Insulin-like Growth Factor-I Extends in Vitro Replicative LifeSpan of Skeletal Muscle Satellite Cells by Enhancing G1/S CellCycle Progression via the Activation of Phosphatidylinositol3*-Kinase/Akt Signaling Pathway*

Received for publication, July 3, 2000, and in revised form, August 9, 2000Published, JBC Papers in Press, August 28, 2000, DOI 10.1074/jbc.M005832200

Manu V. Chakravarthy‡, Tsghe W. Abraha§, Robert J. Schwartz¶, Marta L. Fiorottoi,and Frank W. Booth‡§**

From the ‡Department of Integrative Biology, University of Texas Medical School, Houston, Texas 77030, the ¶Departmentof Cell Biology, Baylor College of Medicine, Houston, Texas 77030, the iDepartment of Pediatrics, United StatesDepartment of Agriculture/Agricultural Research Station Children’s Nutrition Research Center, Houston, Texas 77030,and the §Department of Veterinary Biomedical Sciences, Department of Physiology, and Dalton Cardiovascular Institute,College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211

Interest is growing in methods to extend replicativelife span of non-immortalized stem cells. Using the insu-lin-like growth factor I (IGF-I) transgenic mouse inwhich the IGF-I transgene is expressed during skeletalmuscle development and maturation prior to isolationand during culture of satellite cells (the myogenic stemcells of mature skeletal muscle fibers) as a model system,we elucidated the underlying molecular mechanisms ofIGF-I-mediated enhancement of proliferative potentialof these cells. Satellite cells from IGF-I transgenic mus-cles achieved at least five additional population dou-blings above the maximum that was attained by wildtype satellite cells. This IGF-I-induced increase in pro-liferative potential was mediated via activation of thephosphatidylinositol 3*-kinase/Akt pathway, independ-ent of mitogen-activated protein kinase activity, facili-tating G1/S cell cycle progression via a down-regulationof p27Kip1. Adenovirally mediated ectopic overexpres-sion of p27Kip1 in exponentially growing IGF-I trans-genic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin Aprotein abundance, thereby implicating an importantrole for p27Kip1 in promoting satellite cell senescence.These observations provide a more complete dissectionof molecular events by which increased local expressionof a growth factor in mature skeletal muscle fibers ex-tends replicative life span of primary stem cells thanpreviously known.

Repair or growth of skeletal muscle requires satellite cells fornew myonuclei because myonuclei are postmitotic (1). Satellitecells are a population of mononuclear myogenic precursor cellswedged between the basal lamina and muscle fiber sarcolemma

(2). Satellite cells differ from immortalized myogenic cell linesin that, like other normal diploid cells, they have a limitedcapacity to divide, and after a finite number of cell divisionsenter a state of irreversible growth arrest, termed replicativesenescence (3). Indeed, proliferative potential of these cells hasbeen shown to decrease in muscular dystrophies that are char-acterized by multiple rounds of regeneration (4), ultimatelyresulting in their cellular senescence. Therefore, when satellitecells exhaust their finite proliferative reserves, the effective-ness of regeneration, hypertrophy, and myoblast-mediatedgene therapy for the reconstruction of muscle is constrained.

Given the previously documented effects of insulin-likegrowth factor-I (IGF-I)1 to stimulate myoblast proliferation,myogenic differentiation, and myotube hypertrophy in culturedimmortalized myogenic cell lines (5), and its ability to induceskeletal muscle hypertrophy in both young and old rodents(6–9), we asked if IGF-I might enhance the proliferative poten-tial of the resident satellite cells within animal skeletal mus-cles. Such understanding, which is presently lacking in non-immortalized primary satellite cells, could be used tocounteract many intractable muscle-wasting conditions.

A mouse lacking the igf-1 gene in a liver-specific mannerdemonstrated that IGF-Is action on growth and development islargely autocrine/paracrine (10). Therefore, a transgenic mouseexpressing an IGF-I transgene driven by a skeletal a-actinpromoter was selected (6) to allow the local overexpression ofIGF-I in skeletal muscles in order to mimic its autocrine effectin the resident satellite cells. We hypothesized that satellitecells from the IGF-I transgenic (IGF-I Tg) skeletal musclesexposed to continuous high levels of IGF-I might possess aprolonged ability to proliferate in culture, reflecting an in-creased proliferative potential, relative to those cells isolatedfrom their FVB wild type (WT) littermates.

We next asked how the IGF-I Tg satellite cells were able tohave an enhanced replicative life span, when the correspondingWT satellite cells were growth-arrested? Based upon studies inother cell types (11, 12), it seemed likely that this growth arrestin WT satellite cells might be at the G1/S boundary of the cellcycle, a nodal point suggested as one of the biochemical hall-

* This work was supported by National Institutes of Health GrantsAR 19393 and AG18780 (to F. B.), a NASA NSRBI grant (to F. B. andR. S.), American Federation for Aging Research Glenn Fellowship (toM. C.), and the M.D./Ph.D. program at the University of Texas, HealthScience Center at Houston, and the M. D. Anderson Cancer Center (toM. C.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

** To whom correspondence should be addressed: University of Mis-souri, Columbia, Dept. of Veterinary Biomedical Sciences, E102 Veter-inary Medical Bldg., 1600 East Rollins Rd., Columbia, MO 65211. Tel.:573-882-6652; Fax: 573-884-6890; E-mail: boothf@missouri.edu.

1 The abbreviations used are: IGF-I, insulin-like growth factor I; PI3-kinase, phosphatidylinositol 3-kinase; BrdUrd, bromodeoxyuridine;PD, population doubling; FACS, fluorescence-activated cell sorter;MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gelelectrophoresis; WT, wild type; MEK, mitogen-activated extracellularsignal-regulate kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 46, Issue of November 17, pp. 35942–35952, 2000Printed in U.S.A.

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mark of replicative senescence. While senescent cells are re-fractory to mitogens and are terminally non-dividing even un-der the most optimal growth conditions, quiescent cells can beinduced to initiate DNA synthesis by mitogenic stimulation orsubcultivation (13). Recent findings have demonstrated an ac-cumulation of cdk-inhibitors such as p16Ink4, p21Cip1/Waf1, andp27Kip1 (14, 15) in various senescent cell types, further impli-cating a defect in G1/S transition in cellular senescence. SinceIGFs act as progression factors stimulating progression fromG1 to S phase of the cell cycle in other cell types (5), wereasoned that IGF-I might extend the in vitro replicative lifespan of satellite cells by modulating cell cycle regulatory mol-ecules at the G1/S boundary. IGF-I exerts its pleiotropic effectsby binding to the type I IGF receptor (IGF-IR) (16), which leadsto the activation of both PI 39-kinase and MAPK pathways (forreview, see Ref. 17), in turn producing an increase in cellularproliferation in immortalized myogenic cell lines (18, 19) andother tumor cell lines (20). Therefore, we hypothesized that theIGF-I induced enhancement of cell cycle progression via mod-ulation of G1/S regulatory molecules in the IGF-I Tg satellite cellsmight be signaled by either one, or both, of these pathways.

Here we report that IGF-I Tg satellite cells have an en-hanced in vitro replicative life span, and continued to maintaincell cycle progression via activation of the PI 39-kinase/Aktpathway, which in turn mediated the down-regulation ofp27Kip1 even in late-passage (the point at which WT cells weresenesced). Adenovirally mediated ectopic overexpression ofp27Kip1 in growing late-passage IGF-I Tg satellite cells induceda G1 arrest, and mimicked the biochemical events seen insenesced late-passage WT satellite cells, indicating thatp27Kip1 has a critical role in the regulation of satellite cellsenescence.

