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• W2022062435 abstract "Cell cycle regulators such as E2F1 and retinoblastoma (RB) play crucial roles in the control of adipogenesis, mostly by controlling the transition between preadipocyte proliferation and adipocyte differentiation. The serine-threonine kinase cyclin-dependent kinase 4 (cdk4) works in a complex with D-type cyclins to phosphorylate RB, mediating the entry of cells into the cell cycle in response to external stimuli. Because cdk4 is an upstream regulator of the E2F-RB pathway, we tested whether cdk4 was a target for new factors that regulate adipogenesis. Here we find that cdk4 inhibition impairs adipocyte differentiation and function. Disruption of cdk4 or activating mutations in cdk4 in primary mouse embryonic fibroblasts results in reduced and increased adipogenic potential, respectively, of these cells. We show that the effects of cdk4 are not limited to the control of differentiation; cdk4 also participates in adipocyte function through activation of PPARγ. Cell cycle regulators such as E2F1 and retinoblastoma (RB) play crucial roles in the control of adipogenesis, mostly by controlling the transition between preadipocyte proliferation and adipocyte differentiation. The serine-threonine kinase cyclin-dependent kinase 4 (cdk4) works in a complex with D-type cyclins to phosphorylate RB, mediating the entry of cells into the cell cycle in response to external stimuli. Because cdk4 is an upstream regulator of the E2F-RB pathway, we tested whether cdk4 was a target for new factors that regulate adipogenesis. Here we find that cdk4 inhibition impairs adipocyte differentiation and function. Disruption of cdk4 or activating mutations in cdk4 in primary mouse embryonic fibroblasts results in reduced and increased adipogenic potential, respectively, of these cells. We show that the effects of cdk4 are not limited to the control of differentiation; cdk4 also participates in adipocyte function through activation of PPARγ. Cell proliferation and differentiation have been considered to be mutually exclusive events; however, a close relationship has been established between both cell processes during the adipocyte differentiation program (reviewed in Fajas, 2003Fajas L. Adipogenesis: a cross-talk between cell proliferation and cell differentiation.Ann. Med. 2003; 35: 79-85Crossref PubMed Scopus (102) Google Scholar). Reentry into the cell cycle is one of the key events taking place in early adipogenesis, since inhibition of DNA synthesis at this stage blocks differentiation (Patel and Lane, 2000Patel Y.M. Lane M.D. Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27.J. Biol. Chem. 2000; 275: 17653-17660Crossref PubMed Scopus (157) Google Scholar, Reichert and Eick, 1999Reichert M. Eick D. Analysis of cell cycle arrest in adipocyte differentiation.Oncogene. 1999; 18: 459-466Crossref PubMed Scopus (95) Google Scholar). Like in most cells, the transition from growth arrested preadipocytes into S phase likely depends on the reactivation of the G1 cyclins/cdks and the retinoblastoma protein RB-E2F pathway that controls the G1/S transition of the cell cycle. Association of E2Fs with proteins of the RB family facilitates active repression through recruitment of histone deacetylases (Brehm et al., 1998Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Retinoblastoma protein recruits histone deacetylase to repress transcription.Nature. 1998; 391: 597-601Crossref PubMed Scopus (1053) Google Scholar, Magnaghi-Jaulin et al., 1998Magnaghi-Jaulin L. Groisman R. Naguibneva I. Robin P. Lorain S. Le Villain J.P. Troalen F. Trouche D. Harel-Bellan A. Retinoblastoma protein represses transcription by recruiting a histone deacetylase.Nature. 