Download - Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: A dual role for IGFBP-5

JOURNAL OF CELLULAR PHYSIOLOGY 177:47–57 (1998)

Modulation of Insulin-Like Growth FactorActions in L6A1 Myoblasts by Insulin-LikeGrowth Factor Binding Protein (IGFBP)-4

and IGFBP-5: A Dual Role for IGFBP-5DAINA Z. EWTON,1* SHARON A. COOLICAN,1 SUBBURAMAN MOHAN,2

STEVEN D. CHERNAUSEK,3 AND JAMES R. FLORINI1

1Department of Biology, Syracuse University, Syracuse, New York2Department of Medicine, Biochemistry, and Physiology, Loma Linda University,

Loma Linda, California3Department of Endocrinology, Children’s Hospital Medical Center, Cincinnati, Ohio

We have previously shown that the insulin-like growth factors (IGFs) stimulateboth proliferation and differentiation of skeletal muscle cells in culture, and thatthese actions in L6A1 muscle cells may be modulated by three secreted IGFbinding proteins (IGFBPs), IGFBP-4, -5, and -6. Since we found that the temporalexpression pattern of IGFBP-4 and IGFBP-5 differed dramatically during the transi-tion from proliferating myoblasts to differentiated myotubes, we undertook thecurrent study to examine the effects of purified IGFBP-4 and IGFBP-5 on IGF-stimulated actions in L6A1 muscle cells. As has been shown for other cell types,we found that IGFBP-4 had only inhibitory actions, inhibiting IGF-I and IGF-II-stimulated proliferation and differentiation. In contrast, IGFBP-5 exhibited bothinhibitory and stimulatory actions. When added in the presence of 30 ng/ml IGF-I, IGFBP-5 (250 ng/ml) inhibited all markers of the early proliferative response:the tyrosine phosphorylation of the cytoplasmic signaling molecules IRS-1 andShc, the activation of the MAP kinases, ERK1 and 2, the elevation of c-fos mRNA,the early inhibition of the elevation in myogenin mRNA, and the increase in cellnumber. In contrast, IGFBP-5 stimulated all aspects of the myogenic response toIGF-I: the later rise in myogenin mRNA, the elevation of creatine kinase activity,and the fusion of myoblasts into myotubes. This dual response to IGFBP-5 wasgreatest when it was added at a molar ratio of IGFBP-5 to IGF-I of 2:1. In contrast,when IGFBP-5 was added in the presence of IGF-II, it inhibited both proliferationand differentiation. Neither IGFBP had any effect when added in the presenceof R3 IGF-I, an analog with substantially reduced affinity for IGFBPs. Our resultssuggest that the role of IGFBP-4 is mainly to sequester excess IGFs, and thusinhibit all actions. IGFBP-5, however, is capable of eliciting a dual response,possibly due to its unique ability to associate with the cell membrane. J. Cell.Physiol. 177:47–57, 1998. q 1998 Wiley-Liss, Inc.

The insulin-like growth factors (IGF-I and IGF-II) ily of IGF binding proteins (IGFBPs; reviewed inare unique among mitogens in that they not only stimu- Rajaram et al., 1997; Jones and Clemmons, 1995;late proliferation of skeletal muscle cells in culture, but Bach and Rechler, 1995). L6 muscle cells have beenthey also stimulate myogenic differentiation (reviewed shown to secrete three IGFBPs, IGFBP-4, -5, and -6in Florini et al., 1996). Even in the absence of exogenous (McCusker et al., 1989; McCusker and Clemmons,IGFs, they have an established role as autocrine/para- 1994; Ewton et al., 1994a; Silverman et al., 1995; Ew-crine modulators of skeletal muscle cell growth and ton and Florini, 1995). In our studies, we found thatdifferentiation. IGFs have been shown to be expressed each of these IGFBPs displayed a unique pattern ofby several muscle cell lines in culture (Tollefsen et al.,1989a,b; Florini et al., 1991a; Brown et al., 1992; Rosenet al., 1993; Magri et al., 1994). Evidence from studies

Contract grant sponsor: NIH; Contract grant number: HL11551;measuring relative potencies of IGF-I and IGF-II and Contract grant sponsor: USDA; Contract grant number: 9603268.from studies with IGF analogs with altered receptor*Correspondence to: Dr. Daina Z. Ewton, Biology Department,affinities suggests that the type I IGF-I receptor medi-Syracuse University, 130 College Place, Syracuse, NY 13244.ates both proliferation and differentiation in L6A1E-mail: [email protected] cells (Ewton et al., 1987, 1994b).

The actions of IGFs are further modulated by a fam- Received 26 August 1997; Accepted 13 March 1998

q 1998 WILEY-LISS, INC.

JCP-10193/ 893f$$0193 07-20-98 20:16:31 wlcpa W Liss: JCP

EWTON ET AL.48

expression during the various stages of L6A1 muscle sponse (Coolican et al., 1997). Our studies showed thatIGF-I treatment of L6A1 myoblasts resulted in thecell growth and differentiation (Ewton and Florini,

1995). IGFBP-4 mRNA levels were highest in prolifer- early phosphorylation of both MAP kinase isoforms,ERK1 and ERK2. This phosphorylation was inhibitedating myoblasts but decreased during IGF-stimulated

differentiation. No IGFBP-5 mRNA could be detected by the MEK inhibitor PD098059, resulting not only ininhibition of proliferation, but also in a dramatic in-in proliferating myoblasts, but levels increased dramat-

ically during differentiation. IGFBP-6 mRNA levels increase in the subsequent myogenic response.The recent availability of purified IGFBPs promptedcreased in serum-free medium as cultures became qui-

escent in the absence of IGFs. These observations sug- our study to investigate the actions of each of theIGFBPs secreted by L6A1 cells on the mitogenic andgested to us that each of the three IGFBPs may play

a specialized role during myogenesis. In addition, ana- myogenic actions of the IGFs, including effects on theIGF-stimulated signal transduction pathway. Bach etlogs of IGF-I with reduced affinity for IGFBPs were

about 10 times more potent than native IGF-I in stimu- al. (1994, 1995) have shown that purified IGFBP-6 in-hibited IGF-II-stimulated, but not IGF-I-stimulated,lating L6A1 proliferation, but about 100 times more

potent in stimulating differentiation, suggesting that proliferation and differentiation of L6A1 myoblasts. Inthe present study, we describe the effects of purifiedthe IGFBPs secreted by the cells were inhibitory to

these actions of IGF-I (Ewton et al., 1994a; Silverman IGFBP-4 and -5 on IGF-I and IGF-II-stimulated pro-cesses in L6A1 cells, and find that while IGFBP-4 iset al., 1995; Ewton and Florini, 1995).

