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Transcriptional control of adipogenesis
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Transcriptional control of adipogenesis Lluis Fajas, Jean-Charles Fruchart and Johan Auwerx
Adipocyte differentiation is coordinatedly regulated by several transcription factors. C/EBPI3, C/EBP8 and ADD-1/SREBP-1 are active early during the differentiation process and induce the expression and/or activity of the peroxisome proliferator activated receptor-y (PPARy), the pivotal coordinator of the adipocyte differentiation process. Activated PPARy induces exit from the cell cycle and triggers the expression of adipocyte-specific genes, resulting in increased delivery of energy to the cells. C/EBPc~, whose expression coincides with the later stages of differentiation, cooperates with PPAR7 in inducing additional target genes and sustains a high level of PPARy in the mature adipocyte as part of a feedforward loop. Altered activity and/or expression of these transcription factors might underlie the pathogenesis of disorders characterized by increased or decreased adipose tissue depots.
Addresses INSERM U 325, D6partement d'Ath~roscl~rose, Institut Pasteur, 1 Rue Calmette, F-59019 Lille, France Correspondence: Johan Auwerx: e-mail: [email protected] Current Opinion in Cell Biology 1998, 10:165-173 http://biomednet.com/elecref/095506 7401000165
© Current Biology Ltd ISSN 0955-0674 Abbreviations ADD adipose differentiation and determination factor bHLH basic helix-loop-helix C/EBP CCAAT-enhancer-binding protein FAS fatty acid synthase GR glucocorticoid receptor LPL lipoproteinlipase PKB proteinkinase B PKC~] protein kinase Crl PPAR peroxisomeproliferator activated receptor RA retinoic acid RAR RA receptor Rb retinoblastoma RXR retinoicX receptor SREBP sterol regulatory binding protein STAT signaltransducer and activator of transcription TNF tumor necrosis factor WAT white adipose tissue
Introduction White adipose tissue (WAT) is mainly composed of adipocytes, cells that store energy in the form of triglycerides in times of nutritional affluence and release free fatty acids during nutritional deprivation [1]. WAT mass is determined by the balance between energy intake and expenditure and is controlled by genetic neuroendocrine and environmental factors [1]. Perturbances of this steady state can lead to increased or decreased amounts of
WAT, such as seen, respectively, in obesity or in the lipodystrophic syndromes [1]. Obesity affects between 25% and 30% of the population in industrialized countries and its frequency is still rising [2,3]. It is an independent risk factor for insulin resistance, non-insulin-dependent diabetes mellitus and coronary artery disease. Conversely, decreased amounts of adipose tissue, such as seen in malnutrition, cachexia and anorexia nervosa are also associated with significant levels of morbidity and mortality. Unlike brown adipose tissue, WAT is not present during embryonic life [4] and the adipocyte lineage derives from an embryonic stem cell precursor which has the capacity to differentiate to mesodermal cell types such as adipocytes and myocytes (reviewed in [5-7]). Alteration in WAT mass can involve alterations in both adipocyte volume and number. Increased adipocyte number is the result of a continuous and highly regulated differentiation process, whereas decreased adipocyte number is the consequence of apoptosis and adipocyte dedifferentiation (for review see [8]). This review will be limited to intrinsic regulatory mechanisms determining adipocyte differentiation, which are coordinated by transcription factors. For more general information on adipose tissue biology and obesity we refer the reader to some recent reviews [2-4,8-12]. Cell culture models, like the 3T3-L1 [13] or ob 1771 [14] immortalized mouse preadipocyte cell lines, have been instrumental in the understanding of adipocyte differentiation. The first step of adipocyte differentiation in these models involves arrest of cell proliferation [15]. At this stage, when appropriately induced with hormonal agents, preadipocytes differentiate into adipocytes, in a process orchestrated by a set of interdependently acting transcription factors, including peroxisome-proliferator-activated receptors (PPARs) [16,17], the CCAAT-enhancerbinding proteins (C/EBPs) [18-20], and the adipocyte differentiation and determination factor (ADD)-I, also known as the sterol regulatory clement binding protein (SREBP)-I [21,22°°]. Together these transcription factors stimulate the expression of adipocyte-specific genes such as those for lipoprotein lipase (LPL), leptin and the fatty acid binding protein, aP2 (reviewed in [23]), thereby leading to the characteristic phenotype of the mature adipocyte (Figure 1). These changes in gene transcription are tightly coordinated with changes in the cell cycle. Efforts to identify the master regulators triggering adipocytc differentiation arc driven by the assumption that controlling or reversing adipocyte differentiation might ultimately lead to a way to cure obesity and associated pathologies.
