearance of LDL/oxAAT from plaque

Lipid accumulation/foam cell formation

Elastin

Elastin peptide

ELASTASEActivation of MMPs

Plaque ruptureVessel wall remodellingMonocyte transmigrationSMC migration

TIMP1

Hardening of vessel wallsIncreased blood pressure

Reactive oxygen species (ROS)

Suicide cleavage

AAT

C-36

Activation of monocytesFoam cell formation

Oxidised AAT+LDL

?

Clearance of LDL/oxAAT from plaque

Lipid accumulation/foam cell formation

Elastin

Elastin peptide

ELASTASEActivation of MMPs

Plaque ruptureVessel wall remodellingMonocyte transmigrationSMC migration

TIMP1

Hardening of vessel wallsIncreased blood pressure

Reactive oxygen species (ROS)

Suicide cleavage

Identification of novel polymorphisms in Identification of novel polymorphisms in the promoter of the promoter of AATAAT gene gene

Chromosome 14, within gene cluster of SERPINSChromosome 14, within gene cluster of SERPINS 2 mutually exclusive promoters in liver (endoderm) and 2 mutually exclusive promoters in liver (endoderm) and

macrophages (mesoderm)macrophages (mesoderm) Alternative splicing of macrophage mRNAAlternative splicing of macrophage mRNA 3’ UTR in liver3’ UTR in liver

1AIA

Identification of novel polymorphisms in Identification of novel polymorphisms in the promoter of the promoter of AATAAT gene gene

1AIA

• Sequence databases (Ensembl, NCBI UC)-blast-predicted polys/ conflicts

• Sequencing (PCR, sequencing)

• Validation of polys-RFLP MADGE (PCR, RE, gel)-TaqMan (labeled probes)

Novel polymorphism

Genotyping of novel polys in Genotyping of novel polys in association studiesassociation studies

In linkage disequilibrium with each other and In linkage disequilibrium with each other and some other polyssome other polys

As such, association with increased progression As such, association with increased progression of athersclerosis in one study. No significant of athersclerosis in one study. No significant association seen in other studies (with different association seen in other studies (with different endpoints).endpoints). VV

213a213aGluGlu264 S264 S

LysLys342 Z342 Z

Glu Glu 376as376as

3 new 3 new snpssnps

ArgArg101101

****** ******

VV213a213a

****** ****** ****

GluGlu264 S264 S

**

LysLys342 Z342 Z

**

Glu3Glu37676

*** <0.0005** <0.005* <0.05

Functional analysis of novel polysFunctional analysis of novel polys Transplorer software: predicts Transplorer software: predicts

transcription factor binding sites on transcription factor binding sites on DNA sequencesDNA sequences

YY1 Oct 1CDP CR1 CDP CR3+HD

A allele G allele

In vitroIn vitro functional studies for new polymorphisms: functional studies for new polymorphisms:Splicing profile of AAT mRNA transcripts in stimulated Splicing profile of AAT mRNA transcripts in stimulated

macrophages of different genotypesmacrophages of different genotypes

RT-PCR spanning intronsRT-PCR spanning introns

1A 1B 1C II III IV V1A 1B 1C II III IV V

5’5’ 3’3’

1A1B1C Both1A1B1C Both1A1C1A1C

• mRNA transcript mRNA transcript profile visualised on profile visualised on agarose gelagarose gel

IGFBPs: role in the ovaryIGFBPs: role in the ovaryAims:Aims:To investigate the role of IGFBP-2 and -4 in folliculogenesisBackground:Background: The insulin-like growth factors (IGFs) are potent stimulators of ovarian

follicle function. They are modulated in their actions by a range of IGF binding proteins

(IGFBPs). Amongst the IGFBPS, IGFBP-2 and -4 are uniquely correlated with the

health of a follicle. They are preferentially proteolysed in healthy follicles but remain intact in atretic follicles.

IGFBP-4 caused potent inhibition of gonadotrophin-mediated steroidogenesis and its proteolysis is therefore implicated as a requirement for selection of a dominant follicle.