It is not obvious how the IGFs can stimulate two inhibitory under all conditions tested, IGFBP-5 ap-pears to have a dual role when added in the presencemutually exclusive processes such as proliferation and

differentiation, or what role the secreted IGFBPs play of IGF-I, inhibiting all aspects of the proliferative re-sponse, but stimulating the myogenic response.in modulating the effects of the IGFs. It is also unclear

how one receptor can mediate signaling for two suchMATERIALS AND METHODSantagonistic processes, and the question still remains:

MaterialsWhere do the signaling pathways for IGF-stimulatedproliferation and differentiation diverge? It now seems Recombinant human IGF-I was a gift from Ciba-

Geigy (Summit, NJ), and the analog R3 IGF-I was aclear that the stimulatory effects on proliferation anddifferentiation are temporally separated. Initially, IGF- gift from Paul Walton and John Ballard (GroPep Pty

Ltd., Adelaide, Australia). Recombinant human IGF-I stimulates the proliferative response and inhibits themyogenic response; this is followed by withdrawal from II was kindly provided by Eli Lilly (Indianapolis, IN).

IGFBP-5 was purified from human bone extract as pre-the cell cycle and upregulation of myogenic signals anddifferentiation (Ewton et al., 1994b; Rosenthal and viously described (Bautista et al., 1991; Mohan et al.,

1995), and the homogeneity of the preparation was es-Cheng, 1995; Engert et al., 1996). In other cell types,the mitogenic signal transduction pathway from the tablished by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) and amino-terminal se-IGF-I receptor, a tyrosine kinase, has been shown toinvolve tyrosine phosphorylation of several intracellu- quence analysis. No detectable levels of IGF-I or IGF-

II were found in the purified preparations of IGFBP-5.lar substrates (reviewed in Jones and Clemmons, 1995;LeRoith et al., 1995), including IRS-1 and Shc, the acti- Rat IGFBP-4 was isolated from media conditioned by

the B104s cell line (Cheung et al., 1991) as previouslyvation of the Ras-Raf-MAP kinase pathway, followedby the activation of nuclear transcription factors (see described (Cheung et al., 1994). It contained mainly

the 24 kDa species, but a small amount (õ10%) of 28review by LeRoith et al., 1995). In addition, phosphory-lated IRS-1 associates with PI 3-kinase, leading to the kDa glycosylated IGFBP-4 was present. Purity was ver-

ified by silver staining after SDS-PAGE and by electro-downstream activation of p70 S6 kinase. Similar earlyevents associated with the mitogenic response to IGF- spray mass spectrometry which indicated a single spe-

cies of correct mass. Tissue culture medium compo-I in muscle cells have also been described: stimulationof tyrosine phosphorylation of IRS-1 and Shc (Lamph- nents were purchased from Gibco/BRL (Grand Island,

NY). The c-fos cDNA probe was obtained from ATCCere and Lienhard, 1992; Giorgino and Smith, 1995),induction of the nuclear transcription factor c-fos (Ong (Rockville, MD) and the myogenin cDNA probe was a

gift from Eric N. Olson (University of Texas, South-et al., 1987; Giorgino and Smith, 1995), suppression ofmyogenin gene expression, inhibition of retinoblastoma western Medical Center, Dallas, TX). The anti-rat car-

boxy-terminal IRS-1 polyclonal antibody, anti-humanprotein (Rb) dephosphorylation, and upregulation ofthe gene expression of cyclin-dependent kinase 4 and Shc polyclonal antibody, and recombinant Protein A

agarose were from Upstate Biotechnology, Inc. (Lakecyclin D1 (Rosenthal and Cheng, 1995; Engert et al.,1996). This early mitogenic response is followed by Placid, NY). Horseradish peroxidase conjugated anti-

phosphotyrosine monoclonal antibody RC20H was ob-stimulation of differentiation characterized by eleva-tion in the steady-state levels of myogenin mRNA (Flo- tained from Transduction Laboratories (Lexington,

KY). The antibody to phospho-MAPK was purchasedrini et al., 1991b), an increase in the levels of creatinekinase (CK) activity, and fusion of myoblasts into myo- from New England Biolabs, Inc. (Beverly, MA). Goat

anti-rabbit horseradish peroxidase conjugated IgG, thetubes (Ewton and Florini, 1981).By using specific inhibitors of signaling molecules of BCA protein assay reagents, and the SuperSignalTM

CL-HRP substrate system for enhanced chemilumines-the IGF-I signal transduction pathway, we have re-cently shown that the activation of the MAP kinase cence were purchased from Pierce (Rockford, IL). The

MEK inhibitor PD098059 was purchased from New En-pathway is necessary for IGF-I-stimulated L6A1 my-oblast proliferation, while the PI 3-kinase/p70 S6 ki- gland BioLabs. All other chemicals were from Sigma

(St. Louis, MO).nase pathway plays a critical role in the myogenic re-


ROLE OF IGFBPs IN MYOGENESIS 49

dase conjugated anti-phosphotyrosine monoclonal anti-Cell culturebody RC20H (1:2500) in blocking buffer. The blots wereL6A1 myoblasts were grown in Dulbecco’s modified washed six times for 5 minutes each in TBST at roomEagle’s medium (DMEM) containing 10% horse serum, temperature, and immunolabeling detected by en-1% chick embryo extract, and 1% antibiotic antimycotic hanced chemiluminescence according to the manufac-solution (Sigma). Cultures were plated at 4 1 104 cells/ turer’s directions. The blots were stripped by incuba-2 cm2 well for cell number and CK determination, and tion for 1 hour at 507C in 62.5 mM Tris-HCl, pH 6.7,at 4–5 1 105 cells/60 mm dish for RNA extraction or 2% SDS, and 0.7% ß-mercaptoethanol, washed for 1

immunoprecipitation of proteins. After incubation hour at room temperature in four changes of TBST,overnight, the cultures were washed with DMEM be- and reblocked in blocking buffer containing 3% nonfatfore the addition of IGFs and/or IGFBPs in DMEM dry milk in TBST. The blots were immunolabeled withcontaining 0.1% bovine serum albumin (BSA). polyclonal antibodies to IRS-1 or Shc (1 mg/ml in TBST

containing 3% nonfat dry milk) overnight at 47C, fol-Quantitation of proliferationlowed by washing and detection as described above.and differentiation