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Figure 1
Adipocyte differentiation
P Cell proliferation
Cell contact Growth arrest
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A scheme illustrating the expression of some regulatory proteins and target genes (indicated in italics) during the different stages of adipocyte differentiation.
PPARs, C / E B P s and A D D - 1 / S R E B P - 1 , the three tenors that orchestrate a d i p o g e n e s i s Peroxisome proliferator-activated receptors (PPARs) Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. After dimerization with the retinoic X receptor (RXR) and ligand activation, they control the expression of genes containing specific response elements called PPREs (reviewed in [24-28]). One of the PPARs, PPARy was elegantly shown by Tontonoz et al. to be a key player in adipocyte differentiation [16,17]. Indeed, infection of fibroblast [16] and muscle [29] cells with a retroviral vector expressing PPARy could induce adipocyte differentiation. Initially two different PPARy isoforms, PPARy1 and PPARy2, were identified [30], both derived from the same gene by alternative promoter usage and splicing. PPARy2 is identical to PPARy1 except for 28 additional amino acid at its amino-terminus [30,31°,32-34]. Recently, a third PPARy m R N A , PPARy3, was identified. PPARy3 mRNA produces a protein identical to PPARy1, but is transcribed from an independent promoter region, which could control the regulation of the entire PPARy locus (L Fajas and J Auwerx, unpublished data). So far, little is known about PPARy3 expression, PPARy1 seems ubiquitously expressed, whereas PPARy2 expression is mainly restricted to adipose tissue, suggesting that the PPARy2 form is most important for adipocyte physiology [31°,35]. A recent report that the anaino-terminus of PPARy2 enhances the activity of the ligand-independent transactivation domain, making PPARy2 a better transactivator ([361; L Fajas and
J Auwerx, unpublished data), supports the importance of PPARy2 relative to PPARy1 in regulating gene expression T h e transcriptional activity of the PPARy protein can be modulated by the binding of ligands, by posttranslational modifications and by interaction with other nuclear receptors and various cofactors. Thiazolidinedione antidiabetic agents [37] and prostaglandin J2 derivatives [38,39], which are synthetic and natural ligands of PPARy respectively, all facilitate adipogenesis. T h e fact that the insulin-sensitizing thiazolidinediones are potent PPARy activators suggests that this receptor has a key role not only in adipocyte biology but also in glucose homeostasis (reviewed in [9,28]). In addition to regulation by ligands, PPARy activity is also controlled by post-translational modifications [40°°,41,42]. MAP kinase-mediated phosphorylation of PPARy leads to its inactivation, which could underlie the well known inhibitory effects of growth factors and certain cytokines on adipogenesis [40°°,41,42]. However, other authors have not confirmed this observation [43]. Interaction with corepressors and coactivators, which form a bridge between nuclear receptors and the basal transcription machinery and influence chromatin structure, is another important way of modulating their transcriptional activity (for review see [44]). Recently, it was demonstrated that the transcriptional activity of PPARy was enhanced by ligand-dependent interactions of the receptor with the cofactors steroid receptor coactivator-1 (SRC-1) [45 °°] and p300 (L Gelman, L Fajas, J Auwerx, unpublished data). Furthermore,
Transcriptional control of adipogenesis Fajas, Fruchart and Auwerx
Spiegelman's group has demonstrated that interaction with PGC-1, a novel cofactor, could enhance PPARy transcriptional activity specifically in brown adipose tissue (personal communication). Finally, PPARy activity can be modulated by its absolute requirement for RXR in order to bind to DNA (Figure 2). On a DR-1 response element (such as a PPRE), RXR is a nonpermissive inactive partner in heterodimers with RAR, whereas it is a permissive partner in the PPAR-RXR heterodimer. Because the levels of RAR and PPARy change considerably during adipocyte differentiation, a switch in partners by RXR will result in differential recruitment of corepressors and coactivators to the DR-1 element, which is ultimately translated into an alteration of the activity of the gene from repressed to activated (Figure 2) [45°°]. Such a switch might underlie the mechanism by which not only PPARy agonists, but also RXR agonists, exert their effects on adipogenesis and glucose homeostasis. Figure 2
I Coactivator 1
PPAR
DR-1
Active
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Inactive CurrentOpinionin CellBiology
PPAR-RXR and RAR-RXR heterodimers respectively activate and repress DR-l-containing promoters. When the RAR-RXR heterodimer is bound to a DR-1 site, RAR blocks the binding of ligands to RXR, keeping the heterodimer in an inactive state due to the binding of corepressors such as N-CoR or SMRT. In contrast, the PPAR-RXR heterodimer configuration permits ligand binding to RXR, allowing for activation of both partners in the PPAR-RXR heterodimer.
