Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts
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JOURNAL OF CELLULAR PHYSIOLOGY 195:7079 (2003)
Role of Insulin-Like Growth Factor Binding Protein-3(IGFBP-3) in the Differentiation of Primary
Human Adult Skeletal Myoblasts
EMILY J. FOULSTONE,* PAUL B. SAVAGE, ANNA L. CROWN, JEFF M.P. HOLLY, ANDCLAIRE E.H. STEWART
Division of Surgery, University of Bristol, Bristol Royal Infirmary, Bristol, England
Although muscle satellite cells were identified almost 40 years ago, little is knownabout the induction of their proliferation and differentiation in response to physio-logical/pathological stimuli or to growth factors/cytokines. In order to investigatethe role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system inadult humanmyoblast differentiationwe have developed a primary human skeletalmuscle cell model. We show that under low serum media (LSM) differentiatingconditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis.IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially byproteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer ofcells into LSMbut then increasedwithmyotube formation. Treatmentwith 20ng/mltumour necrosis factor-alpha (TNFa), which inhibits myoblast differentiation,blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, whichstimulatesmyoblast differentiation, increased IGFBP-3 secretion. The TNFa-induc-ed decrease in IGFBP-3 production and inhibition of differentiation could not berescuedby addition of IGF-I. LongR3IGF-I,which does not bind to the IGFBPs, had asimilar effect on differentiation and IGFBP-3 secretion as IGF-I, both with andwithout TNFa, confirming that increased IGFBP-3 is not purely due to increasedstability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretionusing antisense oligonucleotides led to an inhibition of differentiation. Taken to-gether these data indicate that IGFBP-3 supports myoblast differentiation. J. Cell.Physiol. 195: 7079, 2003. 2003 Wiley-Liss, Inc.
Muscle degeneration is recognised as a significantcause of patient disability and death underlying manychronic catabolic illnesses (Calman, 1982), but as yet noeffective therapeutic interventions are available totackle this problem. The normal rate of protein turnoveris high (250300 g/day) so any small but persistentchange in catabolism/anabolism could culminate insignificant muscle loss. Muscle myofibres are terminallydifferentiated and incapable of replication, thereforeregeneration is dependent on a small population of re-sident cells generally referred to as satellite cells (Olson,1992; Olson and Rosenthal, 1994). In order to elucidatepotential therapeutic targets for muscle wasting, it isessential to understand how these muscle satellite cellsproliferate and differentiate. A thorough understandingof human myoblast growth and differentiation wouldalso have potential relevance to skeletal muscle stemcell therapies, allowing manipulation of the system fortherapeutic effect.
Although many studies have been carried out on thegrowth and differentiation of muscle cells, the majorityof in vitro studies use immortalised rat and mousemuscle cell lines. These studies have highlighted thecritical role of the insulin-like growth factors IGF-Iand IGF-II and their binding proteins in the con-trol of proliferation, differentiation and metabolism
(Florini et al., 1991; Stewart and Rotwein, 1996). Todate, six high affinity IGF binding proteins (IGFBPs)have been identified (Jones and Clemmons, 1995). Theseproteins modulate the activity of the IGFs by extendingtheir half-life and by either potentiating or inhibitingbinding of the IGFs to their receptors (Conover et al.,1990, 1993). Importantly, however, the muscle cell linesused to date differ in which IGF binding proteins theyproduce (James et al., 1993; McCusker and Clemmons,1998; Crown et al., 2000) and are not identical in theirresponses to IGFs (Florini et al., 1991). We havetherefore developed a more physiologically relevant,primary adult human muscle cell model in which toaddress our questions (Crown et al., 2000).
2003 WILEY-LISS, INC.
Contract grant sponsor: AICR; Contract grant number: 99-134.
*Correspondence to: Emily J. Foulstone, Division of Surgery,University of Bristol, Bristol Royal Infirmary, Bristol, BS2 8HW,England. E-mail: firstname.lastname@example.org
Received 6 August 2002; Accepted 5 November 2002
One major difference between these cultures and thesecondary cell lines that have been studied to date is thatthe adult human muscle cell cultures produce andsecrete large amounts of IGFBP-3 (Crown et al., 2000).Although IGFBP-3 is the major IGFBP found in serum,it is also produced locally in many tissues (Kim et al.,1997). Its activity is modulated not only by its rate ofsynthesis but also by post-translational modificationsand by proteolysis (Collett-Solberg and Cohen, 1996).Recently IGFBP-3 has been shown to have effects oncell growth and apoptosis that are independent of IGFaction (Gill et al., 1997; Fowler et al., 2000; Hollowoodet al., 2000) and high levels of IGFBP-3 decrease theproliferation of chondrocytes (Spagnoli et al., 2001). Inaddition, IGFBP-3 has also been implicated in porcinemuscle cell differentiation (Johnson et al., 1999).Interestingly, IGFBP-5, which we have shown inhibitsdifferentiation and acts as a potent survival factor formurine C2 myoblasts (James et al., 1996; Meadowset al., 2000; Foulstone et al., 2001) is closer to IGFBP-3in sequence than any other IGFBP suggesting poten-tial for overlapping/related actions. Data derived fromother cell models would therefore suggest a potentialand important role for IGFBP-3 in skeletal musclehomeostasis with manipulation of IGFBP-3 possiblyacting as an important tool in influencing skeletalmuscle maintenance and repair in vivo. We thereforesought to investigate what role IGFBP-3 may play inprimary skeletal muscle differentiation, proliferationand survival.