For phospho-MAPK detection, total cell lysates wereTo quantitate cell proliferation, cells were trypsin- prepared by washing the cell monolayer twice with ice-

ized and counted in a Model ZBI Coulter Counter 24 cold PBS followed by lysis in boiling 11 Laemmli sam-hours after the addition of growth factors. Differentia- ple buffer containing 100 mM DTT, 1 mM Na3VO4, 1tion was quantified 72 hours after the addition of mM NaF, and analyzed by SDS-PAGE and transferredgrowth factors by measuring the elevation in CK activ- to nitrocellulose as described above. The blots wereity using the NAD-coupled microtiter assay (Florini, blocked for 1 hour at room temperature in blocking1989). Statistical analysis was performed by using Stu- buffer containing 5% nonfat dry milk in PBST and incu-dent’s t-test. bated overnight at 47C with the antibody against phos-

pho-MAPK (1:1,000) in 5% milk, PBST. After washingNorthern blot analyses six times in PBST, the membranes were incubated for 1hour at room temperature with horseradish peroxidaseTotal RNA was isolated from cultures using theconjugated goat-anti rabbit IgG diluted 1:3,000 in 5%RNeasy total RNA kit (Qiagen, Chatsworth, CA) ac-milk, PBST. The blots were washed six times in PBSTcording to the manufacturer’s instructions. Five to tenat room temperature, and immunolabeling was de-microgram samples were analyzed on Northern blots.tected as described above.Consistency of RNA loading was verified by visualiza-

tion of ethidium bromide-stained ribosomal RNARESULTSbands. The 32P-labeled probe for c-fos mRNA was pre-

Effect of IGFBP-4 on IGF-I-stimulated L6A1pared by random priming of the 1 kb Pst I restrictionmyoblast proliferation and differentiationfragment, and for myogenin, the 1.1 kb BamH I/EcoR

I restriction fragment. Since IGFBP-4 is the major IGFBP secreted by prolif-erating L6 myoblasts (McCusker et al., 1989; McCuskerImmunoprecipitation and Western blottingand Clemmons, 1994; Ewton and Florini, 1995), we de-The cell monolayers in 60 mm dishes were washed termined its role in modulating the mitogenic and myo-twice with ice-cold phosphate-buffered saline (PBS) genic actions of IGF-I on L6A1 myoblasts. Figure 1Aand the cells were lysed by incubating the cultures for shows that the addition of exogenous IGFBP-4 to L6A130 minutes in 400 ml cold modified radioimmunoprecip- myoblasts in the presence of 30 ng/ml (4 nM) IGF-Iitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, gave a concentration-dependent inhibition of cell prolif-1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, eration. IGF-I alone caused almost a doubling (93%1 mM EGTA, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml increase) of cell number over DMEM controls, whileleupeptin, 1 mg/ml pepstatin, 1 mM Na3VO4, 1 mM250 ng/ml (10.4 nM) IGFBP-4 inhibited this IGF-I effectNaF). The lysates were centrifuged for 10 minutes in aby 80%. IGFBP-4 had no effect on cell number in themicrocentrifuge to remove any insoluble material. Theabsence of IGF-I, or in the presence of R3 IGF-I, anprotein content of the supernatants was determined byanalog with reduced affinity for IGFBPs; there was athe Bicinchoninic Acid (BCA) method. Two hundred71% increase over DMEM cell numbers 24 hours afterfifty micrograms of protein from each dish was immu-addition of R3 IGF-I (3 ng/ml, a level that gives a maxi-noprecipitated overnight at 47C with 3 mg of IRS-1 ormal response), and a 68% increase in cells incubatedShc polyclonal antibody. The immunocomplex was cap-with R3 IGF-I in the presence of 250 ng/ml IGFBP-4.tured by incubating with Protein A agarose for 2 hours

Figure 1B shows that IGFBP-4 also dramatically in-at 47C. Immunoprecipitates were washed three timeshibited L6A1 myoblast differentiation (as measured bywith ice-cold RIPA buffer. The pellets were boiled inelevation of CK activity) in a concentration-dependentLaemmli buffer with 100 mM dithiothreitol (DTT) formanner. In the absence of IGFs, there was little or5 minutes. Proteins were separated by SDS-PAGE andno differentiation, but the addition of 30 ng/ml IGF-Itransferred onto nitrocellulose (Millipore, Bedford, MA)resulted in fusion of myoblasts into myotubes accompa-using transfer buffer containing 48 mM Tris base, 39nied by a 20-fold increase in CK activity. Addition ofmM glycine, 0.037% SDS, and 20% methanol at 0.8250 ng/ml IGFBP-4 inhibited this IGF-I effect by 90%.mA/cm2 for 1 hour.Similar to the lack of effect on proliferation, IGFBP-4For phosphotyrosine blotting, the nitrocellulosehad no effect on CK activity when added in the absencemembranes were blocked for 20 minutes at 377C inof IGF-I or in the presence of R3 IGF-I (31.6 vs. 31.2blocking buffer containing 1% BSA in TBST (10 mMmU CK per well for 3 ng/ml R3 IGF-I in the absence andTris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) and

incubated overnight at 47C with the horseradish peroxi- presence of 250 ng/ml IGFBP-4, respectively). These


EWTON ET AL.50

Fig. 1. Effect of IGFBP-4 on IGF-I-stimulated L6A1 myoblast prolif- Fig. 3. Effect of IGF-I concentration on modulation of L6A1 myoblastproliferation or differentiation by IGFBP-5. L6A1 myoblasts were in-eration and differentiation. L6A1 myoblasts were incubated with 30

ng/ml IGF-I in the presence of the indicated concentrations of IGFBP- cubated with the indicated concentrations of IGF-I in the absence orpresence of IGFBP-5 (250 ng/ml). A: Proliferation (cells/well 1 1003)4. A: Proliferation (cells/well 1 1003) was measured after 24 hours.