1998; 391: 601-605Crossref PubMed Scopus (794) Google Scholar). Upon reentry into cell cycle of these growth-arrested preadipocytes, the members of the retinoblastoma family are phosphorylated by the cyclin/cdk holoenzymes, releasing the E2F complex, resulting in the activation of the E2F target genes (Richon et al., 1997Richon V. Lyle R.E. McGehee R.E.J. Regulation and expression of retinoblastoma proteins p107 and p130 during 3T3-L1 adipocyte differentiation.J. Biol. Chem. 1997; 272: 10117-10124Crossref PubMed Scopus (123) Google Scholar). We have previously shown that cell cycle regulators such as E2F1 and RB play crucial roles in the control of adipogenesis, mostly through the control of the transition between preadipocyte proliferation and adipocyte differentiation. E2F1 positively regulates the expression of peroxisome proliferator-activated receptor γ (PPARγ; Fajas et al., 2002bFajas L. Landsberg R.L. Huss-Garcia Y. Sardet C. Lees J.A. Auwerx J. E2Fs regulate adipogenesis.Dev. Cell. 2002; 3: 39-49Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), which is the master regulator of adipocyte differentiation, whereas RB inhibits PPARγ activity through direct protein-protein interaction (Fajas et al., 2002aFajas L. Egler V. Reiter R. Hansen J. Kristiansen K. Miard S. Auwerx J. The retinoblastoma-histone deacetylase 3 complex inhibits the peroxisome proliferator-activated receptor gamma and adipocyte differentiation.Dev. Cell. 2002; 3: 903-910Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). PPARγ is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. PPARγ is preferentially expressed in the adipose tissue, and upon activation by fatty acids derivatives or antidiabetic thiazolidinediones, PPARγ drives the expression of several adipocyte-specific genes, such as the fatty acid binding protein (aP2; for review see Debril et al., 2001Debril M.B. Renaud J.P. Fajas L. Auwerx J. The pleiotropic functions of peroxisome proliferator-activated receptor gamma.J. Mol. Med. 2001; 79: 30-47Crossref PubMed Scopus (210) Google Scholar). Since cyclin-dependent kinase 4 (cdk4), which mediates the commitment of the cells to enter the cell cycle in response to external stimuli (reviewed in Ortega et al., 2002Ortega S. Malumbres M. Barbacid M. Cyclin D-dependent kinases, INK4 inhibitors and cancer.Biochim. Biophys. Acta. 2002; 1602: 73-87PubMed Google Scholar) is an upstream regulator of the E2F-RB pathway, we hypothesized that cdk4 would be a good target in the search for new factors that regulate adipogenesis. Cdk4−/− mice develop insulin-deficient diabetes due to reduced β cell pancreatic mass. These mice have reduced body weight and are smaller (Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (599) Google Scholar, Tsutsui et al., 1999Tsutsui T. Hesabi B. Moons D.S. Pandolfi P.P. Hansel K.S. Koff A. Kiyokawa H. Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity.Mol. Cell. Biol. 1999; 19: 7011-7019Crossref PubMed Scopus (350) Google Scholar). Furthermore, mice expressing an activating mutation of cdk4 (R24C) that cannot bind the cell cycle inhibitor P16INK4a show pancreatic hyperplasia and increased body weight. We show in this study that cdk4 participates in adipose tissue biology, not only through the control of the clonal expansion phase of adipogenesis but also regulating PPARγ activity and terminal differentiation and function of adipocytes. While evaluating the expression of cell cycle regulators implicated in the transition between preadipocyte proliferation and adipocyte differentiation, we found that cdk4 protein was expressed at similar levels at all stages of differentiation of 3T3-L1 preadipocytes as measured by immunofluorescence assays (Figure 1A ). Interestingly, we observed changes in the cellular localization of cdk4 by confocal microscopy. At confluence (day 0), cdk4 expression was mainly cytoplasmic, whereas differentiated adipocytes (day 5) expressed mainly nuclear cdk4 (Figure 1A). Interestingly, nuclear colocalization of cdk4 and PPARγ could be observed by confocal microscopy in differentiated 3T3-L1 adipocytes (Figure 1B). Differentiation of the cells was quantified by measuring the mRNA expression of the adipogenic marker aP2 (Figure 1C). Similar to 3T3-L1 cells, no significant changes in the expression of cdk4 mRNA were observed during differentiation of primary human preadipocytes, whereas PPARγ expression was increased (Figure 1D). Consistent with these experiments we