EXPERIMENTAL PROCEDURES

Materials——The IGF-I analog, long R3-IGF-I (LR3) was purchasedfrom GroPep Ltd., Adelaide, Australia. PI 39-kinase inhibitor(LY294002), MEK inhibitor (PD098059), bromodeoxyuridine (BrdUrd),and goat polyclonal anti-IGF-I receptor antibody, were all obtainedfrom Sigma, fluorescein isothiocyanate-conjugated anti-BrdUrd mono-clonal antibody was purchased from Caltag (Burlington, CA), and Hoe-scht 33342 and propidium iodide were from Molecular Probes (Eugene,OR). Cell culture media and reagents were purchased from Life Tech-nologies, Inc. (Rockville, MD). The human p27Kip1 adenoviral constructwas a generous gift from Dr. Perry Nisen, Abbott Laboratories (Chica-go, IL). Hybridoma cells secreting anti-desmin (clone D3) and anti-sarcomeric myosin (clone MF20) antibodies were obtained from theDevelopmental Studies Hybridoma Bank, Iowa City, IA.

Animals—FVB WT mice (aged ;1 month) and their littermatesharboring a single copy of a skeletal a-actin/hIGF-I hybrid transgene(IGF-I Tg), which express IGF-I exclusively in striated muscle havebeen previously described (6). Animals were housed in pathogen-freefacilities and fed ad libitum. Protocols were approved by the Institu-tional Animal Care and Use Committees.

Satellite Cell Isolation and Culture—Immediately after the animalswere euthanized by cervical dislocation, gastrocnemius muscles fromboth hindlimbs were removed, trimmed of excess connective tissue andfat, and weighed. Muscles from 5–6 animals from each of the twogroups were pooled to provide sufficient tissue for satellite cell isolation;the methods for their isolation have been described in detail previously(9). These cells were cultivated on collagen-coated tissue culture platesusing high serum conditions (Ham’s F-10 nutrient mixture containing20% fetal bovine serum, 1.0% chicken embryo extract, with 1% penicil-lin/streptomycin antibiotic mixture, and 1% L-glutamine) to minimizeany loss of proliferative cells due to either apoptosis or terminal differ-entiation. Cultures were maintained at 37 °C in a humid air atmo-sphere containing 5% CO2, and were passaged before they reachedconfluence. Both IGF-I Tg and WT satellite cell cultures at late-pas-sages were monitored for myogenic purity, apoptosis, and terminaldifferentiation using previously described methods by staining thesecells with anti-desmin antibody (9, 21), Hoescht 33342 (9, 22), andanti-sarcomeric myosin antibody (9), respectively.

Determination of Cumulative Population Doublings—Cumulative

number of population doublings attained over the course of the in vitroculture life span of WT and IGF-I Tg satellite cells was determined asdescribed previously (9, 22). At the time of cell isolation from theanimal, cell populations from both groups were considered to be at 1population doubling (PD). The number of PDs at every passage there-after was calculated as logN/log2, where N is the number of cellsharvested divided by the number of cells inoculated. Cell number wasdetermined in triplicate using a Coulter counter. Cultures were deemedto have reached the end of their in vitro life span when they failed toreach subconfluence after 3 weeks of refeeding, at which point theywere reassessed again with MF20 and Hoescht 33342 to ensure the lossof proliferation was not due to differentiation or apoptosis, respectively.Lack of proliferation was also assessed by BrdUrd labeling (see below).

Colony Size Distribution Assays (23)—Non-confluent late-passageWT and IGF-I Tg satellite cells were trypsinized and counted using ahemacytometer to calculate cell number. About 100 cells from eachgroup were inoculated into each of several collagen-coated 100-mmtissue culture dishes, and left for ;7 weeks at 37 °C, 5% CO2 atmo-sphere in serum-rich proliferation medium. Fresh growth medium wasgiven to the cells weekly. Our previous protocol was then followed (9).Each of the groups was cloned in triplicate and the colony sizes attainedwere averaged. A total of three such separate experiments were done foreach of the two groups. Parallel set of cultures at the end of the 7-weekgrowth period were stained with anti-sarcomeric myosin and anti-desmin, as described above to ensure that the cells constituting thecolonies were undifferentiated and myogenic.

Cell Synchronization and BrdUrd Labeling Studies—Late-passage(day 76 in culture) satellite cells from the IGF-I Tg and WT groups wereestablished overnight in proliferation media at 1 3 105 and 1 3 107 cellsper 100-mm tissue culture dish, respectively. The cells were washed inDulbecco’s modified Eagle’s medium and then maintained in serum-freemedia (Dulbecco’s modified Eagle’s medium with 1% penicillin-strepto-mycin) for 48 h to arrest any cycling cells at G0. A set of plates from eachgroup was processed for subsequent analysis immediately at the end ofthe 48-h period (time 0) or after serum stimulation for an additional 6,12, or 24 h. During the last 60 min of each incubation period, thecultures were pulsed with 10 mM BrdUrd. The cells were then analyzedby flow cytometry (see below) to determine the percentage of BrdUrd-positive cells (labeling index). To further document that the lack ofproliferation seen in the late-passaged WT cells was due to an irrevers-ible growth arrest, early passage (day 23 in culture) and late-passage(day 76 in culture) IGF-I Tg and WT cells were treated for 48 h with thelong R3-IGF-I (LR3) in serum-free media. The response to LR3 wasmeasured by determining the BrdUrd labeling index after cultureswere pulsed with 10 mM BrdUrd during the last 24 h of the LR3incubation. Percent of nuclei labeled with BrdUrd was quantitated asdescribed below.

Flow Cytometric Analysis of Cell Cycle Distribution—Late-passageWT and IGF-I Tg satellite cells from either synchronized or asynchro-nous cultures were pulse-labeled with 10 mM BrdUrd for 60 min at37 °C, 5% CO2. Cells were then harvested by trypsinization, and pre-pared for fluorescence-activated cell sorting (FACS) analysis as de-scribed previously (24, 25). BrdUrd-labeled nuclei were detected usingfluorescein isothiocyanate-conjugated anti-BrdUrd monoclonal anti-body (1 mg/ml) and counterstained with propidium iodide (20 mg/ml).Stained nuclei were analyzed in a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). DNA histograms were modeled usingthe Modfit-LT software (Verity, Topsham, ME).

Adenoviral Infection with p27Kip1—Replication-defective adenovirusdirecting expression of human p27Kip1 (Adp27) under the transcrip-tional control of the cytomegalovirus promoter have been previouslydescribed (26, 27). Exponentially growing late-passage cultures of IGF-ITg cells were cultured in proliferation media to ;50% confluence, andthen incubated with the indicated amounts of adenovirus for 48 h (27).Cells were then washed with phosphate-buffered saline and processedfor either FACS analysis for cell cycle distribution as detailed above, orharvested for the preparation of whole cell extracts. For mock infection,cells were infected with control adenovirus with empty vector.

Immunoblot and Immunoprecipitation Analyses—Whole cell extractsof late-passage IGF-I Tg and WT satellite cells were prepared by lysingcells with 3 times packed cell volume of lysis buffer (50 mM Tris, pH 7.5,150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 10% glycerol,200 mM NaF, 20 mM sodium pyrophosphate, 10 mg/ml leupeptin, 10mg/ml aprotinin, 200 mM phenylmethylsulfonyl fluoride, and 1 mM

sodium orthovanadate) on ice for 30 min. Protein yield was quantifiedby Bio-Rad DC protein assay kit (Bio-Rad). Equal amounts of totalprotein were resolved by SDS-polyacrylamide gel electrophoresis,transferred to nitrocellulose membranes, and probed with appropriate

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antibodies. The antibodies against p27Kip1 (06-445), cdk2 (06-505), cdk4(06-139), cyclin E (06-459), cyclin A (06-138), cyclin D1 (5D4), p85subunit of PI 39-kinase (06-195), phosphotyrosine (4G10), andp21Cip1/Waf1 (05-345) were purchased from Upstate Biotechnology, LakePlacid, NY. Antibody kits to detect phosphorylated Erk1/Erk2 (9100)and Ser473-Akt (9270) were obtained from New England Biolabs, Bev-erly, MA. Antibodies against pRb (G3–245) and p57Kip2 (E-17) wereacquired from Pharmingen (San Diego, CA) and Santa Cruz (SantaCruz, CA), respectively. Bound antibodies were detected using horse-radish peroxidase-linked sheep anti-mouse or donkey anti-rabbit anti-bodies (Amersham Pharmacia Biotech), and visualized by enhancedchemiluminescence (PerkinElmer Life Sciences) after exposure to x-rayfilm (Kodak X-Omat AR).