B: Differentiation (elevation of CK activity ) was measured after 72 was measured after 24 hours. B: Differentiation (elevation of CK activ-ity) was measured after 72 hours. Data represent the mean { SDhours. Cell number or CK activity in cultures incubated in the absence

of IGF-I is indicated as DMEM. Data represent the mean { SD of of triplicate determinations. Asterisks indicate significant differencefrom cells treated with the indicated concentration of IGF-I in thetriplicate determinations. Asterisks indicate significant difference

from cells treated with 30 ng/ml IGF-I in the absence of IGFBP-4; absence of IGFBP-5; *P õ 0.05; **P õ 0.01. Similar results wereobtained in two other independent experiments.**P õ 0.01. Similar results were obtained in two other independent

experiments.

measured as an increase in CK activity after 72 hours(Fig. 2B). When 250 ng/ml IGFBP-5 was added in thepresence of 30 ng/ml IGF-I, it caused a 35% stimulationof CK activity. This stimulatory effect of IGFBP-5 ondifferentiation was not limited solely to increased CKactivity since more extensive fusion of myoblasts intomyotubes was also observed in IGFBP-5-treated cul-tures (data not shown). IGFBP-5 had no effect on eitherproliferation or differentiation when added to L6A1myoblasts in the absence of IGF-I or in the presence ofR3 IGF-I (71 vs. 70% increase in cell number overDMEM controls, and 31.6 vs. 32.5 mU CK per well forFig. 2. Effect of IGFBP-5 on IGF-I-stimulated L6A1 myoblast prolif-

eration and differentiation. L6A1 myoblasts were incubated with 30 3 ng/ml R3 IGF-I in the absence and presence of 250 ng/ng/ml IGF-I in the presence of the indicated concentrations of IGFBP- ml IGFBP-4, respectively). Similar to the observations5. A: Proliferation (cells/well 1 1003) was measured after 24 hours. with IGFBP-4, these results demonstrate that the ef-B: Differentiation (elevation of CK activity ) was measured after 72

fects of IGFBP-5 on proliferation and differentiationhours. Cell number or CK activity in cultures incubated in the absenceof IGF-I is indicated as DMEM. Data represent the mean { SD of are specific for IGF-I, and require the binding oftriplicate determinations. Asterisks indicate significant difference IGFBP-5 to IGF-I.from cells treated with 30 ng/ml IGF-I in the absence of IGFBP-5;*P õ 0.05; **P õ 0.01. Similar results were obtained in two other Effect of IGF-I concentration on the opposingindependent experiments. IGFBP-5 actions on proliferation

and differentiationSince the above study demonstrating opposite effectsresults demonstrate that the inhibitory effects of

of IGFBP-5 on IGF-I-stimulated proliferation and dif-IGFBP-4 on proliferation and differentiation are spe-ferentiation of L6A1 was performed in the presence ofcific for IGF-I, and require the binding of IGFBP-4 toonly one concentration of IGF-I (30 ng/ml), we deter-IGF-I.mined whether IGFBP-5 had similar effects when

Effect of IGFBP-5 on IGF-I-stimulated L6A1 added in the presence of different levels of IGF-I. Wemyoblast proliferation and differentiation found that the inhibitory effect on proliferation (Fig.

3A) and the stimulatory effects on differentiation (Fig.We had earlier shown that the expression of IGFBP-3B) of 250 ng/ml ( 8.3 nM) IGFBP-5 were evident only5 by L6A1 cells increases dramatically during myogen-at IGF-I concentrations of 30–100 ng/ml (4–13 nM).esis, while that of IGFBP-4 declines (Ewton and Flo-The maximal effect was seen when the molar ratio ofrini, 1995). Because of this inverse relationship in theIGFBP-5 to IGF-I was approximately 2:1.expression of these two IGFBPs, it was of interest to

determine whether exogenous IGFBP-5 would have the Effect of IGFBP-5 on IGF-I stimulationsame inhibitory actions as IGFBP-4. Figure 2A shows of c-fos mRNAthat IGFBP-5, like IGFBP-4, caused a concentration-dependent decrease of L6A1 myoblast proliferation It has previously been shown that induction of the

proto-oncogene c-fos is associated with the IGF-I-stimu-when added in the presence of 30 ng/ml (4 nM) IGF-I.IGF-I-stimulated cell proliferation was inhibited ap- lated mitogenic response in L6 myoblasts (Ong et al.,

1987; Giorgino and Smith, 1995; Coolican et al., 1997). Toproximately 65% by 250 ng/ml (8.3 nM) IGFBP-5. Sur-prisingly, however, the addition of IGFBP-5 caused a investigate whether the effects of IGFBP-5 in inhibiting

L6A1 proliferation were also associated with a decreaseconcentration-dependent stimulation of differentiation,



tion of mRNA levels of the muscle regulatory gene,myogenin, reaching a maximum after 48 hours (Floriniet al., 1991b). However, it was later found that uponremoval of serum-containing growth medium and uponaddition of IGF-I, the myoblasts are initially stimu-lated to undergo cell division before the onset of differ-entiation (Ewton et al., 1994b). It was also found duringthis early proliferative phase that myogenin mRNA lev-els are markedly decreased by IGF-I when comparedto controls (Rosenthal and Cheng, 1995; Engert et al.,1996). Thus, IGF-I treatment results first in a decreasein myogenin mRNA during the proliferative phase, fol-lowed by an elevation during the later myogenic phase.The initial increase in myogenin mRNA abundanceafter serum removal is not a true indication of the myo-genic potential of L6A1 muscle cells used in this study.Cultures in DMEM alone contain high levels of myo-genin mRNA, but do not fully complete the myogenicprocess; there is no elevation of CK activity, and what-ever myotubes do form are very small. IGFs are re-quired for maximal expression of subsequent myogenicsignals. Thus, elevated myogenin mRNA expression isassociated with myogenesis, but is not by itself suffi-cient to stimulate all processes associated with termi-nal differentiation.

We wanted to examine the effects of IGFBP-5 onthese two diverging actions of IGF-I on myogeninmRNA. Figure 5 shows that elevated levels of myogeninmRNA were found in control L6A1 cultures in whichgrowth medium had been replaced by DMEM for 24hours. In contrast, treatment with 30 ng/ml IGF-I for24 hours inhibited this elevation of myogenin mRNAlevels by about 80%. However, the addition of 250 ng/ml IGFBP-5 in the presence of IGF-I reversed this in-hibitory effect. Thus, by inhibiting this early prolifera-tive response to IGF-I, IGFBP-5 permitted the earlyexpression of myogenin.

