Key points are not available for this paper at this time.
Terminal differentiation of stem cells is characterized by cessation of cell proliferation as well as changes in cell morphology associated with the differentiated state. For adipocyte differentiation, independent lines of evidence show that the transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) as well as the tumor suppressor retinoblastoma (Rb) protein are essential. How these proteins promote adipocyte conversion and how they function cooperatively during the differentiation process remain unclear. We have used retinoic acid (RA) inhibition of adipogenesis to investigate these issues. RA blocked adipogenesis of 3T3-L1 cells induced to differentiate by ectopic expression of PPARγ and C/EBPα independently or together. However, under these circumstances RA was only effective at preventing adipogenesis when added prior to confluence, suggesting that factors involved in regulation of the cell cycle might play a role in establishing the commitment state of adipogenesis that is insensitive to RA. During differentiation of wild type 3T3 L1 preadipocytes, we found that Rb protein is hyperphosphorylated early in adipogenesis, corresponding to previously quiescent cells re-entering the cell cycle, and later becomes hypophosphorylated. The data suggest that, together with the coexpression of PPARγ and C/EBPα, permanent exit from the cell cycle establishes the irreversible commitment to adipocyte differentiation. Terminal differentiation of stem cells is characterized by cessation of cell proliferation as well as changes in cell morphology associated with the differentiated state. For adipocyte differentiation, independent lines of evidence show that the transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) as well as the tumor suppressor retinoblastoma (Rb) protein are essential. How these proteins promote adipocyte conversion and how they function cooperatively during the differentiation process remain unclear. We have used retinoic acid (RA) inhibition of adipogenesis to investigate these issues. RA blocked adipogenesis of 3T3-L1 cells induced to differentiate by ectopic expression of PPARγ and C/EBPα independently or together. However, under these circumstances RA was only effective at preventing adipogenesis when added prior to confluence, suggesting that factors involved in regulation of the cell cycle might play a role in establishing the commitment state of adipogenesis that is insensitive to RA. During differentiation of wild type 3T3 L1 preadipocytes, we found that Rb protein is hyperphosphorylated early in adipogenesis, corresponding to previously quiescent cells re-entering the cell cycle, and later becomes hypophosphorylated. The data suggest that, together with the coexpression of PPARγ and C/EBPα, permanent exit from the cell cycle establishes the irreversible commitment to adipocyte differentiation. The molecular mechanisms relating to cell proliferation and cell differentiation are inadequately understood. Adipocyte conversion provides an excellent model system to study terminal differentiation. In the case of 3T3-L1 cells, differentiation is induced upon exposure of cells to a mixture of hormonal stimulants including dexamethasone, isobutylmethylxanthine, insulin, and fetal calf serum (1Green H. Meuth M. Cell. 1974; 3: 127-133Abstract Full Text PDF PubMed Scopus (801) Google Scholar, 2Green H. Kehinde O. Cell. 1975; 5: 19-27Abstract Full Text PDF PubMed Scopus (1076) Google Scholar). These pharmacological stimuli, or alternatives such as thiazolidinediones or other activators of peroxisome proliferator activated receptors (PPARs) 1The abbreviations used are: PPAR, peroxisome proliferator activated receptor; C/EBP, CCAAT/enhancer-binding protein; RA, retinoic acid; GM, growth medium; differentiation medium; Rb, retinoblastoma; BrdUrd, bromodeoxyuridine.1The abbreviations used are: PPAR, peroxisome proliferator activated receptor; C/EBP, CCAAT/enhancer-binding protein; RA, retinoic acid; GM, growth medium; differentiation medium; Rb, retinoblastoma; BrdUrd, bromodeoxyuridine. (3Chawla A. Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1786-1790Crossref PubMed Scopus (217) Google Scholar, 4Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes 8: 1224-1234Crossref PubMed Scopus (1977) Google Scholar, 5Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3435) Google Scholar, 6Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2701) Google Scholar), are necessary for adipocyte differentiation of 3T3-L1 cells. During adipocyte conversion, a variety of transcription factors are induced, including C/EBPβ, PPARγ, and C/EBPα (reviewed in Ref. 7Mandrup S. Lane M.D. J. Biol. Chem. 1997; 272: 5367-5370Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Enforced expression of PPARγ (8Tontonoz P. Hu E. Devine J. Beale E.G. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 351-357Crossref PubMed Google Scholar), C/EBPα (9Freytag S.O. Paielli D.L. Gilbert J.D. Genes 8: 1654-1663Crossref PubMed Scopus (390) Google Scholar, 10Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (802) Google Scholar, 11Lin F.-T. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8757-8761Crossref PubMed Scopus (378) Google Scholar), or C/EBPβ (10Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (802) Google Scholar,12Wu Z. Bucher N.L.R. Farmer S.R. Genes 9: 2350-2363Crossref PubMed Scopus (474) Google Scholar, 13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar) stimulates adipogenesis in NIH 3T3 fibroblasts, suggesting the essential roles of these transcription factors in regulating adipogenesis. Furthermore, combined expression of PPARγ and C/EBPα has synergistic effects on promoting fat cell conversion in myoblasts (14Hu E. Tontonoz P. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9856-9860Crossref PubMed Scopus (578) Google Scholar). Therefore, it is likely that PPARγ and C/EBPα function cooperatively to establish terminal adipocyte differentiation. In addition to the expression of differentiated marker genes, terminal differentiation is characterized by permanent withdrawal of cells from the cell cycle. One protein that is involved in cell cycle progression is the retinoblastoma susceptibility gene product, Rb (15Weinberg R.A. Trends Biochem. Sci. 1990; 15: 199-202Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Hypophosphorylation of Rb inhibits cell cycle progression, and this inhibitory effect of Rb is lost upon phosphorylation of the protein (16Chen P.L. Scully P. Shew J.Y. Wang J.Y. Lee W.H. Cell. 1989; 58: 1193-1198Abstract Full Text PDF PubMed Scopus (788) Google Scholar, 17DeCaprio J.A. Ludlow J.W. Lynch D. Furukawa Y. Griffin J. Piwnica-Worms H. Huang C.M. Livingston D.M. Cell. 1989; 58: 1085-1095Abstract Full Text PDF PubMed Scopus (687) Google Scholar). The involvement of Rb in adipocyte differentiation is suggested by the observation that ectopic expression of protein kinase Cη in quiescent NIH 3T3 cells induces hypophosphorylation of Rb and promotes adipogenesis (18Livneh E. Shimon T. Bechor E. Doki Y. Schieren I. Weinstein I.B. Oncogene. 1995; 12: 1545-1555Google Scholar), whereas Rb binding by SV40 large T antigen interferes with adipogenic differentiation (19Higgins C. Chatterjee S. Cherington V. J. Virol. 1996; 70: 745-752Crossref PubMed Google Scholar). Moreover, Rb−/− mouse embryonic lung fibroblasts failed to undergo adipocyte differentiation under appropriate conditions, and ectopic expression of Rb restored the adipogenic phenotype of the Rb−/− cells (20Chen P.-L. Riley D.J. Chen Y. Lee W.H. Genes 10: 2794-2804Crossref PubMed Scopus (386) Google Scholar). Together, these observations suggest an essential role of Rb in adipocyte differentiation. To explore early events in adipogenesis, we have used retinoic acid (RA), which normally inhibits adipocyte differentiation of 3T3-L1 cells (21Kuri-Harcuch W. Wise L.S. Green H. Cell. 1978; 14: 53-59Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 22Stone R.L. Bernlohr D.A. Differentiation. 1990; 45: 119-127Crossref PubMed Scopus (72) Google Scholar, 23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar). Liganded RAR blocks C/EBP-stimulated gene transcription, and RA also prevents adipogenesis due to ectopic expression of C/EBPα or -β (13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar). However, during normal adipogenesis RA exerts its inhibitory function only when added in the first 24–48 h after exposure to differentiating stimuli that are applied postconfluence. The inhibitory function of RA is mediated by RA receptors (RARs), which are down-regulated early in adipocyte differentiation (23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar). However, ectopic expression of RAR in 3T3 L1 cells only extends the period during which RA is effective in preventing adipogenesis by an additional 24–48 h. Interestingly, at this time, although PPARγ is already induced, the level of PPARγ protein is diminished upon RA treatment. This could be explained by the fact that RA/RAR inhibits C/EBP function, which may be responsible for maintaining the level of PPAR during differentiation (23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar). We first examined the effect of RA on adipocyte differentiation due to incubation of wild type 3T3-L1 cells with the PPARγ ligand BRL49653, as well as in 3T3-L1 cells that ectopically express PPARγ. RA inhibited PPARγ activator-mediated fat cell conversion, suggesting that RA blocks adipogenesis due to endogenous PPARγ. The inhibitory effects of RA were also dominant over ectopic co-expression of both PPARγ and C/EBPα when the cells were maintained in RA at the time of gene transduction and