In addition to the IGFs, other growth factors andcytokines can influence muscle growth and mainte-nance (Hawke and Garry, 2001). Production of theinflammatory cytokine tumour necrosis factor a (TNFa)occurs as an important pathophysiological responseduring many critical illnesses and TNFa has beenimplicated in the aetiology of muscle wasting (Matthysand Billiau, 1997). We have shown that TNFa blocks C2myoblast differentiation and decreases IGFBP-5 secre-tion while at the same time increasing proliferation andinducing cell death (Meadows et al., 2000; Foulstoneet al., 2001). We therefore investigated whether TNFahad similar effects in the primary human skeletalmuscle cells with regard to differentiation, death andimpact on the IGFBPs. In light of their importancein differentiation, growth and survival in many cellsystems, including muscle, we also investigated the roleof the IGFs, alone or in combination with TNFa, ondifferentiation and IGFBP-3 secretion in the primarycells. These studies identify the IGFBPs secreted byhuman primary myoblasts during differentiation, theimpact of physiologically important growth factor/cytokine family members on their regulation and im-plicate IGFBP-3 as a potent and positive differentiationfactor.
MATERIALS AND METHODSMaterials
Unless otherwise stated all chemicals were fromSigma (Poole, UK). Hams F-10 media was from BioWhittaker (Wokingham, UK) and heat inactivatedfoetal bovine serum (hiFBS) was from GIBCO (Paisley,UK). TNFa was purchased from Bachem (St. HelensMerseyside, UK). Recombinant human IGF-I and Long-
R3IGF-I were from GroPep (Adelaide, Australia).Recombinant human non-glycosylated IGFBP-3 wasfrom Dr. C. Moot (Brogrowth, CA). Trypsin and gelatinwere from Difco (Oxford, UK). IGFBP-3 anti-sense(CATGACGCCTGCAACCGGGG) and sense (CCCC-GGTTGCAGGCGTCATG) oligonucleotides were pur-chased from Sigma Genosys. Anti-human IGFBP-5antibody was from IBT-Immunological and BiochemicalTestsystems, GmbH (Reutlingen, Germany).
The study was approved by the Local Research EthicsCommittee and all patients gave written, informedconsent. Perioperative muscle biopsies were taken fromthe anterior abdominal wall of patients undergoingelective surgery. Biopsies were collected from 41 sub-jects, 24 male and 17 female. Average age was 56 years,(range 3685 years); BMI 26.71, (range 16.540.5).
Cells were isolated following a modified method ofBlau and Webster (1981) (Crown et al., 2000). Briefly,biopsies were dissected into 1 mm3 pieces and digestedin TE (0.05% trypsin, 0.02% EDTA in phosphatebuffered saline (PBS)) for 15 min with gentle mixing.The supernatant was removed and added to hiFBS toneutralize the trypsin before centrifugation at 1,000 rpmfor 5 min. The resultant cell pellet was resuspendedin growth medium (Hams F-10 supplemented with20% hiFBS, 50 IU/ml penicillin, 5 mg/ml streptomycinand2.5mg/mlamphotericin B).The processwasrepeateda further two times and the cell suspensions pooled andplated onto T75 flasks coated with 0.2% gelatin. Cellswere grown in a humidified 5% CO2 atmosphere at378C and passaged when 8090% confluent. Cells wereinduced to differentiate when approximately 80% con-fluent by washing twice in PBS and placing into lowserum media (LSM) (minimum essential medium(MEM) supplemented with 2% hiFBS, 1% L-glutamine,50 IU/ml penicillin, 5 mg/ml streptomycin and 2.5 mg/mlamphotericin B). Experimental cells were dosed attransfer into LSM as described in the text and all ex-periments were terminated after 7 days unless other-wise stated. By day 7, cultures had reached maximumdifferentiation as assessed by measuring increasingcreatine kinase (CK) activity. Experiments were per-formed on cells between passages 2 and 6. The culturesare not homogenous populations of myoblasts butinclude other cell types that were present in the originalbiopsy. The proportion of myoblasts was determined foreach biopsy by desmin staining and cell counting (Crownet al., 2000). Cultures on average contained 482.4%myoblasts. Cell death was assessed using Trypan Blueexclusion. Aliquots of cells (50 ml) were loaded onto ahaemocytometer 1:1 with Trypan Blue (50 ml). Total cellnumber was counted and dead cells were identified asthose that had taken up the blue dye. The percentage ofdead cells could then be calculated.