Although the mechanisms by which PPAR7 activation drives adipogenesis are not fully elucidated, one particular feature is that it induces the formation of small-sized adipocytes [46]. T h e enhanced adipocyte differentiation most likely involves the regulation of adipocyte-specific gene expression. Indeed, PPARy controls the expression of several crucial adipocyte genes, including those for LPL, acyl coenzyme A synthase, fatty acid synthase (FAS) and phospho-enol pyruvate carboxykinase, which are all involved in coordinating fatty acid uptake and storage (reviewed in [23,28]). Furthermore, PPAR¥ decreases the expression of the adipocyte-derived signaling molecule leptin, which results in an increase in energy intake
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and optimization of energy usage, contributing further to PPARy's adipogenic effect [47-50]. PPARy-mediated activation of adipocyte gene expression and differentiation most probably also underlies its antidiabetic effects. Indeed the low level of expression of PPARy in muscle, the main tissue for glucose metabolism, makes a direct effect of PPARy activators in this tissue unlikely and suggests rather that they modify the production of adipocyte-derived mediators of insulin resistance, such as free fatty acids or the cytokine tumor necrosis factor (TNF) cc PPAR 7 activation will decrease production of TNF(x by adipocytes [51,52], reduce TNF's antidifferentiation effects [52-54] and interfere with its inhibitory effect on insulin signaling [55°']. In addition, because of its tissue-selective effects on genes involved in fatty acid uptake, PPARy activation will induce repartitioning of fatty acids in the body, with enhanced accumulation of fatty acids in adipose tissue at the expense of a relative depletion of muscle fatty acids [56°°]. T h e relative lipid depletion of muscle cells will improve their glucose metabolism and result in an improvement in insulin sensitivity [57].
The C/EBP family of transcription factors Three members of the C/EBP family of transcription factors--C/EBPct, 13 and ~i- - although not specific for the adipocyte lineage, are expressed at defined times during adipogenesis, and each has a specific regulatory role. Adipogenic hormones such as glucocorticoids, cyclic AMP (cAMP) and insulin induce a transient increase in the expression of C/EBP13 and 8 early in adipocyte differentiation [58-60]. C/EBPI3, in synergy with C/EBP~5, then induces PPARy expression in the preadipocyte [61,62°], subsequently triggering full-blown adipocyte differentiation. Supporting this idea is the fact that there are at least two binding sites for C/EBP in the human PPARy2 promoter [30,31°].
In contrast to the early effects of C/EBPI3 and 8 on PPARy expression and adipocyte differentiation, C/EBPct seems to play an important part in the later stages of differentiation by sustaining high levels of PPARy expression and by maintaining the differentiated adipocyte phenotype. Several lines of evidence support an important role for C/EBP(x in adipocyte differentiation. First, temporal activation of C/EBPc~ expression occurs immediately before the coordinate expression of a group of adipocyte-specific genes, suggesting its involvement in their regulation [18]. Second, antisense C/EBP(x RNA can inhibit adipocyte differentiation [63]. Third, premature induction or overexpression of C/EBPct triggers adipocyte differentiation [20,63]. And finally, adipocytes from mice in which the C/EBP(x gene was disrupted by homologous recombination failed to accumulate lipids in adipose tissue [64].