thereafter. However, if added after gene transduction but rather at the time of confluency, RA was no longer effective at blocking differentiation of 3T3-L1 cells that ectopically expressed these adipogenic transcription factors. Thus, co-expression of PPARγ and C/EBPα in cells was to cells to undergo differentiation in the of RA the cells is associated with withdrawal from the cell cycle, we that the of irreversible commitment to adipocyte differentiation exit from the cell cycle as well as the co-expression of both PPARγ and during adipocyte differentiation of wild type 3T3-L1 cells, cells undergo by permanent withdrawal from the cell cycle that at the time RA in preventing differentiation. During this process Rb protein from a state to its We that the state of commitment to adipocyte differentiation only expression of PPARγ and C/EBPα but also hypophosphorylation of Rb and withdrawal from the cell cycle. 3T3-L1 cells were from the were in growth fetal serum in For adipocyte differentiation, after cells as cells were to differentiation fetal of insulin, dexamethasone, and for h. were maintained in fetal and of RA was in and used at a of For adipogenesis, cells were maintained in was added to cells on were to for fat cells were For RA, cells were in the cells were maintained in the of RA from the time of and to the or of at or cells were in the of RA and to RA and The first of these to that previously (13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar). was by of a of mouse the of 1989; Google Scholar). was by of a of C/EBPα of the were To cells, cells were with of and and J. Virol. PubMed Google Scholar). h from the cell were used to 3T3 L1 cells. after cells were in or for For cells were with for h. were in growth were with by in for h at cells were with and with a of in of for at were with for and 3T3-L1 cells were in cell for the and for the and cells were on for by at at for was and protein was by protein of protein was to were to and was to The was first with (23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google for by for h. were and by exposure to were with for h and in and with on for or cells were with and cells were by were with of for h and and in of and on for prior to the addition of of was a and in a During 3T3-L1 differentiation, RA is only effective in preventing adipogenesis when added during the first 24–48 h and when PPARγ has induced S. Lane M.D. J. Biol. Chem. 1997; 272: 5367-5370Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). under circumstances when PPARγ is already RA to the of expression of PPARγ, with its to the of C/EBPβ, which induces PPARγ during adipogenesis Z. Bucher N.L.R. Farmer S.R. Genes 9: 2350-2363Crossref PubMed Scopus (474) Google Scholar, 23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar). To the of RA as a for the of adipocyte differentiation, we first the of RA to the adipogenesis of 3T3-L1 cells that is induced by the induces adipogenesis of 3T3-L1 cells, of cells to differentiate Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). This is by the of an endogenous level of PPARγ in the (23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar) and the of the differentiation and fat cell These were in fat cell differentiation is by RA was to differentiation that induced both PPARγ and C/EBPα and that, with the this was blocked by RA. that express of C/EBPα, to in this as and from for C/EBPβ P. U. Cell. Full Text PDF PubMed Scopus Google inhibits adipogenesis of 3T3 L1 cells. 3T3-L1 maintained in the growth were to of BRL49653, in the or of RA. were on the and to to the expression of PPARγ and The the PPARγ and as well as the of C/EBPα and RA blocked PPARγ by BRL49653, we examined expression of PPARγ be to establish to RA by the of RA on cells that are ectopically as that these cells express In a of cells differentiated fat cells in the of adipogenic stimuli in due to a level of endogenous ligand or of the ectopic PPARγ Ref. M. Reginato M.J. Shao D. Lazar M.A. Chatterjee J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google and The addition of the PPARγ ligand and the differentiation with of cells differentiating fat cells. This is by in In that the adipocyte and were induced However, when maintained in the of RA, cells that were in with or the PPARγ ligand fat cells, both by and C/EBPα expression and The of RA no effect on the ectopic expression of PPARγ and to and These suggest that RA is to adipogenesis induced by ectopic PPARγ protein and expression of PPARγ is to the inhibitory effect of RA on adipogenesis. we that RA blocks the function of PPARγ to fat cell conversion, suggesting that additional may be necessary to be insensitive to RA. we have that adipogenesis in cells that ectopically express C/EBPα or C/EBPβ is also blocked when cells are maintained in the of RA from the of ectopic gene expression and Ref. 13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar). This was in the ectopic cells are to as Interestingly, when RA was added at the of ectopic expression of PPARγ or C/EBPα, but rather at a later time when cells were it was no longer effective at blocking differentiation expression of C/EBPα in the cells is in and The used to ectopically express C/EBPα express the of C/EBPα, (23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar). Thus, expression as a marker of adipogenesis in these F.