For immunoprecipitations, 500–1000 mg of total protein were incu-bated with antibodies to cyclin E, cyclin A, cyclin D1, cdk2, or p85subunit of PI 39-kinase at 4 °C for 2 h, followed by 1 h incubation withprotein A-agarose (Amersham Pharmacia Biotech) at 4 °C. After 3washes in lysis buffer, the immunocomplexes were resolved by SDS-PAGE, and transferred to nitrocellulose for subsequent immunoblotanalysis.

Measurement of Cyclin-associated cdk Activity—Cyclin E- and cy-clin-A associated kinase activities on the respective immunoprecipi-tates were performed using histone H1 as a substrate, as describedpreviously (12, 24). Reaction mixtures were stopped with 2 times Lae-mmli sample buffer and the phosphorylated histone H1 resolved on 10%SDS-PAGE gels. The gels were subsequently dried and exposed to x-rayfilms. In another set of experiments, the kinase reaction was stopped byspotting 25 ml of the samples onto P81 phosphocellulose squares (Up-state Biotechnology) followed by extensive washing in 1% phosphoricacid. The filters were then dried in acetone, and counted in a b-counterwith scintillation fluid.

Measurement of Phosphatidylinositol 39-Kinase Activity—Using pre-viously described procedures (19), PI 39-kinase activity in anti-phospho-tyrosine and anti-p85 immunoprecipitates of late-passage IGF-I Tg andWT satellite cells was measured using phosphatidylinositol as a sub-strate. The products of the kinase reaction were spotted onto Silica Gel60 plates coated with 1% potassium oxalate, and resolved by thin-layerchromatography in chloroform/methanol/water/ammonium hydroxide(60:47:11.3:2). The phosphorylated lipid products were then visualizedby autoradiography.

Measurement of MAPK/ERK Activity—MAP kinase activity in wholesatellite cell extracts was determined using myelin basic protein as asubstrate provided as part of the MAP kinase assay kit (Upstate Bio-technology), following the manufacturer’s protocol. 25 ml of the reactionmixtures were spotted onto P81 phosphocellulose squares, and theradioactivity incorporated in the filters was determined by liquid scin-tillation counting.

RESULTS

IGF-I Overexpression Enhances the in Vitro Replicative LifeSpan of Satellite Cells Isolated from the Hypertrophied Gastro-cnemius Muscles of IGF-I Tg Mice—The gastrocnemius mus-cles of 1-month-old IGF-I Tg mice were significantly larger(22% increase, p , 0.05) than their WT littermates. Residentsatellite cells from the IGF-I Tg muscles secreted more hIGF-Ipeptide into the media (114.9 6 4.5 ng/ml) compared with WTsatellite cells (values were below detection limits of the assay).Overexpression of the IGF-I transgene did not affect endoge-nous mouse IGF-I peptide levels in the satellite cells fromeither of the two groups, consistent with our previous observa-tions in whole muscle (28). IGF-I Tg satellite cells started toproliferate earlier (4 days) in culture and proliferated ingreater numbers than those from WT muscles (lag period of;10 days before they started to double) (Fig. 1A). Both popu-lations underwent a period of rapid proliferation, after whichthe growth rates slowed, and finally ceased in WT cells afterapproximately 76 days in culture. Satellite cells from the IGF-ITg group had a significantly greater proliferative capacity thanthe WT group, as reflected by the 119% higher (p , 0.0001)cumulative population doublings attained by the IGF-I Tg sat-ellite cells (11.43 6 2.1) compared with WT (5.21 6 1.6) cells,after 76 days in culture. Thus, IGF-I Tg satellite cells achievedat least five additional rounds of divisions above the maximumthat was attained by WT satellite cells. The population of

satellite cells from both IGF-I Tg and WT groups at this 76-daypoint are henceforth referred to as the “late-passage” satellitecells, which were then used for all subsequent analyses de-scribed below. Analysis of these cultures with anti-desmin an-tibody routinely revealed .95% desmin-positive cells, henceensuring that these cultures at late-passages continued to beenriched in muscle-specific cells.

Late-passage satellite cells from the IGF-I Tg group showeda greater preponderance to form large-sized colonies after 7weeks in proliferation media, compared with correspondingcells from the WT group (Fig. 1B, upper panel). Quantitation ofthese colony sizes (Fig. 1B, lower panel) demonstrated a dra-matic shift to the right (i.e. colonies with $256 cells or $8 celldoublings) in IGF-I Tg satellite cells, thereby indicating agreater proliferative potential of individual cells, comparedwith the WT group. Profiles with $8 cell doublings accountedfor 20–25% of the total 200–250 colonies in satellite cells fromthe IGF-I Tg group, in contrast to ,2% for the WT group.

Long-R3 IGF-I (LR3), a synthetic analog of IGF-I is morebiologically potent than unmodified IGF-I as it has low affinityfor IGF-I-binding proteins (18). Addition of LR3 at 100 ng/mlresulted in 27 and 29% incorporation of the BrdUrd label insatellite cells from WT and IGF-I Tg muscles, respectively, inearly passages (day 23 in culture) (Fig. 1C). The percent oflabeled nuclei at all the other doses were also comparable inboth WT and IGF-I Tg satellite cells in early-passage. However,by late-passage (day 76 in culture) (Fig. 1C), BrdUrd incorpo-ration dramatically decreased to ,5% in WT cells, whereas itwas maintained at 26% in the IGF-I Tg cells when stimulatedby 100 ng/ml LR3, thereby confirming that WT satellite cells inlate-passage had exhausted their proliferative reserves. Evenwithout any added LR3 (Fig. 1C), the percentage of BrdUrd-labeled nuclei in late-passage IGF-I Tg satellite cells remainedat levels similar to that seen in early-passage WT and IGF-I Tgcells (;12.5%), validating that satellite cells overexpressingIGF-I possessed a higher proliferative capacity, compared withWT cells. The dramatic increase in proliferation potential seenin IGF-I Tg satellite cells was due to the growth-promotingeffects of IGF-I since cell proliferation (measured with BrdUrdincorporation) was reduced by ;65% when these late-passageIGF-I Tg cells were cultivated in the presence of 1 mg/ml IGF-I-receptor antibody for 48 h, relative to control cultures grownwithout the blocking antibody (data not shown).

Of note, ,3% of late-passage IGF-I Tg and WT satellite cellsdemonstrated sarcomeric myosin and Hoescht 33342 positivecells when cultivated in high serum conditions, indicating thatthe growth arrest seen in WT cells was likely not due to ter-minal differentiation or apoptosis, respectively. Furthermore,IGF-I Tg satellite cells from 1-month-old mice were unlikely tobe immortalized secondary to continuous IGF-I overexpression,because satellite cells cultured from 18-month-old IGF-I Tgmuscles (i.e. these cells were exposed to high levels of IGF-I for18 continuous months) had lesser proliferation capacity thantheir WT littermates, and failed to respond to exogenouslyadded LR3-IGF-I.2

Late-passage Primary Culture of Satellite Cells from WildType (WT) Muscles Are Arrested at G1 Phase of the Cell Cy-cle—We asked if the lack of proliferation seen in these late-passage WT satellite cells was due to a G1 cell cycle arrestsecondary to replicative senescence, since senesced cells char-acteristically arrest at the G1/S boundary (12, 13). FACS anal-ysis of cell cycle distribution of asynchronous populations oflate-passage WT and IGF-I Tg satellite cells in proliferationmedia revealed that WT cells had a 4–5-fold decrease in S-phase cell population and a concomitant accumulation of G0/G1

2 M. V. Chakravarthy and F. W. Booth, manuscript in preparation.

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cells (;85%), compared with late-passage IGF-I Tg satellitecells (Fig. 2A). These results prompted us to investigate thebiochemical basis for this G1 arrest.