found that cdk4 was expressed in mature adipocytes of mouse adipose tissue as assessed by immunohistochemical analysis of mouse adipose tissue sections (Figure 1E). These results suggested a role of cdk4 in adipose tissue biology and differentiation. To further assess the participation of cdk4 in differentiated adipocytes, kinase activity experiments were performed. Immunoprecipitated cdk4 from either differentiated 3T3-L1 adipocytes (Figure 1F, lane 2) or freshly prepared primary mice adipocytes (Figure 1F, lane 5) was able to phosphorylate a purified recombinant retinoblastoma protein in vitro, indicating that cdk4 was indeed active in adipocytes (Figure 1F). No phosphorylation of RB was observed when rabbit antiserum was used to immunoprecipitate the extracts (Figure 1F, lanes 3, 4, and 6). Cdk4 was also inactive in quiescent, nondifferentiated 3T3-L1 preadipocytes (Figure 1F, lane 1). Since cdk4 activity is related to the control of cell cycle, we next evaluated DNA synthesis in 3T3-L1 adipocytes by BrdU incorporation assays. As expected, most of cdk4-expressing 3T3-L1 preadipocytes incorporated BrdU, indicating that these cells were proliferating (Figure 1G). In contrast, only a small proportion of cdk4-expressing 3T3-L1 adipocytes were positive for BrdU incorporation (Figure 1G), demonstrating that cdk4 was expressed in nonproliferating cells and suggesting that the detected cdk4 activity in differentiated cells could be independent of the control of cell cycle (Figures 1F and 1G). Participation of cdk4 in adipogenesis was further studied. Differentiation of 3T3-L1 cells was induced with a typical hormonal mix either in the absence or in the presence of the specific cdk4 inhibitor 2-Bromo-12,13-dihydro-5H-indolo(2,3-a)pyrrolo (3,4)carbazole (IDCX) (Zhu et al., 2003Zhu G. Conner S.E. Zhou X. Shih C. Li T. Anderson B.D. Brooks H.B. Campbell R.M. Considine E. Dempsey J.A. et al.Synthesis, structure-activity relationship, and biological studies of indolocarbazoles as potent cyclin D1–CDK4 inhibitors.J. Med. Chem. 2003; 46: 2027-2030Crossref PubMed Scopus (102) Google Scholar). Oil Red O staining indicated that IDCX was a potent inhibitor of 3T3-L1 adipocyte differentiation (Figure 2A ). This was consistent with a decrease in the expression of the adipogenic markers PPARγ and aP2 in the presence of the inhibitor (Figure 2B). Similar results were observed when the cdk4 inhibitor I3M was used (data not shown). The inhibition of cdk4 kinase activity in these cells was demonstrated by in vitro kinase assays (data not shown). After hormonal induction, 3T3-L1 preadipocytes reenter the cell cycle before terminally differentiating into adipocytes. This clonal expansion phase (days 1 and 2) is required for the differentiation process of the 3T3-L1 cells. It would be therefore plausible that cdk4 participates in adipogenesis by inducing the proliferative clonal expansion phase. This was consistent with the observation that cdk4 inhibition with IDCX abrogated the clonal expansion of hormonally induced 3T3-L1 cells as analyzed by BrdU incorporation experiments (Figure 2C, day 1). Furthermore, the expression of the cell cycle markers cyclin E and cyclin B was decreased in the presence of IDCX, consistent with the BrdU incorporation experiments (Figure 2D). To further elucidate whether participation of cdk4 was limited to the control of the proliferative phase of the adipocyte differentiation process, we inhibited cdk4 activity with IDCX after completion of clonal expansion at day 2. Interestingly, adipocyte differentiation was also inhibited under these conditions as assessed by Oil Red O staining (Figure 2E) and by the expression of adipocyte marker genes (Figure 2F). BrdU incorporation studies demonstrated that the cells were not proliferating at the time of IDCX incubation (Figure 2G). These results suggested that active cdk4, in addition to its role in the control of the cell cycle during the clonal expansion phase, is also required during terminal differentiation in a cell cycle-independent manner. A second cellular model was next used to further