Hypothesis:Hypothesis:IGFBP-2 and -4 are acting as brakes on folliculogenesis. Local regulation of

production and proteolysis occurs within the follicular and ovarian micro-environment

Some pretty picturesSome pretty pictures

Effects of IGFBP-2 and -4 on Effects of IGFBP-2 and -4 on FSH-mediated steroidogenesisFSH-mediated steroidogenesis

0

20

40

60

E2

fm

ol/1

000c

ells

/48h

t t+BP-4 t+FSH t+FSH+ BP-4

0

0.5

1.5

2.5

3.5

4.5

t t+FSH 0.5 5.0 50 BP-4 (ng/ml)

+ t + FSH

d

cbcb

a

a

t t+ BP-2 BP-3 BP-4 FSH t+ FSH+

0

0.5

1

1.5

2

2.5

3

a

b

c

E2

nm

ol/1

000

cell

s/48

h

Cells cultured +/- gonadotrophin+/- BP-2,-4 for 48h[Steroid] determined in the medium by RIASimilar results for lactate production (as measured by an ELISA kit)

Status of IGFBP-4 within Status of IGFBP-4 within follicular cellsfollicular cells

granulosa theca

IGFBP-4 WIB of GCCM (visualized by ECL)

lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

NHS

30

28

24

kDa

Mechanism of IGFBP-4 effectsMechanism of IGFBP-4 effects

0

20

40

60

0

20

40

60

6h

3h

C LH LH+IGFBP-4

Pnmol/1000 cells/48h

(i)

a

b

a

b

c

c

t FSH FSH+ FSH+ FSH+ 8-br 8-br+ 8-br+ 8-br+ BP-4 BP-4 BP-4 BP-4 BP-4 BP-4 (50) (5) (0.5ng/ml) (50) (5) (0.5ng/ml)

Pn

mol

/100

0 ce

lls/

48h

0

15

30

45

a

d

c

b

c c

(i) GC (3-7mm follicles)

Timecourse

Effect on cAMP-mediated steroidogenesis

Localisation of IGFBP-4 in Localisation of IGFBP-4 in follicular cellsfollicular cells

28 kDa(glycosylated,Intact)

16.5 kDa(fragment)

48 kDa(dimers)

CM AW N M C

LegendCM=conditioned mediumAW=cell surface associated fraction

N=nuclear fraction M=membrane fractionC=cytosolic fraction

Fig. 8a

(i) (ii)

(iii) (iv)

(v) (vi)

5 m 5 m

5 m 5 m

10 m

20 m

(iv)(iii)

(ii)(i)

5 m

5 m 5 m

5 m

(v) (vi)

10 m

20 m

Possible mechanisms by which Possible mechanisms by which IGFBP-4 inhibits gonadotrophin-IGFBP-4 inhibits gonadotrophin-

mediated steroidogenesismediated steroidogenesis

PutativeBP-4receptor

PKA

Gn

cAMP

ATP

GDPGTP

AC

steroidogenesismitogenesis

Extracellular

Intracellular

GTP

GsGs

PKA

Gn

cAMP

ATP

GDPGTP

AC

steroidogenesismitogenesis

Extracellular

Intracellular

GTP

GsGs

IGFBP-4

IGFBP-4

(i) Schematic model of the Gn signal transduction pathway in follicular cells leading toincreased steroidogenesis.

(ii) Possible sites of action of IGFBP-4 in the Gn signal transduction pathway leadingto inhibition of steroidogenesis.

Gn receptor

ActivatedGs protein

-subunitlinked toGTP

Figure 14. Postulated schematic model of the mechanism bywhich IGFBP-4 inhibits Gn-stimulated steroidogenesis.

See legend overleaf.

Possible mechanisms by which Possible mechanisms by which IGFBP-4 inhibits gonadotrophin-IGFBP-4 inhibits gonadotrophin-

mediated steroidogenesismediated steroidogenesis

PutativeBP-4receptor

PKA

Gn

cAMP

ATP

GDPGTP

AC

steroidogenesismitogenesis

Extracellular

Intracellular

GTP

GsGs

PKA

Gn

cAMP

ATP

GDPGTP

AC

steroidogenesismitogenesis

Extracellular

Intracellular

GTP

GsGs

IGFBP-4

IGFBP-4

(i) Schematic model of the Gn signal transduction pathway in follicular cells leading toincreased steroidogenesis.