Analysis of asynchronous populations of late-passage WTsatellite cells (Fig. 2B), revealed the characteristic profiles (29–31) expected of G1/S arrested cells (lack of phosphorylated pRband a decrease in cyclin A protein levels), whereas the corre-sponding IGF-I Tg cells revealed a pattern more consistentwith that of cycling cells (hyperphosphorylated pRb and in-crease in cyclin A protein). In contrast, there was an insignif-icant difference in protein levels of the early-mid G1 cyclins(cyclin D1 and E), and cdk2 (Fig. 2B), consistent with an inter-pretation (32) that WT cells were likely arrested in the G1

phase of the cell cycle. Furthermore, hypophosphorylation ofpRb and the dramatic decrease in cyclin A protein levels inlate-passage WT cells supports a potential arrest at the R-pointin these cells since phosphorylation of pRb is required forpassage through this point (12, 31), and consequently for syn-thesis of late-G1 genes (29).

To confirm that the G1/S arrest was due to cellular senes-cence, and not an extended state of quiescence, we analyzed thesame cell cycle regulatory molecules after defined intervalsfollowing release from serum starvation in late-passage WTand IGF-I Tg cells, since it has been previously shown thatsenescent cells are refractory to further mitogenic stimulation(13). Mean generation time for the IGF-I Tg cultures weredetermined to be between 24 and 29 h, and hence the timeintervals chosen approximated early-mid G1 (6 h), late G1/S (12h), and G2/M (24 h) phases of the cell cycle (12). Approximately80–85% of late-passage WT satellite cells remained in G0/G1

throughout 24 h of serum stimulation (Fig. 2C, upper panel),whereas the IGF-I Tg cells showed a time dependent decreasein the percentage of G0/G1 cells, and a corresponding increasein the percentage of S phase cells (,10% at time 0 to ;35% atthe end of 24 h) with serum stimulation, indicating cell cycleprogression. The failure of late-passage WT satellite cells to berecruited into S-phase confirms a G1 cell cycle arrest secondaryto replicative senescence, and is supported by the following. (i)An unchanged predominance of the unphosphorylated form ofpRb, before (lane 1, Fig. 2C, lower panel) and after 24 h ofserum stimulation (lane 4 versus IGF-I Tg cells in lane 8, Fig.2C, lower panel). (ii) Cyclin A levels were extremely low andindistinguishable from amounts seen in unstimulated IGF-I Tgcells (lane 1 versus lane 5, Fig. 2C, lower panel). Serum stim-ulation resulted in no change in cyclin A in late-passage WTsatellite cells (lane 1 versus lane 4), whereas in IGF-I Tg cells,there was ;15-fold increase in cyclin A by G2 phase (24 h) (lane5 versus lane 8). (iii) In marked contrast, there were highconstitutive abundance of both cyclin D1 and cyclin E proteinsin unstimulated (lane 1) WT cells (15–20-fold increase relative

triplicate. B, colony size distribution of late-passage IGF-I Tg and WTsatellite cells (i.e. cells from 76-day cultures from Fig. 1A). Satellite cellsfrom each group were seeded at clonal density and grown in prolifera-tion media for 7 weeks. A representative set of such plates after stainingwith 0.5% crystal violet is shown in the upper panel. Colony size wasthen estimated by counting the number of cells per colony for up to 256cells (representing 8-cell doublings). The percent of total colonies thatwere able to attain a specified size, denoted by the number of celldoublings attained in culture over the 7-week growth period is shown inthe lower panel. A total of 200–250 colonies were scored for eachdistribution and the results are representative of three separate exper-iments. C, late-passage WT satellite cells were unable to be furtherstimulated with exogenous IGF-I-LR3. Early-passage (day 23 in cul-ture) and late-passage (day 76 in culture) satellite cell cultures fromIGF-I Tg and WT muscles were treated with the indicated concentra-tions of LR3 for 48 h, following which the labeling index was determinedby scoring the percentage of BrdUrd-positive nuclei. Results aremean 6 S.E. of three independent experiments in triplicate.

FIG. 1. Overexpression of IGF-I in skeletal muscle enhances invitro replicative life span of the resident satellite cells. A, cumu-lative population doublings of satellite cells derived from IGF-I Tg andWT gastrocnemius muscles attained in culture from the time of isola-tion from the animal (time 0) over the duration of their in vitro culturedlife span. Each point represents the time at which cells were passagedbefore they reached confluence. Symbols (E and ●) denote cumulativepopulation doublings attained from WT and IGF-I Tg satellite cells,respectively. Results are mean 6 S.E. from four parallel cultures in

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to unstimulated IGF-I Tg cells (lane 5); Fig. 2C, lower panel).Serum stimulation resulted in only a modest further accumu-lation of these cyclins (15–20% for cyclin D1 and ;10% forcyclin E in WT cells (lanes 2–4)), whereas serum stimulation ofIGF-I Tg cells resulted in a dramatic accumulation (;10-fold)of cyclin D1 by mid-G1 phase (6 h) and of cyclin E (5–7-foldincrease) by mid- to late-G1 phase (12 h) (Fig. 2C, lower panel,lanes 6 and 7). (iv) The abundance of total cdk2 protein re-

mained unchanged with serum stimulation and was similar inboth WT and IGF-I Tg satellite cells. The above molecularevents delineated in WT satellite cells are consistent withearlier findings reported in serum-stimulated senescent hu-man fibroblasts (12, 33). Hence, the ability of IGF-I Tg satellitecells to continue doubling even in late-passages suggest thatIGF-I may help overcome this G1/S arrest to subsequentlyenhance their in vitro replicative life span.

FIG. 2. Late-passage wild type satel-lite cells are arrested in the G1 phaseof the cell cycle. A, flow cytometry anal-ysis of cell cycle distribution. Asynchro-nous populations of late-passage (day 76in culture) satellite cells from IGF-I Tgand WT groups were pulse-labeled with10 mM BrdUrd for 1 h. BrdUrd incorpora-tion was analyzed by fluorescein-conju-gated anti-BrdUrd antibody and DNAcontent was measured by propidium io-dide staining. The percentage of cells in Sphase (upper boxes), G0/G1 phase (lower-left boxes), and G2/M phase (lower-rightboxes) are as indicated. Results are a rep-resentative set from four separate exper-iments. B, Western blot analysis of G1/Scell cycle regulatory molecules in late-passage asynchronous WT and IGF-I Tgsatellite cells. 35 mg of total protein wasresolved by SDS-PAGE and immuno-blotted with the indicated antibodies. Theantibody used against retinoblastomaprotein detects both the phosphorylated(slower-migrating band; ppRb) and un-phosphorylated (faster-migrating band;pRb) forms. For normalization, the re-solved proteins were subsequently probedwith anti-actin antibody that recognizesall isoforms from skeletal, cardiac, andsmooth muscle actin. Representativeblots of three independent experimentsare shown. C, failure of cell cycle progres-sion upon release from serum starvationin late-passage WT satellite cells. After48 h of serum starvation, late-passageWT and IGF-I Tg satellite cell cultureswere stimulated with serum-rich mediafor the indicated times, at the end ofwhich, the cells were prepared for FACSanalysis for cell cycle distribution (upperpanel) or were analyzed by Western blot-ting with the indicated antibodies (lowerpanel). The cell cycle profiles (percentag-es) are means of three separate experi-ments performed in duplicate. Each lanecontains 35 mg of total protein from thewhole cell lysates of the late-passage WTand IGF-I Tg satellite cells. Representa-tive blots from three independent experi-ments are shown.