demonstrate the implication of cdk4 in adipocyte differentiation. F442A cells were induced to differentiate into adipocytes in the absence or in the presence of IDCX. Lipid incorporation (data not shown), as well as the expression of adipocyte markers, indicated that cdk4 inhibition resulted in decreased adipocyte differentiation when compared with F442A differentiated in the absence of IDCX (Figure 2H). As for 3T3-L1 cells, F442A preadipocytes reentered the cell cycle after insulin induction of differentiation. Surprisingly, IDCX did not prevent reentry into the cell cycle of differentiating cells as measured by BrdU incorporation (Figure 2I) and expression of cyclin E and cyclin B (Figure 2J). Yet, IDCX decreased differentiation to a similar level to that observed in 3T3-L1 cells, suggesting that, in F442A cells, cdk4 inhibition prevents adipogenesis independently of the control of the cell cycle. To test the hypothesis that cdk4 is a positive factor for adipocyte differentiation, a 3T3-L1 stable cell line constitutively expressing cdk4 was generated by retroviral infection (pBabe-cdk4). Oil Red O staining indicated an increased capacity to differentiate into adipocytes of pBabe-cdk4 cells compared to 3T3-L1 cells infected with an empty retrovirus (pBabe) 4 days after induction of adipogenesis (Figure 3A ). Overexpression of cdk4 could not bypass, however, the requirement of differentiation medium in order to differentiate (data not shown). Interestingly, 3T3-L1 cells infected with a retrovirus expressing a kinase-dead cdk4 mutant (pBabe-K35M) lost their capacity to differentiate (Figure 3A), suggesting that the positive effects on adipogenesis are mediated by the kinase activity of cdk4. Cdk4 protein was highly expressed, as expected, in pBabe-cdk4 and pBabe-K35M cells, compared to pBabe 3T3-L1 cells (Figure 3B). Consistent with the Oil Red O assays, real-time PCR analyses showed increased levels of aP2 up to 2-fold in pBabe-cdk4-3T3-L1 cells, whereas no such increase was observed in cells infected with pBabe-K35M (Figure 3B). Mice deficient for cdk4 have decreased body weight (Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (599) Google Scholar, Tsutsui et al., 1999Tsutsui T. Hesabi B. Moons D.S. Pandolfi P.P. Hansel K.S. Koff A. Kiyokawa H. Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity.Mol. Cell. Biol. 1999; 19: 7011-7019Crossref PubMed Scopus (350) Google Scholar), decreased fat mass, and smaller adipocytes (our unpublished data). In contrast, mice expressing a hyperactive cdk4 mutant have increased weight (Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (599) Google Scholar), increased fat mass, and bigger adipocytes (our unpublished data). The phenotypes of cdk4−/− and cdk4R24C mice suggest a defect in adipose tissue homeostasis. However, cdk4−/− mice are hypoinsulinemic as a result of diabetes, and cdk4R24C mice develop pancreatic islet β cell hyperplasia with increased production of insulin. It could be therefore possible that the effects on adipogenesis in these mice are secondary to insulin signaling and not directly related to adipose tissue development. To test this notion, we compared the capacity of cdk4−/− or cdk4R24C primary isolated MEFs to differentiate into adipocytes in vitro in response to hormone stimulation. Adipocytes were scored using Oil Red O staining to detect lipid droplets and mRNA expression of adipogenic markers was quantified. Consistent with a direct effect of cdk4 in adipogenesis, the capacity of cdk4−/− MEFs to differentiate into adipocytes was totally inhibited (Figure 3C). Conversely, hormonally stimulated cdk4R24C MEFs showed a robust increase in their capacity to differentiate into adipocytes in vitro when compared to wild-type MEFs (Figure 3C). Furthermore, when expression of cdk4 was rescued by retroviral infection, cdk4−/− MEFs could normally differentiate, demonstrating that inhibition of differentiation in cdk4−/− MEFs was directly the result of the lack of cdk4 (Figure 3C). Reexpression of cdk4 in cdk4−/− MEFs was verified by QPCR analysis (Figure 3D). Gene expression analysis of