However, with time, myogenin mRNA levels in-creased in response to IGF-I stimulation, as shown inFigure 6. After 30 hours, myogenin mRNA levels wereabout equal in DMEM and IGF-I-treated cultures, andthe addition of IGFBP-5 in the presence of IGF-I re-sulted in an even greater elevation in myogenin mRNA.Fig. 4. Effect of IGFBP-5 on c-fos mRNA. L6A1 myoblasts were incu-

bated with DMEM or IGF-I (30 ng/ml) in the absence or presence of By 46 hours, IGF-I treated cultures contained higherIGFBP-5 (250 ng/ml) for 30 minutes. Total RNA was extracted and myogenin mRNA levels than DMEM controls, almost10 mg analyzed on Northern blots for c-fos mRNA (upper panel) equaling those of IGFBP-5-treated cells. These resultsand quantitated by scanning laser densitometry (lower panel); equal

provide further evidence that IGFBP-5 inhibits the pro-loading was verified with ethidium bromide-stained ribosomal 28 SRNA (middle panel). Similar results were obtained in another inde- liferative response to IGF-I (reversing the early inhibi-pendent experiment. tion of myogenin mRNA elevation by IGF-I), but stimu-

lates the myogenic response (stimulating the subse-quent elevation of myogenin mRNA by IGF-I).in this early marker of the proliferative response, we

performed Northern analyses of RNA isolated from Effect of IGFBP-5 on IGF-I-stimulatedIGFBP-5-treated myoblasts (Fig. 4). A 30- minute incuba- IRS-1 tyrosine phosphorylationtion with IGF-I (30 ng/ml) caused a 85% increase in the

It has been shown in other cell types that mitogenicsteady-state levels of c-fos mRNA. The addition of IGFBP-signaling by the type I receptor is accompanied by the5 (250 ng/ml) in the presence of IGF-I inhibited the eleva-tyrosine phosphorylation of several intracellular sub-tion of c-fos mRNA by 77%. Thus, these results are instrates (reviewed in Jones and Clemmons, 1995), in-agreement with those obtained from cell proliferationcluding that of a Ç170–185 kDa protein designatedstudies indicating that IGFBP-5 inhibits the early mito-IRS-1. Since IRS-1 has also been shown to be tyrosinegenic response of L6A1 cells to IGF-I.phosphorylated in L6 myoblasts after IGF-I treatment

Effect of IGFBP-5 on early and later actions (Lamphere and Lienhard, 1992; Giorgino and Smith,of IGF-I on myogenin mRNA 1995), we examined the effects of IGFBP-5 on IGF-I-

stimulated IRS-1 phosphorylation in L6A1 myoblastsWe had earlier reported that IGF-stimulated differ-entiation of L6A1 myoblasts is accompanied by eleva- (Fig. 7). Tyrosine phosphorylation of IRS-1 was mark-


EWTON ET AL.52

Fig. 6. Effect of IGFBP-5 on the later expression of myogenin mRNA.L6A1 myoblasts were incubated with DMEM or IGF-I (30 ng/ml) inthe absence or presence of IGFBP-5 (250 ng/ml) for 30 or 46 hours.Total RNA was extracted and 5 mg analyzed on Northern blots formyogenin mRNA (upper panel) and quantitated by scanning laserdensitometry (lower panel); equal loading was verified with ethid-ium bromide-stained ribosomal 28 S RNA (middle panel). Similarresults were obtained in another independent experiment.

Fig. 5. Effect of IGFBP-5 on the early expression of myogenin mRNA.L6A1 myoblasts were incubated with DMEM or IGF-I (30 ng/ml) inthe absence or presence of IGFBP-5 (250 ng/ml) for 24 hours. TotalRNA was extracted and 10 mg analyzed on Northern blots for myo-genin mRNA (upper panel) and quantitated by scanning laser densi-tometry (lower panel); equal loading was verified with ethidium bro-mide-stained ribosomal 28 S RNA (middle panel). Similar resultswere obtained in two other independent experiments.

edly increased 1 minute after IGF-I treatment. Theaddition of IGFBP-5 in the presence of IGF-I preventedthe IGF-I stimulation of IRS-1 phosphorylation. Theseresults demonstrate that IGFBP-5 inhibits anothercomponent of the proliferative response of L6A1 myo-blasts to IGF-I: the tyrosine phosphorylation of IRS-1.

Effect of IGFBP-5 on tyrosinephosphorylation of Shc

Shc is another substrate for the tyrosine kinase activ-Fig. 7. Effect of IGFBP-5 on tyrosine phosphorylation of IRS-1. L6A1ity of the IGF-I receptor involved in mitogenic signalingmyoblasts were incubated for 1 minute with DMEM or IGF-I (30 ng/(Giorgetti et al., 1994). IGF-I treatment of L6 myoblastsml) in the absence or presence of IGFBP-5 (250 ng/ml). Cell lysateshas recently been reported to cause the tyrosine phos- were immunoprecipitated (IP) with an antibody to IRS-1 and analyzed

phorylation of three Shc protein species, 46, 52, and by Western immunoblotting (IB) with an antibody to phosphotyrosine.Similar results were obtained in another independent experiment.66 kDa (Giorgino and Smith, 1995). Accordingly, we



Fig. 9. Effect of IGFBP-5 on phosphorylation of MAP kinase. L6A1myoblasts were incubated for 1 hour with DMEM or IGF-I (30 ng/ml)in the absence or presence of IGFBP-5 (250 ng/ml) or PD098059 (10mM). Cell lysates were prepared and 10 mg protein was separated bySDS-PAGE and immunoblotted (IB) with anti-phospho-MAPK. TheFig. 8. Effect of IGFBP-5 on tyrosine phosphorylation of Shc. L6A1 phosphorylated forms of ERK 1 and ERK 2 are shown as pp44 andmyoblasts were incubated for 2 minutes with DMEM or IGF-I (30 ng/ pp42. Similar results were obtained in another independent experi-ml) in the absence or presence of IGFBP-5 (250 ng/ml). Cell lysates ment.were immunoprecipitated (IP) with an antibody to Shc and analyzed

by Western immunoblotting (IB) with an antibody to phosphotyrosine.Similar results were obtained in another independent experiment.

determined the effects of IGFBP-5 on IGF-I-stimulatedShc phosphorylation in L6A1 myoblasts. As shown inFigure 8, IGF-I (30 ng/ml) treatment of L6A1 myoblastsfor 2 minutes caused the tyrosine phosphorylation ofall three Shc isoforms. The addition of 250 ng/ml ofIGFBP-5 in the presence of IGF-I inhibited this phos-phorylation of the Shc proteins.