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The ADD-1/SREBP-1 family of transcription factors ADD-1 and SREBP-1, which are rodent and human homologs, were independently cloned and characterized as transcription factors implicated, respectively, in the control of adipocyte differentiation [21] and in cholesterol control of gene expression [65]. T h e protein is synthesized as a membrane-bound precursor localized in the nuclear envelope and the endoplasmic reticulum. Upon sterol depletion the precursor is proteolytically cleaved to generate a soluble amino-terminal fragment, which contains an acidic activation domain and basic helix-loop-helix (bHLH) region mediating protein dimerization and DNA binding (reviewed in [66]). This mature form translocates to the nucleus and has a dual DNA-binding specificity. It can bind either to a cis element containing variations on the core sequence C A N N T G (where N represents any of the four nucleotides), also referred to as the E box, which is the b H L H consensus response element, or to a sequence motif identified as the sterol regulatory element (SRE). T h e expression of ADD-1/SREBP-1, is induced during differentiation of adipocytes, where it activates transcription of target genes involved in both cholesterol metabolism and fatty acid metabolism [22°']. Direct evidence of the adipogenic effect of ADD1/SREBP-1 was provided by its capacity to induce fat accumulation and adipocyte differentiation when overexpressed in fibroblasts with a retroviral vector [22°°]. The adipogenic effect of ADD-1/SREBP-1 is enhanced by PPARy and its activators [22°°]. Furthermore, overexpressing SREBP-1 in the liver of transgenic animals resulted in a massive fat accumulation in the liver subsequent to the induction of genes involved in lipid uptake and biosynthesis [67]. Part of the adipogenic effect of ADD-1/SREBP-1 might involve direct activation of the expression of PPARy through interaction with a response element in the PPARy3 promoter (L Fajas and J Auwerx, unpublished data). In addition, SREBP-1/ADD-1 controls the expression of genes involved in lipid metabolism, such as those for L P L [22"°,67], acetyl coenzyme A carboxylase [68], and FAS [22°*,67,69], which could lead to the generation of natural activators and ligands for PPARy. O t h e r factors i n v o l v e d in a d i p o g e n e s i s In addition to the proteins discussed above, recent preliminary evidence implicates other factors in adipogenesis (reviewed in [23]). For all of these factors, however, further studies are required to identify the molecular pathways in which they operate. Signal transducers and activators of transcription T h e signal transducer and activator of trascription (STAT) family of transcription factors consists of six members that are phosphorylated in response to various cytokines and peptide hormones (reviewed in [70,71]). After phosphorylation and translocation to the nucleus, STATs regulate the transcription of specific target genes. Recently it has
been shown that three members of the STAT family, STAT-1, -3 and -5 are strongly induced during adipocyte differentiation in a manner similar to C/EBPc~ and PPARy [72°]. Furthermore, inhibition of adipogenesis by TNFc~ correlates with the repression of STATs in 3T3-L1 cells [72°]. Which signaling molecules and receptors are involved in the activations of these STATs is currently unknown. Adipose tissue, however, contains several receptors of the cytokine receptor superfamily, such as the growth hormone receptor and the prolactin receptor, both of which are induced during differentiation [73,74] and which have been shown to control the expression of adipocyte genes such as LPL [75,76]. Altogether, these data suggest that cytokines and peptide hormones, through their effects on STAT-mediated gene expression could have a significant role in the development and/or maintenance of the adipose phenotype. HMGI-C
T h e chromosomal protein HMGI-C belongs to the high mobility group family of architectural DNA-binding proteins that are abundant, heterogeneous nonhistone components of chromatin [77]. Although they do not possess transcriptional activity by themselves, they change the conformation of DNA and may hence influence transcription. Support for the involvement of HMGI-C in adipogenesis is provided by the observation that the mouse mutant pygmy, characterized by its small size and disproportionally reduced body fat content, was found to be a null allele of the HMGI-C gene [78]. Further evidence comes from the finding that in certain lipomas gene rearrangements were found in which the HMGI-C DNA-binding domain was fused to either a LIM domain or an acidic transactivation domain [79,80]. In the first case, the LIM domain of the translocation partner could recruit transcriptional activators to the DNA site, whereas in the second case, the fusion of the HMGI-C protein to an acidic transactivation domain could turn the chimeric protein into a powerful transcriptional activator. Thus, the lack or reduced expression of HMGI-C could predispose to leaness, whereas the juxtaposition of its DNA-binding motif to transcriptional regulatory domains could promote adipogenesis. Glucocorticoid receptor Activated glucocorticoid receptor (GR) is thought to have multiple pro-adipogenic effects, several of them enhancing PPARy activity. First, activated GR will increase the expression of early adipogenic genes, including those for the C/EBPs [58-60], which in their turn will switch on the PPARy gene [62°]. It has also been suggested that GR might directly stimulate the expression of PPARy [62°], although further studies are required to elucidate whether there is a functional binding site for GR in the PPAR7 promoter. In addition, glucocorticoids are known to activate phospholipase A2, hence stimulating the production of prostanoid ligands of PPARy [81].