the adipose tissue-specific gene aP2 further demonstrated the decreased and increased number of cdk4−/− and cdk4R24C MEF-derived adipocytes, respectively, as well as the increased number of adipocytes in rescued cdk4−/− MEFs (Figure 3E). Taken together, these data suggest that cdk4 directly stimulates adipogenesis. In addition to its participation in adipocyte differentiation, we next wanted to analyze whether cdk4 have a role in adipocyte biology. First, we performed glucose uptake experiments in fully differentiated 3T3-L1 adipocytes in response to cdk4 inhibitors. Twenty-four hour IDCX treatment inhibited in a dose-dependent manner the incorporation of radioactive glucose into adipocytes in response to insulin (Figure 4A ). Interestingly, basal glucose uptake was increased upon incubation of high doses of IDCX when compared to cells treated with vehicle only (Figure 4A). To exclude the possibility that the observed differences in the response of the cells to insulin were the result of a distinct stage of differentiation of the cells, the expression of aP2 mRNA was measured, and no differences were found (Figure 4B). Consistent with decreased insulin sensitivity, the expression of genes such as Glut-4, insulin receptor (IR), insulin receptor substrate-1 (IRS1), IRS2, and PI3K, which are key proteins in glucose transport and glucose homeostasis, was also decreased in the presence of IDCX (Figure 4C). Strikingly, Glut-1 mRNA expression was increased upon incubation with IDCX, which was consistent with the increased basal glucose uptake observed under these conditions. Finally, the expression of genes implicated in lipogenesis was analyzed. Fatty acid synthase (FAS) and phosphoenol pyruvate carboxy kinase (PEPCK) mRNA expression was decreased in IDCX-treated adipocytes (Figure 4D), which was consistent with decreased lipid load in these cells. No changes in the expression of genes implicated in lipolysis was observed in these cells (data not shown). These results suggested that cdk4 inhibition impaired adipocyte function decreasing lipogenesis and glucose transport and metabolism in these cells. PPARγ plays a crucial role in the adipocyte differentiation process. Any factor modulating PPARγ activity has major effects on adipogenesis. We therefore tested the ability of cdk4 to activate PPARγ in transient transfection experiments in COS cells, using a PPARγ-responsive luciferase reporter (PPRE-TK-Luc) and PPARγ and cdk4 expression vectors. A 3-fold induction of luciferase activity was observed upon transfection of limiting concentrations of PPARγ in the presence of pioglitazone. This induction was significantly increased up to 6-fold by cotransfection of cdk4 (Figure 5A ), suggesting that cdk4 activates PPARγ. Interestingly, the cdk4-K35M kinase-dead mutant was not able to increase PPARγ activity (Figure 5A), which was consistent with the lack of effects of this mutant on adipogenesis (Figure 3A). Furthermore, cdk4 inactivation by the specific cdk4 inhibitor IDCX resulted in the attenuation, in a dose-dependent manner, of PPARγ activation by pioglitazone (from 3- to 1-fold; Figure 5B). Consistent with these results, we found that either pioglitazone or rosiglitazone treatment of cdk4−/− MEFs in which cdk4 expression was rescued by retroviral infection resulted in a 3-fold induction of the PPARγ target aP2 mRNA, whereas only a 1.5-fold induction was observed when cdk4−/− MEFs infected with empty retrovirus were treated (Figure 5C, left panel). PPARγ was equally expressed in both cell lines (Figure 5C, right panel). Next, to test whether the increase in PPARγ activity in the presence of cdk4 was the consequence of an interaction with PPARγ, total cell extracts from PPARγ- and cdk4-transfected COS cells were immunoprecipitated with an anti-PPARγ antibody. A 33 kDa protein was recognized in the immunoprecipitates by an anti-cdk4 antibody, indicating that cdk4 interacted in vivo with PPARγ (Figure 5D). Furthermore, when extracts from cdk4-expressing differentiated 3T3-L1 adipocytes were immunoprecipitated using an anti-PPARγ antibody, endogenous cdk4 