Effect of IGFBP-5 on MAP kinaseIn most cell types, IGF stimulation of the phosphory-

Fig. 10. Effect of IGFBP-4 on IGF-II-stimulated L6A1 myoblast pro-lation of IRS-1 and Shc is followed by the activation of liferation and differentiation. L6A1 myoblasts were incubated withthe Ras-Raf-MAP kinase pathway and the initiation of 100 ng/ml IGF-II in the presence of the indicated concentrations ofthe subsequent mitogenic response. We have recently IGFBP-4. A: Proliferation (cells/well 1 1003) was measured after 24

hours. B: Differentiation (elevation of CK activity) was measured aftershown that IGF-I treatment of L6A1 myoblasts re-53 hours. Cell number or CK activity in cultures incubated in thesulted in the phosphorylation of both isoforms of MAP absence of IGF-II is indicated as DMEM. Data represent the mean {kinases, ERK1 and ERK2, and that this activation was SD of triplicate determinations. Asterisks indicate significant differ-

inhibited by the MEK inhibitor PD098059 (Coolican ence from cells treated with 100 ng/ml IGF-II in the absence of IGFBP-4; **P õ 0.01. Similar results were obtained in another independentet al., 1997). Since treatment with either IGFBP-5 orexperiment.PD098059 inhibited L6A1 myoblast proliferation, we

compared the effects of IGFBP-5 and PD098059 onIGF-I-stimulated MAP kinase activation. Figure 9shows that a 1-hour incubation with IGF-I resulted in of interest to determine what the effects of exogenous

IGFBP-4 and -5 would be on L6A1 myoblasts whensignificant stimulation of the phosphorylation of bothERK1 and ERK2 (pp44 and pp42). This stimulatory added in the presence of this ligand. Since we had

shown that IGF-II is less potent than IGF-I in stimulat-effect was inhibited by both IGFBP-5 and PD098059.The inhibition by PD098059 was less dramatic than ing mitogenic and myogenic responses in L6A1 myo-

blasts, IGF-II was added at 100 ng/ml (13.3 nM), athat by IGFBP-5, possibly because concentrations ofPD098059 higher than 10 mM are required to com- concentration that gives approximately the same stim-

ulation of proliferation and differentiation as IGF-I atpletely inhibit the proliferative response to 30 ng/mlIGF-I used in this study as opposed to 3 ng/ml R3 IGF- 30 ng/ml (4 nM) (Ewton et al., 1987). Figure 10 shows

that in the presence of IGF-II, IGFBP-4 caused a con-I used in our previous study (Coolican et al., 1997).centration-dependent inhibition of both proliferation

Effect of IGFBP-4 and IGFBP-5 and differentiation. After 24 hours, IGF-II alone in-on IGF-II-stimulated L6A1 proliferation creased cell number by 74% over DMEM controls, while

and differentiation 250 ng/ml IGFBP-4 inhibited this IGF-II effect by 57%.Similarly, IGF-II-stimulated elevation of CK activitySince significant levels of IGF-II have been reported

to be secreted by differentiating skeletal muscle cells was inhibited by 48%. Thus, it appears that IGFBP-4has only inhibitory actions on L6A1 myoblasts.(Tollefsen et al., 1989b; Florini et al., 1991a), it was


EWTON ET AL.54

tory actions in all other cell types, it was not surprisingthat it also inhibited IGF-I and IGF-II-stimulated pro-liferation and differentiation in L6A1 muscle cells. Al-though we do not have confirming data from bindingstudies, it is likely that IGFBP-4 inhibits IGF actionin L6A1 cells by inhibiting binding of IGF-I and IGF-II to the IGF receptor, as has been demonstrated forthe inhibitory actions of IGFBP-4 in neuronal (Cheunget al., 1991) and bone cells (Mohan et al., 1995). Ourfindings of the inhibitory actions of IGFBP-4 on skeletalmuscle cell differentiation have recently been con-firmed by Damon et al. (1998) who demonstrated thatoverexpression of IGFBP-4 in C2 myoblasts resulted inFig. 11. Effect of IGFBP-5 on IGF-II-stimulated L6A1 myoblast pro-

liferation and differentiation. L6A1 myoblasts were incubated with inhibition of proliferation and differentiation, and that100 ng/ml IGF-II in the presence of the indicated concentrations of this inhibition could be restored by exogenous IGFs.IGFBP-5. A: Proliferation (cells/well 1 1003) was measured after 24 Similarly, Bach et al. (1994) reported only inhibitoryhours. B: Differentiation (elevation of CK activity ) was measured

actions in L6A1 cells for exogenous IGFBP-6. In thisafter 53 hours. Cell number or CK activity in cultures incubated inthe absence of IGF-II is indicated as DMEM. Data represent the case, however, IGFBP-6 inhibited only IGF-II-, but notmean { SD of triplicate determinations. Asterisks indicate significant IGF-I-, stimulated actions; this preferential inhibitiondifference from cells treated with 100 ng/ml IGF-II in the absence of of IGF-II action is probably due to the much higherIGFBP-5; *P õ 0.05; **Põ 0.01. Similar results were obtained in two

affinity of IGFBP-6 for IGF-II than for IGF-I.other independent experiments.IGFBP-5, on the other hand, has been reported to

both inhibit and to potentiate IGF actions (reviewed inJones and Clemmons, 1995; Bach and Rechler, 1995;

Surprisingly, while IGFBP-5 also inhibited IGF-II- Rajaram et al., 1997) and to have IGF-independent ac-stimulated proliferation (23% decrease in the presence tions (Andress and Birnbaum, 1992; Andress et al.,of 250 ng/ml IGFBP-5) (Fig. 11A), it did not stimulate 1993; Mohan et al., 1995). Such stimulatory actionsdifferentiation as was seen in the presence of IGF-I, have been shown only for IGFBPs which have beenbut caused a slight but statistically significant decrease found to be associated with the cell surface and in thein CK activity (9% inhibition of the IGF-II effect) (Fig. extracellular matrix, such as IGFBP-3 and IGFBP-5. In11B). These results demonstate that IGFBP-4 and our study, we found that IGFBP-5 could also function inIGFBP-5 have similar actions when added in the pres- both an inhibitory and a stimulatory capacity, de-ence of IGF-II, inhibiting both mitogenic and myogenic pending on the culture conditions. It is interesting toprocesses. However, the inhibitory effect in the pres- note that in our earlier studies on IGFBP secretion byence of IGFBP-4 was greater than that of IGFBP-5 L6A1 cells (Ewton and Florini, 1995), we detected someunder similar experimental conditions. IGFBP-5 associated with the cell surface (unpublished

data). We did not find any effect of IGFBP-5 in theDISCUSSION absence of IGF-I or IGF-II or in the presence of R3 IGF-I, an analog with reduced affinity for IGFBPs, indicat-We have previously demonstrated that the IGFs are

the primary growth factors that control both growth ing that the effects of IGFBP-5 involve binding to IGF-I or IGF-II. In the presence of IGF-II, IGFBP-5, similarand differentiation of muscle cells in culture (reviewed

in Florini et al., 1996). We have also shown that L6A1 to IGFBP-4, had only inhibitory effects on both prolifer-ation and differentiation. However, in the presence ofmuscle cells secrete three IGFBPs: IGFBP-4, -5, and