-T. Lane M.D. Genes 16: 1567-1575Crossref PubMed Google Scholar). C/EBPα (13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar) and C/EBPβ PPARγ Z. Bucher N.L.R. Farmer S.R. Genes 9: 2350-2363Crossref PubMed Scopus (474) Google Scholar, 13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar), we the that PPARγ and C/EBPα may after time to a state that is to RA inhibition of adipogenesis. To PPARγ is to C/EBPα gene cells were prior to differentiation and to that a but level of C/EBPα protein was in This is from wild type 3T3-L1 cells, which express C/EBPα on Thus, ectopic PPARγ expression induced a level of expression of The C/EBPα expression was on with the adipocyte phenotype of these cells. Interestingly, RA blocked expression of C/EBPα expression of the ectopic PPARγ, suggesting that RA inhibits PPARγ of This be with the of that RAR with gene transcription S. J.A. Wang S. J. R.A. Evans R.M. Genes 9: PubMed Scopus Google Scholar). In a we examined the expression of endogenous PPARγ in cells. that endogenous PPARγ was expressed in the whereas expression of PPARγ was in of PPARγ in the cells was by with RA from the time of C/EBPα expression and with the of RA to by C/EBPα (13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar). The that PPARγ and C/EBPα Therefore, it is likely that the of RA to fat cell conversion in or C/EBPα cells when added is due to the coexpression of PPARγ and We the for could be by cells to express both PPARγ and C/EBPα at For these we 3T3 L1 cells with and These cells to growth of C/EBPα in other cell lines M. M. Genes 10: PubMed Scopus Google cells that express only ectopic C/EBPα but to normal ectopically both PPARγ and C/EBPα, suggesting that PPARγ a in 3T3-L1 cells. In the cells C/EBPα and PPARγ differentiated during of prior to However, when cells were and maintained in the of RA, they were to to at normal and no fat cells were in the or of The of RA to differentiation of cells that ectopically express both PPARγ and C/EBPα suggested that expression of these proteins was to establish a commitment state to adipogenesis that is no longer to RA. We that RA is to 3T3-L1 adipogenesis due to C/EBPα and PPARγ, or in that RA is added at the time of gene In RA have an effect when added to cells is associated with withdrawal of cells from the cell cycle and cell proliferation and differentiation are we that both cell cycle and the expression of adipogenic are necessary for the commitment to fat cell differentiation. is that differentiation induces of quiescent 3T3-L1 cells prior to cell cycle withdrawal and of differentiation adipogenic RA blocks differentiation, it the and in cell due to the adipogenic R.L. Bernlohr D.A. Differentiation. 1990; 45: 119-127Crossref PubMed Scopus (72) Google Scholar). the time of the to adipogenic in the and in the of RA, as a of In both a in on and adipogenic Thus, the cells on and These data that, although RA adipocyte differentiation, it the that adipogenic although it to be that in the or of RA, the cells to a quiescent state characterized by However, these are as in The cells are quiescent due to at to the cell cycle. In that differentiated by the in the of RA are and after at These suggest that after the that in the early of adipogenesis, cells exit from the cell cycle. Interestingly, the time after adipogenic at which the cells are to the time at which cells to the effects of RA, and h after the differentiation (23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar). This suggested the role of cell cycle events in adipocyte differentiation. The Rb protein a role in the withdrawal of cells from the cell cycle, and it was that Rb is for adipogenic differentiation in a well characterized model embryonic lung fibroblasts (20Chen P.