protein was associated to PPARγ (Figure 5E). Deletion experiments in the cdk4 protein indicated that the K35 amino acid in the ATP binding domain of cdk4 was not implicated in the PPARγ interaction. However, deletion of aa 203–295 abolished binding to PPARγ (Figure 5F). Interestingly, GST pull-down experiments indicated that cdk4 is able to bind both, the AB domain of PPARγ and the DEF domain, which contain the ligand-independent and the ligand-dependent transactivation domains, respectively (Figure 5G). Finally, to further prove that cdk4 is associated with PPARγ and that it activates PPARγ-mediated transcription, chromatin immunoprecipitation studies of the aP2 promoter were performed in both nondifferentiated and differentiated 3T3-L1 adipocytes. A 200 bp fragment of the mouse aP2 promoter containing the binding site of PPARγ was amplified by PCR when anti-cdk4, anti-PPARγ, or anti-acetylated histone H4 antibodies were used to immunoprecipitate chromatin from differentiated 3T3-L1 cells (Figure 5H). No amplification product was observed when immunoprecipitated chromatin from confluent, nondifferentiated 3T3-L1 preadipocytes was used as template (data not shown). The results of the ChIP assays demonstrate that the complex cdk4-PPARγ is present in the promoter of PPARγ target genes. Moreover, the presence of acetylated histone H4 on the PPARγ binding site of the aP2 promoter suggests that in the presence of cdk4 this promoter is active. These results suggest that the positive effects of cdk4 on adipogenesis are the result of cdk4-mediated increase of PPARγ activity through cdk4 kinase activity. Cdk4 is the catalytic subunit of the cyclin D-cdk holoenzyme. The kinase activity of this complex is induced in response to extracellular signals, including growth factors, and translates signals from extracellular environment into cell cycle activation (Matsushime et al., 1991Matsushime H. Roussel M.F. Ashmun R.A. Sherr C.J. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle.Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (985) Google Scholar). We have recently shown that the transcription factor E2F1, which is the effector of the cyclin/cdk pathway in cell cycle regulation, mediates the transition between preadipocyte proliferation and adipocyte differentiation through activation of the expression of PPARγ, which is the master regulator of adipogenesis. The most studied role of cdk4 is phosphorylation of the retinoblastoma family proteins that repress E2F1 activity, thereby facilitating the release of E2F1 from this complex, resulting in the activation of E2F1 target genes. Since cdk4 is an upstream regulator of the E2F1/RB pathway, it is therefore likely that cdk4 also participates in adipocyte differentiation. Consistent with this, we found that cdk4 activity is required for adipogenesis. We propose that cdk4 plays a dual role in this process (Figure 6). On the one hand, cdk4 participates in the clonal expansion phase of adipocyte differentiation phosphorylating RB and therefore facilitating activation of E2F complexes, which will trigger the transcription of PPARγ and likely other factors implicated in terminal differentiation. This is so far consistent with the known functions of cdk4. On the other hand, we found surprisingly that participation of cdk4 was not limited to the control of cell cycle and E2F1 activity in the proliferative phase of adipocyte differentiation, but we found that cdk4 has also a positive, cell cycle-independent role in terminal differentiation and function of adipocytes. This is supported by three main observations. First, cdk4 is expressed in fully differentiated adipocytes in both human and mouse adipose tissue (Figure 1; Phelps and Xiong, 1998Phelps D.E. Xiong Y. Regulation of cyclin-dependent kinase 4 during adipogenesis involves switching of cyclin D subunits and concurrent binding of p18INK4c and p27Kip1.Cell Growth Differ. 