-6 (Ewton et al., 1994a; Ewton and Florini, 1995), but IGF-I, IGFBP-5 exhibited a dual function, inhibitingproliferation and stimulating differentiation. Thewe had no information as to what function each of these

binding proteins had in modulating IGF actions. In higher affinity of IGFBP-5 for IGF-II (Rajaram et al.,1997) may be a factor in its greater activity in inhibitingother cell types, IGFBPs have been shown to both in-

hibit and stimulate IGF action, or to have actions inde- IGF-II- mediated processes.The dual actions of IGFBP-5 in the presence of 30pendent of IGF binding (reviewed in Jones and Clem-

mons, 1995; Bach and Rechler, 1995; Rajaram et al., ng/ml IGF-I were observed only when IGFBP-5 wasadded in a 2:1 molar excess (250 ng/ml or 8 nM). This1997). The much greater potency of IGF-I analogs with

reduced affinity for IGFBPs suggested that the IGFBPs quantity of IGFBP-5 is within the range of endogenousIGFBP-5 secreted by L6A1 myoblasts under the culturesecreted by L6A1 cells inhibited IGF-I actions (Ewton

and Florini, 1995; Silverman et al., 1995). However, we conditions used in the present study. We estimate byWestern blot band intensity of IGFBP-5 in conditionedhad shown that each of the three IGFBPs exhibits a

unique expression pattern during the different stages medium from IGF-I-treated cells that L6A1 culturessecrete approximately 100 ng/ml (data not shown).of muscle development (Ewton et al., 1994a; Ewton and

Florini, 1995). Thus, it seemed unlikely that all three Since concentrations in the microenvironment adjacentto the cell surface could be much higher, the effects weIGFBPs would have identical effects. Indeed, earlier

studies by Bach et al. (1994, 1995) with recombinant see with 250 ng/ml IGFBP-5 appear to be physiologi-cally relevant. Previous studies of IGFBP actions onIGFBP-6 and our present report with IGFBP-4 and -5

show that the three IGFBPs secreted by L6A1 cells bone cells reported significant effects of IGFBP-5 whenit was added at concentrations up to 100 ng/ml (Mohanhave different effects on IGF- stimulated proliferation

and differentiation. et al., 1995) and 230 ng/ml (Andress and Birnbaum,1992). We and others (McCusker and Clemmons, 1994)Since IGFBP-4 has been reported to have only inhibi-



could detect little or no fragments due to proteolytic several studies (reviewed in Florini et al., 1996), thenwhere does the myogenic signaling pathway divergedegradation of IGFBP-5 secreted by L6 myoblasts.

Some 21–23 kDa fragments could be detected in condi- from the mitogenic signaling pathway? The tyrosinephosphorylation of IRS-1 and Shc are well-establishedtioned medium from cultures incubated with exogenous

IGFBP-5, suggesting that some degradation had oc- actions of the activated type I receptor. IGFBP-5 inhib-ited the phosphorylation of both of these signaling in-curred, but there was also a much greater increase in

the 31 kDa band, indicating that most of the added termediates, suggesting that high levels of IRS-1 andShc phosphorylation are associated with the mitogenicIGFBP-5 remained intact (data not shown).

The inhibitory effects on proliferation and the stimu- pathway, but may not be necessary or may be requiredonly at very low levels for myogenesis. Indeed, very lowlatory effects on differentiation of IGFBP-5 in the pres-

ence of IGF-I were evident not only at a morphological levels of phosphorylation, undetected by our methods,may be sufficient for activating downstream events, aslevel of increased fusion of myoblasts into myotubes,

but could also be assessed by examining several mark- has been reported by Myers et al. (1994) for the stimu-lation of the p70 S6 kinase. We have recently showners of the mitogenic and myogenic responses. IGFBP-

5 inhibited all aspects of the early proliferative re- that activation of the p70 S6 kinase is required fordifferentiation and its activity is elevated by agentssponse of L6A1 cells to IGF-I: the tyrosine phosphoryla-

tion of the cytoplasmic signaling molecules, IRS-1 and that promote myogenesis (Coolican et al., 1997). It isinteresting to note that PD098059, a pharmacologic in-Shc, the activation of the MAP kinases, ERK1 and

ERK2, the elevation in steady-state levels of the mRNA hibitor of MEK, and IGFBP-5, a physiological memberof the IGF system, have similar effects on certain signalof the nuclear transcription factor c-fos, the early inhi-

bition of elevation of myogenin mRNA, and finally, the tranduction molecules, inhibiting phosphorylation ofMAP kinases, with the resulting inhibition of prolifera-increase in cell number. In contrast, IGFBP-5 stimu-

lated all markers of the myogenic response to IGF-I: tion and stimulation of differentiation.A different approach was taken by James et al. (1996)the later rise in myogenin mRNA, the elevation in CK

activity, and finally, the fusion of myoblasts into myo- to study the function of IGFBP-5 during myogenesis.They found that overexpressing IGFBP-5 in the sensetubes.