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G1 Cyclin-associated Kinase Activities Are Decreased in Pri-mary Cultures of Late-passage WT Satellite Cells—To under-stand in greater detail the regulatory events contributing tothe G1/S arrest observed in late-passage WT satellite cells, weanalyzed cdk2 as well as cyclin E- and cyclin A-associatedkinase activities. These kinase activities were reduced 8–12-fold in late-passage asynchronous WT satellite cells, comparedwith IGF-I Tg cells (Fig. 3A). High levels of cyclin E protein inlate-passage WT satellite cells (Fig. 2C, lower panel, lanes 1–4)were not coordinated with increased cyclin E-associated kinaseactivity up to 24 h (Fig. 3B, upper panel), or 72 h after serumstimulation (data not shown for 48–72 h). WT cells also lackedcyclin A-associated kinase activity (Fig. 3B, lower panel), con-sistent with the lack of cyclin A protein in these cells (Fig. 2C,lower panel, lanes 1–4). In contrast, cyclin E-associated kinaseactivity in IGF-I Tg satellite cells (Fig. 3B, upper panel) paral-leled the maximum increase seen in cyclin E protein after 12 hof serum stimulation (Fig. 2C, lower panel, lane 7). CyclinA-associated kinase activity on the other hand peaked at 24 hafter serum stimulation (when cells have progressed past Sphase), and correlated with the maximum accumulation ofcyclin A protein as shown in Fig. 2C (lower panel, lane 8).Furthermore, the observed kinetics of the activation of cyclinE-associated kinase correlated well with the kinetics of pRbphosphorylation (compare Fig. 2C, lower panel, lane 7, withFig. 3B, upper panel, at the 12-h time point). Taken together,these results suggest that IGF-I overexpression likely main-tains cyclin E/cdk2 kinase activity to promote the phosphoryl-ation of pRb, and consequently cell cycle progression in thelate-passage IGF-I Tg satellite cells. Conversely, the lack ofthese kinase activities in late-passage WT satellite cells couldimpair the ability of these cells to pass the R-point, leading tothe observed G1/S arrest. Thus, cyclin E/cdk2 kinase activitymay be a key regulatory mechanism contributing to cell cycletransit beyond the R-point in satellite cells.

Up-regulation of p27Kip1 and Increased Association of theInhibitor with cdk2 in Late-passage WT Satellite Cells—Simi-lar amounts of cyclin E-cdk2 complexes were observed in thelate-passage WT and IGF-I Tg satellite cells (Fig. 4A, upperpanel). However, the kinase activities of these same immuno-complexes were 8–12-fold lower in the WT cells, relative to thatof the IGF-I Tg cells (Fig. 3A, middle panel). Hence, the lowcyclin E-associated kinase activity observed in late-passage WTsatellite cells is due to mechanisms other than a failure ofcyclin E to bind to its kinase partner. In contrast, cyclin Aimmunoprecipitates exhibited a 5–10-fold decrease in the abil-ity of cyclin A to complex with cdk2 in late-passage WT satellitecells (Fig. 4A, middle panel), relative to IGF-I Tg cells, and thesame immunoprecipitates also exhibited a markedly decreasedcyclin A-associated kinase activity (Fig. 3A, lower panel). Thus,the decrease cyclin A-cdk2 association, in part, may explain thedecreased cyclin A-associated kinase activity seen in the late-passage WT cells. Association between cyclin D1 and cdk4 alsowas decreased to similar levels as cyclin A-cdk2 (5–10-fold) inlate-passage WT satellite cells relative to the correspondingIGF-I Tg cells (Fig. 4A, lower panel).

Activities of the cdks can be regulated in part via cdk inhib-itors (34). When the protein abundance of p27Kip1, p21Cip1/Waf1,and p57Ki, all of which are known to play crucial roles in G1/Stransition (35), were analyzed in asynchronous populations oflate-passage WT and IGF-I Tg satellite cells, only the level ofp27Kip1 protein (Fig. 4B, upper panel) was affected (3–4-folddecrease) by IGF-I overexpression, consistent with previousreports (15) seen with other growth factors. On the other hand,protein levels of p21Cip1/Waf1 and p57Kip2 (members of Waf/Kipfamily) remained unchanged in late-passage IGF-I Tg satellite

cells (Fig. 4B, middle and lower panels), relative to WT cells.This was in marked contrast to the high levels of p21Cip1/Waf1

that was reported previously in senescent human fibroblasts(14). Therefore, these results suggest that the cdk inhibitor

FIG. 3. Enhanced histone HI kinase activity in late-passageIGF-I Tg satellite cells. A, histone H1 kinase activity of anti-cdk2,anti-cyclin E, and anti-cyclin A immunoprecipitates (IP) from aliquotsof the same asynchronous, late-passage IGF-I Tg and WT satellite cellextracts used for Fig. 2B. 500 mg of total protein was immunoprecipi-tated with the indicated antibodies, and the kinase assay was per-formed using histone H1 as the substrate (lanes 3 and 4), or by omittingthe substrate (S) (lane 2) to determine background phosphorylation, aswell as with a species-specific immunoglobulin (Ig) for control (lane 1).The products of the kinase reaction were resolved on 10% SDS-PAGEgels and autoradiographed to visualize the 32P-labeled histone H1(HH1). A representative set from one of the triplicate experiments ispresented. B, histone H1 kinase activity of anti-cyclin E (upper panel)and anti-cyclin A (lower panel) immunoprecipitates from aliquots of theserum-stimulated, late-passage IGF-I Tg and WT satellite cells extractsused in Fig. 2C. Results are presented as the radioactivity (cpm) incor-porated into the substrate after correcting for background, and is rep-resentative of three independent experiments.

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expression pattern in senescent cells is cell-type dependent.The binding of cdk inhibitors to their respective cdks leads to

the inhibition of the activities of these kinases (34). Theamount of p27Kip1 bound to cdk2 was ;7-fold higher in late-passage WT anti-cdk2 immunoprecipitates, compared with thecorresponding IGF-I Tg immunoprecipitates (Fig. 4C, upperpanel). These same immunoprecipitates from WT cells had asignificantly decreased histone H1 phosphorylation, relative toIGF-I Tg IPs (similar to that shown in Fig. 3A, upper panel).Thus, the up-regulation of total p27Kip1 protein levels andincreased p27Kip1-cdk2 binding (Fig. 4, B and C, upper panels)could contribute to the decreased cdk2 kinase activity (Fig. 3A,upper panel), and may lead to the G1/S arrest seen in late-passage WT satellite cells. The amount of total cdk2 immuno-precipitated from both WT and IGF-I Tg cells were similar asshown in Fig. 4C (lower panel), and hence it is unlikely that thedecreased cdk2 kinase activity in late-passage WT satellitecells is due to insufficient amounts of total cdk2 precipitates.Also, overexpression of IGF-I in late-passage transgenic satel-lite cells did not change the amount of association ofp21Cip1/Waf1 bound to cdk2 (Fig. 4C, middle panel), nor theassociation of p57Kip2 with cdk2 (data not shown; results areidentical to that seen with p21Cip1/Waf1).