1998; 9: 595-610PubMed Google Scholar). These differentiated adipocytes do not express E2F1 (Fajas et al., 2002bFajas L. Landsberg R.L. Huss-Garcia Y. Sardet C. Lees J.A. Auwerx J. E2Fs regulate adipogenesis.Dev. Cell. 2002; 3: 39-49Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar) and are not proliferating (Figure 1). Furthermore, cdk4 activity can be detected in differentiated 3T3-L1 adipocytes and in mouse adipose tissue (Figure 1; Phelps and Xiong, 1998Phelps D.E. Xiong Y. Regulation of cyclin-dependent kinase 4 during adipogenesis involves switching of cyclin D subunits and concurrent binding of p18INK4c and p27Kip1.Cell Growth Differ. 1998; 9: 595-610PubMed Google Scholar). Second, inhibition of cdk4 activity with specific cdk4 inhibitors impairs adipocyte differentiation regardless of whether the inhibitor is added before or after the proliferative phase of adipogenesis (Figure 2). Moreover, cdk4 inhibitors do not block the proliferative phase of F442A preadipocyte differentiation process and yet inhibit adipogenesis in this system. We cannot explain, at present, why IDCX did not affect the clonal expansion phase of F442A cells. Finally, cdk4 interacts with PPARγ, resulting in increased transactivation activity of PPARγ (Figure 5). Furthermore, cdk4 can be found in the PPARγ-responsive element of the aP2 promoter (Figure 5). We can conclude from our results that cdk4 not only regulates adipocyte differentiation, but also participates in adipocyte biology. Cdk4 might act as a sensor of homeostatic signals. Nutrition and the subsequent hormonal signaling, such as insulin, results in the activation of preadipocyte differentiation and lipogenic pathways in already preexisting adipocytes. In support of this hypothesis, the experiments in F442A cells show that inhibition of cdk4 in these cells results in decreased lipogenesis rather than differentiation. As a proof of the participation of cdk4 in adipocyte biology, we observed decreased insulin sensitivity, reduced glucose uptake, and decreased expression of genes implicated in lipogenesis and insulin signaling such as IRS1 and 2, Glut-4, PI3K, FAS, or PEPCK in 3T3-L1 adipocytes in which cdk4 activity was inhibited. In contrast with this, we found that cdk4 inhibition resulted in increased Glut-1 mRNA expression and enhanced basal glucose uptake in 3T3-L1 adipocytes. We cannot explain at present how cdk4 regulates basal glucose uptake, and further studies are required. The role of cdk4 as a growth signal sensor is underscored in cdk4−/− mice, which have impaired postnatal pancreatic β cell growth resulting in severe atrophy and diabetes (Mettus and Rane, 2003Mettus R.V. Rane S.G. Characterization of the abnormal pancreatic development, reduced growth and infertility in Cdk4 mutant mice.Oncogene. 2003; 22: 8413-8421Crossref PubMed Scopus (78) Google Scholar, Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (599) Google Scholar). Furthermore, cdk4−/− and D-type cyclin−/− mice show reduced body size, stressing the importance of cyclin D/cdk4 complex in the control of tissue growth (Kozar et al., 2004Kozar K. Ciemerych M.A. Rebel V.I. Shigematsu H. Zagozdzon A. Sicinska E. Geng Y. Yu Q. Bhattacharya S. Bronson R.T. et al.Mouse development and cell proliferation in the absence of D-cyclins.Cell. 2004; 118: 477-491Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, Mettus and Rane, 2003Mettus R.V. Rane S.G. Characterization of the abnormal pancreatic development, reduced growth and infertility in Cdk4 mutant mice.Oncogene. 2003; 22: 8413-8421Crossref PubMed Scopus (78) Google Scholar, Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (599) Google Scholar). In contrast, cdk4R24C mice have increased body weight (Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (599) Google Scholar). Cdk4R24C mice express a mutant cdk4 protein (R24C) which is not inhibited by the cdk inhibitor p16, rendering cdk4 hyperactive (Zuo et al., 1996Zuo L. Weger J. Yang Q. Goldstein A.M. Tucker M.A. Walker G" @default.
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• W2022062435 date "2005-10-01" @default.
• W2022062435 modified "2023-10-13" @default.
• W2022062435 title "Cdk4 promotes adipogenesis through PPARγ activation" @default.
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