What is the mechanism by which IGFBP-5 inhibits orientation in C2 myoblasts resulted in cultures thatfailed fo differentiate normally. In contrast, cells trans-one process and stimulates another? It is possible that

by inhibiting the IGF-I-stimulated mitogenic pathway, fected with the IGFBP-5 antisense construct differenti-ated earlier and more extensively than control cells.IGFBP-5 simply allows the cells to survive and to follow

a separate pathway leading to differentiation. If this They concluded that IGFBP-5 normally inhibits muscledifferentiation. Their results appear to be in direct con-were the case, one would expect IGFBP-4 at certain

concentrations to produce similar effects. However, we trast to our findings that IGFBP-5 can also stimulatemyogenesis. However, upon closer examination, thosefound that while exogenous IGFBP-4 also inhibited pro-

liferation, it did not stimulate differentiation; in fact, discrepancies can be explained. C2 muscle cells secretehigh levels of IGF-II (Tollefsen et al., 1989b; Florini etIGFBP-4 inhibited differentiation. Recently, Stewart

and Rotwein (1996) and Stewart et al. (1996) showed al., 1991a), and overexpressed IGFBP-5 could act bysequestering the autocrine IGF-II, preventing its myo-that in C2 myoblasts expressing IGF-II in the antisense

orientation, IGFs act as survival factors during the genic actions. In contrast, L6A1 muscle cultures secretevery little IGF-II, and cells cultured in the absence oftransition from proliferating to differentiating myo-

blasts. These antiapoptotic actions appear to be medi- IGFs undergo very little myogenesis. In our study, wealso found that IGFBP-5 inhibited IGF-II-stimulatedated by the PI 3-kinase pathway, the same pathway

which we have found to be required for stimulation processes; the dual action of IGFBP-5 was manifestedonly in the presence of IGF-I. In vivo, the stimulatoryof differentiation. However, FGF-2 and PDGFbb also

inhibited apoptosis in these cells but did not promote effects of IGFs on muscle growth and differentiationmust be kept in a fine balance in order to permit enoughdifferentiation, demonstrating that a positive signal

from the IGF receptor is required for myogenesis. It is cells to accumulate before the onset of differentiation.IGF-II functions primarily as an autocrine factor,possible that exogenous IGFBP-5 could inhibit prolifer-

ation by binding enough added IGF-I to eliminate the whereas IGF-I is also distributed via paracrine andendocrine pathways. The action of IGFBP-5 could bemitogenic signaling mediated through the MAP kinase

pathway. However, its unique property of being able to determined by the cellular context, modulating the mi-togenic and myogenic actions of IGF-I.associate with the cell membrane or the extracellular

matrix allows it to modulate IGF-I actions, resulting in IGFBP-4 and IGFBP-5 have also been shown to haveopposing actions in bone cells (Mohan et al., 1995).stimulation of PI 3-kinase and subsequent myogenesis

(Coolican et al., 1997; Kaliman et al., 1996, 1998; Pinset IGFBP-4 (100 ng/ml) inhibited, while IGFBP-5 (100 ng/ml) potentiated, bone cell proliferation. In addition,et al., 1997). In fact, we have earlier reported that as

high levels of IGF-I are reached, the myogenic response since IGFBP-5 increased 125I-IGF-I binding separatefrom the IGF-I receptor and potentiated cell prolifera-becomes biphasic and further differentiation is inhib-

ited (Florini et al., 1986). It is clear that IGF-I has to tion even in the presence of IGF-I analogs with reducedaffinity for IGFBPs, Mohan et al. suggested thatbe present to transduce some signal in order for L6A1

myoblasts to differentiate, since cells in the absence of IGFBP-5 may in part stimulate bone cell proliferationby an IGF-independent mechanism.any IGF (DMEM alone) or in the presence of IGFBP-5

without IGF-I did not differentiate. In summary, we have demonstrated that IGFBPs se-creted by L6A1 myoblasts have the potential of signifi-If the IGF-I receptor mediates the stimulation of dif-

ferentiation by the IGFs, as has been concluded from cantly modulating the actions of both IGF-I and IGF-


EWTON ET AL.56

myoblast proliferation and differentiation. J. Endocrinol., 144:539–II. While IGFBP-4 has only an inhibitory function, in-553.hibiting both IGF-I and IGF-II- stimulated prolifera- Ewton, D.Z., Falen, S.L., and Florini, J.R. (1987) The type II IGF

tion and differentiation, IGFBP-5 may play a dual role. receptor has low affinity for IGF-I analogs: Pleiotypic actions ofIGFs on myoblasts are apparently mediated by the type I receptor.IGFBP-5 inhibits proliferation in the presence of bothEndocrinology, 120:115–124.IGFs. While it inhibits IGF-II-stimulated differentia-

Ewton, D.Z., Magri, K.A., and Florini, J.R. (1994a) Roles of IGF-I,tion, under certain conditions it can also potentiate IGF-II, and IGFBPs in myogenesis. In: The insulin-like growth fac-IGF-I- stimulated differentiation. These observations tors and their regulatory proteins. R.C. Baxter, P.D. Gluckman, and

R. G. Rosenfeld, eds. Excerpta Medica, Amsterdam, pp. 283–290.confirm studies by others showing that IGFBP-4 func-Ewton, D.Z., Roof, S.L., Magri, K.A., McWade, F.J., and Florini, J.R.tions primarily as an inhibitor of IGF action, while

(1994b) IGF-II is more active than IGF-I in stimulating L6A1 myo-IGFBP-5 can act as both an inhibitor or potentiator of genesis: Greater mitogenic actions of IGF-I delay differentiation. J.IGF action (reviewed in Jones and Clemmons, 1995; Cell. Physiol., 161:277–284.

Florini, J.R. (1989) Assay of creatine kinase in microtiter plates usingRajaram et al., 1997).thio-NAD to allow monitoring at 405 nM. Anal. Biochem., 182:399–404.ACKNOWLEDGMENTS

Florini, J.R., Ewton, D.Z., Falen, S.L., and VanWyk, J.J. (1986) Bipha-sic concentration dependency of the stimulation of myoblast differ-The authors thank Ciba Geigy for the generous sup-entiation by somatomedins. Am. J. Physiol. (Cell. Physiol.),ply of IGF-I, Eli Lilly for IGF-II, and Paul Walton and 250:771–778.

John Ballard (GroPep) for the R3 IGF-I analog. We also Florini, J., Magri, K., Ewton, D., James, P., Grindstaff, K., and Rotw-ein, P. (1991a) ‘‘Spontaneous’’ differentiation of skeletal myoblaststhank Dr. Eric Olson for the myogenin cDNA probe. Weis controlled by autocrine secretion of insulin-like growth factor-II.are especially grateful to Cathleen Jenney for carefulJ. Biol. Chem., 266:15917–15923.technical assistance, and to Dr. Derina Samuel for per- Florini, J.R., Ewton, D.Z., and Roof, S.L. (1991b) IGF-I stimulates

forming the phospho-MAPK Western analyses. terminal myogenic differentiation by induction of myogenin geneexpression. Mol. Endocrinol., 5:718–724.

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