-L. Riley D.J. Chen Y. Lee W.H. Genes 10: 2794-2804Crossref PubMed Scopus (386) Google Scholar). We examined expression and phosphorylation of Rb protein in 3T3-L1 cells and during differentiation in the and of RA. The in that quiescent 3T3-L1 cells both hyperphosphorylated and of Rb The of Rb protein was during differentiation. to an in of Rb during the first with the process of that be by of adipogenesis as by expression of PPARγ and C/EBPα, the Rb protein to to its corresponding to cells the cell cycle. when adipocyte conversion was Rb protein was hypophosphorylated. The of RA to the time of withdrawal from the cell cycle in addition to the expression of the adipogenic factors PPARγ and C/EBPα that the of RA at the time of adipogenic the phosphorylation state of The of on was with the of cells that Furthermore, in the of RA, Rb was in both and on after exposure to adipogenic with the of adipogenesis, was no of PPARγ or C/EBPα in the of RA These data suggested a terminal differentiation and the hypophosphorylation of Rb with of the role of in RA of cells that ectopically express both PPARγ and C/EBPα, we that irreversible commitment to adipogenic differentiation both the expression of the adipogenic transcription factors and the cell cycle associated with hypophosphorylation of We have used the of RA to adipogenesis as a to explore the molecular events that during the adipogenic differentiation In we were in the observation that at early after exposure of 3T3-L1 cells to adipogenic the cells to the differentiation and no longer to RA. RA was to differentiation in wild type 3T3-L1 cells, as well as adipogenesis due to ectopic expression of C/EBPα, PPARγ, or in the of the PPARγ ligand These suggest that the to RA inhibition that during normal differentiation is due to the expression of PPARγ and However, we have that or cells to the state that after h during wild type 3T3-L1 cell differentiation. with this we found that during 3T3-L1 cell differentiation, at the at which cells normally to the effects of RA, cells are only PPARγ and C/EBPα but are the cell cycle The of RA to adipocyte differentiation that endogenous RAR with the function of PPARγ in addition to its to of (13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar). the of RAR to PPAR be in cells S. J.A. Wang S. J. R.A. Evans R.M. Genes 9: PubMed Scopus Google and data However, during normal adipogenesis, RA is effective at prior to expression of PPARγ, suggesting that its is transcription (13Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar). We have that RA blocks adipocyte differentiation of 3T3-L1 cells when added prior to expression of adipogenic transcription factors and also prevents adipogenesis of cells that express C/EBPα and PPARγ when added prior to Thus, the RAR is (23Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar), a state of commitment to differentiation that is by RA when PPARγ and C/EBPα are expressed and growth cessation has In the of RA, cells that ectopically express PPARγ and C/EBPα undergo adipocyte differentiation cells confluency, that may be necessary for adipogenesis to However, these cells suggesting that expression of adipogenic may to the cessation of cell growth that is also for cells to undergo adipogenic differentiation. PPARγ and C/EBPα of PPARγ and C/EBPα has to have a synergistic effect and promote fat cell differentiation of P. Hu E. Spiegelman B.M. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar) and cell lines (14Hu E. Tontonoz P. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9856-9860Crossref PubMed Scopus (578) Google Scholar). C/EBPα has suggesting that its role in promoting differentiation is at to of cell growth M. M. Genes 10: PubMed Scopus Google Scholar). of C/EBPα in 3T3-L1 cells growth and cells that ectopically expressed both PPARγ and C/EBPα and adipocyte conversion they However, we found that co-expression of these proteins was to cells to undergo differentiation in a that was irreversible by RA the cells were These observations suggest that growth a with the differentiation in cells or During the normal differentiation we found that Rb protein is first hyperphosphorylated and hypophosphorylated. This well with the cell cycle of the cells both during and after this was in also that Rb protein were adipogenesis but that the Rb protein was the differentiation process J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The to be due to the of Rb and the as we have found changes in Rb phosphorylation to The role of Rb in adipogenesis has by other In addition to for the adipogenesis of mouse lung fibroblasts (20Chen P.-L. Riley D.J. Chen Y. Lee W.H. Genes 10: 2794-2804Crossref PubMed Scopus (386) Google Scholar), Rb is necessary for differentiation W. J.W. S. V. Cell. Full Text PDF PubMed Scopus Google Scholar). is evidence that with adipogenic C/EBP proteins P.-L. Riley D.J. S. Lee W.H. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) as well as with the differentiation W. J.W. S. V. Cell. Full Text PDF PubMed Scopus Google Scholar). However, the role of Rb in differentiation be is that cell cycle is a effect of adipogenic differentiation factors expression of C/EBP and PPARγ but the effects of cell growth on the of 3T3-L1 cells to to adipocyte differentiation. suggest that cell cycle is as as the expression of adipogenic in promoting cell differentiation and the irreversible commitment of to this We E. Huang for on the
Shao et al. (Fri,) studied this question.