(ii) Possible sites of action of IGFBP-4 in the Gn signal transduction pathway leadingto inhibition of steroidogenesis.

Gn receptor

ActivatedGs protein

-subunitlinked toGTP

Figure 14. Postulated schematic model of the mechanism bywhich IGFBP-4 inhibits Gn-stimulated steroidogenesis.

See legend overleaf.

Regulation of IGFBP-4 proteinRegulation of IGFBP-4 protein

0

5

10

15

20

25

30

Arb

itra

ry O

D u

nit

s

of 2

8KD

a p

rote

in

c t 10 1ng/ml

t+FSH

25%

48%

(ii) Mean optical density of 28kDa isoform of IGFBP-4

t+FSH t+FSH

28 kDa

NHS

NHS H A A A H H A A A H

NHS H A A HNHS H A A H

Follicular fluid

Granulosa cells

Regulation of IGFBP-4 proteaseRegulation of IGFBP-4 protease

NAS PS H A H H A A

c+tt+LH

Large Follicle

BP-4 PS +++

Intact IGFBP-4

IGFBP-4fragments

Fig. 13b(i) Protease assay of GCCM (19mm)

Follicular fluidGranulosa cells

The effects of cell culture The effects of cell culture on IGFBP profileon IGFBP profile

1 3 4 7 11

30

28

24

kDa

3 4 7 11 1 3 4 7 11day ofincubation

Large follicles Small follicles Large follicles

0

1 00

2 00

1 3 4 7 110

100

200

300

1 3 4 7 11 0

100

200

300

3 4 7 11

OD

Arb

itra

ry u

nit

s

30 kDa

28 kDa

24 kDa

day ofincubation

Role of IGFBP-4 in the ovaryRole of IGFBP-4 in the ovary

FSH LHdue to steroids bydominant follicle

Atresia of remainder of cohort

BP-4 protease inhibitor

Figure 18. Schematic model for the role of IGFBP-4 in the ovary.

The concentration of IGFBP-4 protease in a follicle at the selection stageis dependent on GC number and GC responsiveness to proteaseregulatory factors, i.e. FSH. At selection stage, the follicle with thehighest concentration of IGFBP-4 protease and lowest levels of inhibitor,has the greatest ability to produce steroids in response to Gn and IGF-II.This follicle is able to become dominant over the remaining cohort and bythe increased production of E2, decrease the circulating levels of FSH andacquire receptors to LH on the GC. This results in further proteolysis ofIGFBP-4 and a reduction in the production of IGFBP-4 and proteaseinhibitor.The reduction in FSH levels due to the selected follicle, causes theproduction of IGFBP-4 to increase and proteolysis to decrease. This leadsto increased inhibition of Gn-stimulated steroidogenesis and eventuallyatresia.

proteolysed BP-4 Gn-mediated steroidogenesis

intact BP-4 inhibition ofGn-mediated steroidogenesis

Recruitment Selection

Growth and development ofdominant follicle

TechniquesTechniques Protein: - activity (functional assays, eg protease assays)Protein: - activity (functional assays, eg protease assays)

- concentration (ELISA, RIA)- concentration (ELISA, RIA) -status (WIB)-status (WIB) -localisation (confocal, sub-cell fractionation+WIB)-localisation (confocal, sub-cell fractionation+WIB)

Cells: -micro-dissectionCells: -micro-dissection -primary cells-primary cells -cell lines (immortalised or finite lifespan)-cell lines (immortalised or finite lifespan) -viability (MTT, neutral red)-viability (MTT, neutral red) -cell proliferation (3H-thymidine incorporation, cell lysis, count)-cell proliferation (3H-thymidine incorporation, cell lysis, count)

Signaling: -cAMP analogues Signaling: -cAMP analogues -specific enzyme blockers-specific enzyme blockers

DNA: - determination of genotype (RFLP, TaqMan)DNA: - determination of genotype (RFLP, TaqMan) - detection of genetic polymorphisms (sequencing, databases, SSCP)- detection of genetic polymorphisms (sequencing, databases, SSCP) - measurement of gene expression (RT-PCR)- measurement of gene expression (RT-PCR) - functional analysis of polymorphisms (consensus sequence prediction - functional analysis of polymorphisms (consensus sequence prediction

software, luciferase assayssoftware, luciferase assays