Ectopic Overexpression of p27Kip1 in Late-passage IGF-I TgSatellite Cells Results in Decreased cdk2 Kinase Activity and G1

Arrest—To demonstrate that p27Kip1 is important in mediatingthe cell cycle arrest observed in late-passage WT satellite cells,we infected growing late-passage IGF-I Tg cells with an ade-novirus encoding p27Kip1, or with empty virus as a control(mock infection). Adp27-infection was documented by Westernblot analysis (Fig. 5A, upper panel) and resulted in a dose-de-pendent inhibition of the BrdUrd labeling index, whereas cel-lular proliferation was not inhibited by mock infection (Fig. 5A,lower panel). As shown in Fig. 5B, the mean doubling time ofAdp27-infected IGF-I Tg satellite cells, compared with mock-infected cells was prolonged ;2.5-fold, providing further evi-dence for an inhibition of cellular proliferation with ectopicp27Kip1 overexpression. Indeed, overexpression of p27Kip1 inIGF-I Tg satellite cells was sufficient to abolish the cdk2 kinaseactivity in cdk2 immunoprecipitates, failed to phosphorylate

pRb, and caused a 3–4-fold reduction in cyclin A protein abun-dance (Fig. 5C). In addition, FACS analysis of Adp27-infectedcells demonstrated a significant accumulation in G0/G1, with aconcomitant decrease in the percentage of S phase cells, com-pared with mock-infected cells (Fig. 5D). Thus, overexpressionof p27Kip1 alone is sufficient to reverse the IGF-I-mediatedrescue from G1 arrest in late-passage IGF-I Tg cells, and con-

FIG. 4. Association of G1 cyclins and cdk-inhibitors with cdk2in late-passage WT and IGF-I Tg satellite cells. A, cyclin-cdk com-plexes in WT and IGF-I Tg satellite cells. 350 mg of total protein fromlate-passage asynchronous cultures used in Fig. 2B were first immuno-precipitated with anti-cyclin E (upper panel), anti-cyclin A (middlepanel), or anti-cyclin D1 (lower panel). The immunocomplexes werethen resolved by SDS-PAGE gels and immunoblotted with antibodiesagainst cdk2 or cdk4, as indicated. Representative blots from threeindependent experiments are presented. B, up-regulation of cdk-inhib-itor p27Kip1 in total extracts of late-passage WT satellite cells. 35 mg ofwhole cell lysates were resolved by SDS-PAGE and probed with anti-bodies against p27Kip1 (upper panel), p21Cip1/Waf1 (middle panel), andp57Kip2 (lower panel). Representative blots from three separate exper-iments are shown. C, increased p27Kip1-cdk2 binding in late-passageWT satellite cells. 750 mg of total protein from WT and IGF-I Tg wholecell lysates was first immunoprecipitated (IP) with anti-cdk2 antibody,and then the immunoprecipitate was examined by Western blot anal-ysis using the indicated antibodies. For each immunoprecipitation, anappropriate species-specific immunoglobulin (Ig) was used as back-ground control. A set of representative blots from three independentexperiments is shown.

FIG. 5. Adenovirus-mediated overexpression of p27Kip1 ingrowing late-passage IGF-I Tg satellite cells induces G1 arrest,and inhibits cdk2 kinase activity and phosphorylation of pRb.A, upper panel, Western blot analysis using anti-p27Kip1 antibody todocument p27Kip1 overexpression in IGF-I Tg satellite cells, infected ata multiplicity of infection of 5, 10, or 20 plaque forming units (pfu) percell, or adenovirus with empty vector (mock), at an multiplicity ofinfection of 20 pfu/cell. Results are representative of three independentobservations. During the last hour of the 48-h virus incubation, parallelsets of plates were pulse-labeled with 10 mM BrdUrd, and subsequentlyprocessed to determine the BrdUrd labeling index (lower panel). Thesolid black bar represents mock-infected cells, and the hatched barscorrespond to Adp27 concentrations in the upper panel. Values aremean 6 S.E. from three separate experiments. B, growth curves ofAdp27Kip1- and mock-infected late-passage IGF-I Tg cells. The numberof cells in equivalent subconfluent cultures of mock-infected (l) andAdp27-infected (E) IGF-I Tg satellite cells (both at a multiplicity ofinfection of 20 pfu/cell) were counted daily for 5 days. The averagedoubling time (from three separate experiments) of Adp27-infected cellswas 71 6 2.5 h versus 29 6 1.8 h for mock-infected cells. C, 350 mg oftotal protein from mock- and Adp27-infected cells (at multiplicity ofinfection of 20 pfu/cell) were immunoprecipitated with anti-cdk2 anti-body, and histone H1 kinase activity (upper panel) in the immunopre-cipitates were determined as detailed in the legend to Fig. 3. In parallel,50 mg of total protein from mock- and Adp27-infected IGF-I Tg satellitecells were resolved by SDS-PAGE gels and immunoblotted with anti-bodies against pRb (middle panel) or cyclin A (lower panel). Results arerepresentative of three independent experiments. D, flow cytometricanalysis of cell cycle distribution was performed in mock and Adp27-infected cells (at multiplicity of infection of 20 pfu/cell) that werecultured at low density (5.5 3 104 cells/60-mm dish). A representativeset of histograms of the distribution of the cells with percentages ofcells in G0/G1, S, and G2/M phases from three separate experiments ispresented.

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sequently mimics the biochemical events seen in senesced WTcells. Our findings therefore substantiate a causal role forp27Kip1 in the regulation of critical molecules at the G1/Sboundary, necessitating its down-regulation for cell cycle pro-gression in satellite cells.

Decreased MAP Kinase and PI 39-Kinase Activities in Late-passage WT Satellite Cells—Late-passage IGF-I Tg satellitecells had an 8–10-fold increased phosphorylation of both p44(ERK1) and p42 (ERK2) MAPK isoforms, as well as ;5-foldincrease in total MAPK activity in whole cell extracts, relativeto late-passage WT cells (Fig. 6A, upper and lower panels).Total ERK1 and ERK2 proteins in WT and IGF-I Tg cells weresimilar (Fig. 6A, middle panel), indicating increased phospho-rylation per unit of protein. As shown in Fig. 6B (upper panel),there was a significant accumulation of the phosphorylatedlipid product, phosphatidylinositol phosphate in late-passageIGF-I Tg satellite cells indicating a greater PI 39-kinase activ-ity in these cells, relative to the corresponding WT cells. Sim-ilar results were obtained when whole cell lysates were immu-noprecipitated with anti-p85 antibody (data not shown). Late-passage IGF-I Tg immunoprecipitates demonstrated asignificant increase (6–7-fold) in the amount of tyrosine-phos-phorylated p85 (Fig. 6B, middle panel), relative to the WT cells,indicating that IGF-I-mediated accumulation of the PIP lipidproduct was likely due to an increased activation of the p85regulatory subunit of PI 39-kinase, as the total amount of p85protein by itself was similar in both groups (Fig. 6B, lowerpanel). In addition, late-passage IGF-I Tg cells, relative to WTcells demonstrated ;3-fold increase in the phosphorylation ofthe Ser473 residue of Akt (phosphorylation of which is criticalfor Akt kinase activity (36)), whereas no difference was noted intotal Akt protein between the two groups (Fig. 6C). Therefore,IGF-I overexpression allows transgenic satellite cells to main-

tain MAPK and PI 39-kinase activities, as well as activation ofAkt even in late-passages, when corresponding WT cells have adeficiency in these signaling molecules.