Cells were grown on 35-mm dishes. After treatment,media was removed and stored at808C. The cells werewashed twice with PBS and scraped into 60 ml of TMTbuffer (50 mM Tris-MES, 1% Triton X-100, pH 7.8).
MYOBLAST DIFFERENTIATION: A ROLE FOR IGFBP-3 71
Samples were stored at808C and were assayed within2 weeks of collection using a commercially available kitfollowing the manufacturers instructions (Sigma CK-10). Enzymatic activity was normalised to total proteincontent as determined by the BCA protein assay (Pierce,Chester, UK).
Ligand and Western blotting
Cells were grown on 35-mm dishes. After treatmentmedia was removed and stored at208C and cell lysateswere prepared. Cells were washed twice with PBS andthen scraped into 100 ml lysis buffer (10 mM Tris-HClpH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodiumpyrophosphate, 50 nM NaF, 100 mM Na2VO4, 0.1 mMPMSF). Samples were stored at 208C until used.Binding proteins in conditioned media or cell lysates,were separated under non-reducing conditions using12.5% SDSPAGE, transferred to Hybond-C nitro-cellulose membrane (Amersham, Little Chalfont, UK)and analysed either by ligand or Western blotting. Forligand blots, membranes were probed with a mixtureof radiolabelled IGF-I and IGF-II and visualised byautoradiography. For Western blots, membranes wereprobed with an in-house anti-human IGFBP-3 antibody,(1:5,000) or an anti-human IGFBP-5 antibody (1:1,000)in TBST (10 mM Tris base pH 7.4, 150 mM NaCl, 0.2%tween-20) plus 5% milk. Antibodies were incubated atroom temperature over-night. Appropriate secondaryantibody was used (1:10,000) in TBST plus 5% milk andproteins were visualised using enhanced chemilumines-cence (Amersham).
Secreted IGFBP-3 was quantified by an in-house radioimmunoassay (RIA) as previously described(Yateman et al., 1993), using an in-house antiserum(Cwyfan Hughes et al., 1995) at a final dilution of1:20,000 (interassay % CV 5.1).
Cells were grown on 35-mm dishes. Following treat-ment, dead cells in the media were collected and the liveadherent cells were trypsinised in 0.1 ml TE solution.The cells were pooled and fixed following a PBS wash in1 ml of 70% ethanol. Following a minimum of 24 h at 48C,cells were pelleted (6,500 rpm; 5 min) and washed oncewith PBS (6,500 rpm; 5 min). The cells were resus-pended in Krishans reaction buffer (0.05 mg/ml propi-dium iodide, 0.1% sodium citrate, 0.02 mg/ml RNAseA, 0.3% NP-40, pH 8.3) and incubated at 48C for 30 minin the dark. DNA profiles of all cells were measuredon a FacsCalibur flow cytometer (Beckton-Dickinson,Oxford, UK) by an argon laser at 488 nm and analysedusing Cell Quest (Beckton-Dickinson) and Multicycle(Phoenix flow systems, San Diego, CA) softwarepackages.
Data were analysed using Microsoft Excel version5.0. Results were expressed as meanSEM. Signifi-cant effects were determined using Students t-test(unpaired, 2-tailed). A statistically significant differencewas considered to be present at P
The initial decrease followed by an increase inIGFBP-3 production with differentiation was specificto this binding protein. In contrast to IGFBP-3, intactIGFBP-5, which is seen in the differentiating mediaat day 1, decreased with time over the 7 days in LSM(Fig. 4). IGFBP-5 proteolytic fragments increased overthe same time period such that by day 7 no intactIGFBP-5 was present in the media (Fig. 4).
In contrast to the IGFBPs, both IGF-I and IGF-II wereundetectable by RIA in the conditioned media from theprimary cultures (data not shown).
TNFa blocks myoblast differentiation,IGF-I stimulates differentiation
Having characterised the IGFBPs secreted by theprimary skeletal muscle cultures, we next wished toinvestigate the impact of modulators of skeletal muscledifferentiation in this model. The inhibition of differ-entiation by TNFawas dose dependent, with maximuminhibition occurring at 1020 ng/ml (Fig 5A). Addition of20 ng/ml TNFa significantly inhibited differentiation by54.64.6% (P
Reduction of IGFBP-3 using antisenseoligonucleotides inhibits differentiation
As we had shown that modulation of IGFBP-3 secre-tion with TNFa and IGF-I occurred in parallel withchanges in differentiation, we next wished to establishwhether IGFBP-3 was important for skeletal muscledifferentiation. Following dose and time course experi-ments (data not shown), cells were treated with a singledose of 50 mg/ml antisense or sense IGFBP-3 oligonu-cleotide on transfer into differentiating conditions andcells were extracted for CK activity at day 4. Conditionedmedia from the cells were assayed for IGFBP-3 usingRIA. Additio...