Pharmacological Inhibition of PI 39-Kinase, but Not MAPK,Leads to an Up-regulation of p27Kip1 and G1 Cell Cycle Arrest inIGF-I Tg Satellite Cells—In order to first establish that eachpharmacological inhibitor was specific for its respective path-way, growing late-passage IGF-I Tg satellite cell cultures weretreated for 24 h with 5–100 mM concentrations of eitherPD098059 (PD, a synthetic inhibitor of the MAPK-activatingenzyme (MEK) (37)) or LY294002 (LY, inhibits PI 39-kinase bybinding to the ATP-binding sites of the PI 39-kinase p110 cat-alytic subunit (38)), or both. Control cultures were treated withan equivalent volume of Me2SO. Pilot experiments showed thatmaximum inhibitory effects for PD and LY were 50 and 30 mM,respectively (data not shown). The effects of each inhibitorindividually (Fig. 7A, lanes 2 and 3) was highly specific for theinhibition of its corresponding pathway (i.e. PD reduced ERK 1and 2 phosphorylation (first panel), but did not affect Aktphosphorylation (third panel), while LY inhibited Akt phospho-rylation, but did not affect ERK1 and 2 phosphorylation), andthese compounds in combination (Fig. 7A, lane 4) did not resultin any further significant inhibition of phosphorylation, thaneither one alone. Additionally, at the doses used, there was noeffect of these inhibitors on total protein levels of ERK1/ERK2(second panel) or Akt (fourth panel).

We next determined if the lack of cell cycle progression inlate-passage WT satellite cells was a consequence of a decreasein the activities of MAPK, PI 39-kinase, or both. Treatment ofgrowing, late-passage IGF-I Tg satellite cells with 50 mM PDalone resulted in essentially no change in the cell cycle profilecompared with that of Me2SO-treated control cells (,15% in-crease in the %G0/G1 cells for PD from control) (Fig. 7B).

FIG. 6. Up-regulation of both the MAPK and PI 3*-K pathways in late-passage IGF-I Tg satellite cells. A, phosphorylation status andkinase activity of Erk1/Erk2 MAPK in late-passage WT and IGF-I Tg satellite cells. 35 mg of total protein from each whole cell extract were resolvedby SDS-PAGE and subjected to Western blot analysis to determine phosphorylated (upper panel) and total (middle panel) abundance of Erk1/Erk2proteins in WT and IGF-I Tg cells. Results are representative of three separate experiments. Lower panel, 10 mg of each lysate was also subjectedto MAP kinase assay using myelin basic protein as a substrate. Mean 6 S.E. of the amount of 32P radiolabel incorporated into myelin basic proteinsubstrate from four separate experiments in triplicate are presented. * indicates significantly (p , 0.0001) increased, compared with WT. B, PI39-kinase activity in the anti-phosphotyrosine (4G10 antibody) immunoprecipitated complexes was determined using phosphatidylinositol (PI) asa substrate, and the phosphorylated lipid product, phosphatidylinositol phosphate (PIP) was visualized by autoradiography (upper panel) andquantitated using a PhosphorImager. 350 mg of total protein was immunoprecipitated (IP) with anti-p85 antibody, followed by immunoblot (IB)with 4G10 (middle panel). The same membrane was then stripped and reprobed with a p85 polyclonal antibody to determine the abundance of totalp85 protein (lower panel). Species-specific immunoglobulins (Ig) were used as background control for each of the immunoprecipitations. Results arerepresentative of four independent experiments. C, 35 mg of protein from late-passage IGF-I Tg and WT whole cell extracts were subjected toWestern blot analysis to determine phosphorylation status of Akt on the Ser473 residue (upper panel) as well as total Akt protein levels (lowerpanel). Representative blots from three experiments are presented.

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However, treatment of IGF-I Tg cells with 30 mM LY resulted inan almost doubling of the proportion of cells in G1, coupled with;5-fold reduction in the percentage of S-phase cells, comparedwith control (Me2SO), and nearing the value for the percentageof S-phase cells seen in late-passage WT satellite cells (Fig. 2A).PD in combination with LY did not have a further significantincrease in the percentage of G1 cells in IGF-I Tg cultures(Fig. 7B).

To explore further the effect of this LY-induced cell cyclearrest on p27Kip1 protein, we investigated its expression levelin LY-treated IGF-I Tg satellite cells. The Western blottingresults not only showed a marked increase of 2–4-fold inp27Kip1 protein (Fig. 7C, panel 1, lane 2), but also demonstrateda similar decrease in pRb levels leading to its overall hypophos-phorylation (panel 2), decreased cyclin A expression (panel 3),and resulted in no changes in cyclin D1 levels (panel 4). Incontrast, there was no effect of PD alone (Fig. 7C, lane 3) onthese intracellular mediators, except for cyclin D1 (a modestdecrease of ;30%), from that seen in Me2SO-treated controlcultures (lane 1). Therefore, these results support the notionthat it is a deficiency in the PI 39-kinase activity (rather thanMAPK) that results in the G1/S arrest noted in senesced late-passage WT cells, and signaling through this pathway selec-

tively regulates p27Kip1, pRb, and cyclin A proteins in satellitecells.

DISCUSSION

After 76 days in culture, satellite cells from 1-month-oldIGF-I Tg mice continued to proliferate. In contrast, satellitecells from WT littermates at this time point had exhaustedtheir proliferative reserves, were refractive to exogenous LR3-IGF-I, and demonstrated negligible amounts of sarcomeric my-osin staining, suggesting that this loss in proliferative capacitywas likely due to cellular senescence. Thus, IGF-I overexpres-sion in satellite cells for a period of ;4 months (;1-month inthe animal 1 2.5-months in culture) delayed cellular senes-cence by extending their in vitro replicative life span. Thecumulative number of in vitro population doublings attained byIGF-I Tg satellite cells was ;5 additional rounds of divisions(i.e. on the average each satellite cell could replicate to 32 morecells), above the maximum attained by corresponding late-passage WT satellite cells derived from FVB littermates. Thisdramatic increase in proliferation potential seen in IGF-I Tgsatellite cells was IGF-I-dependent since the IGF-I receptorantibody blocked this proliferation. These observations raisedthe critical question of how IGF-I Tg satellite cells were able to

FIG. 7. Pharmacological inhibition of PI 3*-kinase results in G1 arrest and up-regulation of p27Kip1 in late-passage IGF-I Tgsatellite cells, independent of MAPK. A, MEK1- and PI 39-kinase inhibitors are specific for the MAPK and PI 39-kinase pathways, respectivelyin late-passage IGF-I Tg satellite cells. These cells were treated with either Me2SO (control; lane 1), 50 mM PD098059 (lane 3) or 30 mM LY294002(lane 2), or both (lane 4), for 24 h. Effects of the two compounds were determined by subjecting 35 mg of protein from each of the cell lysates toWestern blot analysis for phospho-specific (panel 1) and total (panel 2) Erk1/Erk2 proteins, as well as Ser473 phosphorylation of Akt (panel 3) andtotal Akt (panel 4). Results for each of the assays are representative of three independent experiments. B, late-passage IGF-I Tg satellite cells werepulsed with 15 mM BrdUrd during the last 30 min of the 24-h treatment with the indicated compounds, and prepared for dual-parameter flowcytometry. The percentage of cells in G0/G1, S, and G2/M are as indicated, and are representative from four independent experiments. C, cells fromsimultaneously treated dishes were harvested and 50 mg of protein/lane were analyzed by Western blot analysis for p27Kip1, pRb, cyclin A, andcyclin D1, as indicated. Blots were also subsequently probed with anti-actin for normalization control. Results shown are representative of fourseparate experiments.

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sustain their proliferative ability even in late-passages to ulti-mately result in the observed extension of their in vitro repli-cative life span.

Analysis of the molecular profile of late-passage IGF-I Tgsatellite cells demonstrated characteristics of cycling cells,whereas that of the corresponding WT cells was consistent witha G1/S arrest characteristic of senescent cells. Upon releasefrom serum starvation, the IGF-I Tg cells demonstrated a time-dependent accumulation of S-phase cells (coincident with theinduction of cyclin D1 and E proteins), phosphorylated pRb,and up-regulated cyclin A protein. In addition, IGF-I overex-pression in late-passage satellite cells down-regulated p27Kip1

protein levels, leading to an associated increase in the cyclinE/cdk2 kinase activity, relative to WT cells (Fig. 8). Cdk2 is oneof the critical kinases required to phosphorylate pRb (39),which in turn has been suggested to be a rate-limiting stepcontrolling cell cycle progression past the R-point to synthesizelate-G1 genes necessary for S phase entry (29, 31). Polyak et al.(40) suggested that p27Kip1 could affect both the activation andkinase activity of cdk2, since the binding of p27Kip1 may inter-fere with phosphorylation of cdk2s activating site (Thr-160).Others have suggested that p27Kip1 could act as a thresholddevice controlling cdk2 kinase activity, increased cyclin D1/cdk4 accumulation sequesters free p27Kip1, decreasing p27Kip1-cdk2 association, and consequently allowing for cdk2 activation(41). Consistent with this notion, we demonstrated an in-creased abundance of the cyclin D1zcdk4 complex in late-pas-sage IGF-I Tg cells, which we speculate may also help seques-ter p27Kip1, effectively decreasing the p27Kip1 protein that isavailable to bind and inactivate cdk2.

Remarkably, adenoviral-mediated ectopic overexpression ofp27Kip1 in exponentially growing cultures of late-passage IGF-ITg satellite cells down-regulated cdk2 kinase activity, hy-pophosphorylated pRb, decreased cyclin A protein, and in vitrocell proliferation, increased cell doubling time by ;2.5-fold, andinduced a G1 arrest, thereby mimicking the changes observedin senesced cultures of late-passage WT satellite cells. Previousstudies have shown that p27Kip1 overexpression (27, 42) re-pressed transcription from the cyclin A promoter regions that

contain an E2F-binding site required for cell cycle-regulatedcyclin A gene expression, indicating that p27Kip1 can regulatetranscription of late G1 genes. Furthermore, recent findings ofBusse et al. (25) showed that antisense p27Kip1 essentiallyreversed both p27Kip1 up-regulation and the G1 arrest in A431cells treated with the tyrosine kinase inhibitor AG-1478. Therole of p27Kip1 as a critical regulator of cellular proliferation isfurther illustrated by p27Kip1 knockout mouse models, whichexhibits gigantism, organomegaly, and enhanced spontaneoustumor formation (43–45). Given that cdk2 kinase activity,phosphorylation of pRb, and up-regulation of cyclin A are crit-ical for cell cycle progression (12, 13), and the fact that all ofthese regulatory factors are inhibited by p27Kip1 overexpres-sion in growing late-passage IGF-I Tg cells, it suggests thatp27Kip1 is a critical mediator through which IGF-I delays G1/Sarrest in these cells. These results also support the possibilitythat p27Kip1 could be an initiator of a cascade of G1/S cell cycleevents, to consequently regulate replicative senescence (Fig. 8).

Interruption of PI 39-kinase activity in IGF-I Tg cells byLY294002 mimicked the molecular events seen in senescedlate-passage WT satellite cells, i.e. increased p27Kip1, decreasedpRb phosphorylation and cyclin A protein, and caused a G1/Scell cycle arrest. In contrast, blockade of the activated MAPKby PD098059 resulted in neither an appreciable accumulationof cells in G1, nor an increase in p27Kip1 protein levels. Theseobservations in IGF-I Tg satellite cells extend the previousfindings of Collado et al. (46), who showed that inhibition of PI39-kinase pathway induced a senescent-like arrest that wasmediated by p27Kip1 in mouse embryo fibroblasts. We thereforeinfer that PI 39-kinase activation induced by IGF-I overexpres-sion in late-passage IGF-I Tg satellite cells are necessary andsufficient for the down-regulation of p27Kip1, and consequentlyfor the enhanced G1 to S-phase cell cycle progression, resultingin the increased in vitro doubling potential of these cells(Fig. 8).

Thus, it is probable that Akt itself or other molecules acti-vated by the PI 39-kinase signaling pathway is essential for G1

cell cycle progression in skeletal muscle satellite cells fromIGF-I Tg mice. Indeed, a requirement of an intact PI 39-kinasesignaling pathway (independent of MAPK activity in many ofthese cells), for G1 to S cell cycle progression has previouslybeen reported in other cell types (20, 24, 25, 47–50). Our resultsare also consistent with Milasincic et al. (19) who had previ-ously shown that the mitogenic response of IGF-I was mediatedvia the activation of the PI 39-kinase pathway, independent ofMAPK in C2C12 murine cell line, but they had not examinedcell cycle markers. However, our results contradict the reportin rat L6A1 immortalized myoblasts, which had demonstratedthat pharmacological inhibition of MAPK activation attenu-ated the proliferative response by LR3-IGF-I in these cells (18).Thus, it appears that the pluripotent effects of factors such asIGF-I and their consequent signaling may depend on the cellcontext in which its receptor is activated.

Clearly, processes as complex as cellular proliferation andreplicative senescence involve multiple mediators and cross-talk mechanisms. The activation of PI 39-kinase/Akt to signalthe down-regulation of p27Kip1 is a crucial phenomenon thatleads to an enhanced proliferative potential, resulting in exten-sion of the in vitro replicative life span of IGF-I Tg satellitecells. However, this need not be the exclusive mechanism.IGF-I-evoked modulation of other regulatory pathways such asthe inhibition of the pro-apoptotic factor BAD via phosphoryl-ation by Akt/PKB to enhance survival (51), up-regulation oftelomerase activity (52), and/or changes in IGF-I-binding pro-tein concentrations (53) are some of the mechanisms that needto be further explored.

FIG. 8. Potential model for IGF-I-mediated enhancement ofcell cycle progression resulting in an extension of the in vitroreplicative life span of satellite cells from IGF-I Tg skeletalmuscle. Signaling events utilized by IGF-I to modulate critical cellcycle regulatory molecules at the G1/S boundary. Directionality of thearrows is relative to senesced late-passage WT satellite cells. Placementof the phosphorylation of pRb at the R point is based on the modelproposed by Dulic et al. (12) as well as the fact that pRb gets phospho-rylated several hours before S phase (30). While the results presented inthis study uniquely support the notion that p27Kip1 is a downstreamtarget of the PI 39-kinase/Akt pathway in skeletal muscle satellite cells,it remains to be determined if it is directly downstream of Akt or if othermolecules activated by PI 39-kinase, independent of Akt directly mod-ulate its down-regulation. Other processes known to be mediated byactivated Akt, but not measured in this study are indicated inparentheses.

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While previous studies have shown that the number of rep-lications can be extended in other cell types by other means (15,23, 54–56), this is the first demonstration of extending thereplicative life span of skeletal muscle stem cells with a growthfactor. Given that satellite cells are absolutely required forpostnatal muscle growth and repair (2), our data provide novelinsights into some of the regulatory events which may serve asattractive tools for potential ex vivo manipulation of stem cellautografts to extend the replicative life span of old satellitecells for gene therapy in muscle wasting conditions.

Acknowledgments—We thank Dr. Jeanie McMillin for critically read-ing the manuscript and helpful suggestions, Louise Barnett for assist-ance with flow cytometry, Bilal Thair and Sandra Higam for assistancewith cell culture. The mouse monoclonal antibodies D3 and MF20developed by Danto and Fischman (57) and Bader et al. (58), respec-tively, were obtained from the Developmental Studies Hybridoma Bankdeveloped under the auspices of the NICHD and maintained by theUniversity of Iowa, Department of Biological Sciences.

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IGF-I Enhances G1/S Progression in Satellite Cells35952

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Frank W. BoothManu V. Chakravarthy, Tsghe W. Abraha, Robert J. Schwartz, Marta L. Fiorotto and

-Kinase/Akt Signaling Pathway ′of Phosphatidylinositol 3/S Cell Cycle Progression via the Activation1Muscle Satellite Cells by Enhancing G

Replicative Life Span of Skeletalin VitroInsulin-like Growth Factor-I Extends

doi: 10.1074/jbc.M005832200 originally published online August 28, 20002000, 275:35942-35952.J. Biol. Chem. 

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