Transcriptionalcontrol of adipogenesis Fajas, Fruchart and Auwerx
Protein kinase B and protein kinase Crl Protein kinase B (PKB or Rac), a serine/threonine kinase which is activated by insulin, has recently been shown to be involved in adipogenesis. As differentiation of 3T3-L1 preadipose cells into adipocytes is dependent on insulin it was postulated that PKB was responsible for transducing the insulin signal to downstream proteins. Experiments in which an ectopic and constitutively activated form of PKB was expressed in 3T3-L1 preadipocytes showed that these cells spontaneously differentiated into adipocytes, pointing to a crucial role of this kinase in differentiation [82]. PKB might achieve this adipogenic effect by activation of pT0 $6 kinases which in previous studies were shown to be the target of rapamycin-mediated inhibition of adipogenesis [83]. Another protein kinase with a potential role in adipocyte differentiation is protein kinase Clq. Overexpression of PKCrl in quiescent NIH-3T3 cells induces hypophosphorylation of the retinoblastoma protein leading to adipocyte differentiation [84]. As the studies with both PKB and PKCrl involved ectopic expression of the kinase, more work is required to provide conclusive evidence for a role of the endogeneous kinases in adipocyte differentiation.
Inhibition of adipogenesis: TNF(~, retinoic acid and Pref-1 In contrast to those factors already discussed, which all seem to enhance adipogenesis, knowledge is accumulating about factors which are either involved in the reversal and/or inhibition of this process. TNFc~ is a potent inhibitor of adipocyte differentiation and exposure of 3T3-L1 adipocytes to TNFo~ results in lipid depletion and a complete reversal of adipocyte differentiation [52]. One important way in which TNFc~ exerts its anti-adipogenic action is by downregulation of the expression of adipogenic factors such as C/EBPc~ [85,86] and PPARy [52,87]. Interestingly, several other polypeptides, such as tumor growth factor-13 (TGFI3) and basic fibroblast growth factor (bFGF), as well as calcium ionophores and agents that activated protein kinase C are also capable of reversing adipocyte differentiation through decreased expression of adipogenic transcription factors [87,88]. How these agents mediate this decrease in expression is unclear but further studies are required to investigate the role of NF-KB, AP1, and other transcription factors in the inhibition and reversal of adipocyte differentiation. Retinoic acid (RA), is another molecule capable of blocking early steps of adipocyte differentiation. T h e effects of RA are mediated by the liganded RA receptor (RAR) and downregulation of RARy1 expression during adipocyte differentiation correlates well with the loss of responsiveness to RA [89]. Ectopic expression of RAR extends the RA-responsive period [89]. One mechanism by which RA might block adipogenesis is by interfering with C/EBPl3-mediated transcription, which is only essential in the early stages of adipogenesis [90"]. This would
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explain why RA is less effective later in the adipocyte differentiation program. In addition, RA can inhibit PPARy action by displacement of the active PPARy-RXR heterodimer from its DR-1 response element. RA favors the formation of the inactive RAR-RXR heterodimer over the PPAR-RXR dimer [45"'] (see also Figure 2). Alternatively, RA has been shown to induce the expression of the repressive orphan receptor COUP-TF [91], which also competes for binding to a DR-1. Another factor with anti-adipogenic activity is Pref-1, a transmembrane protein of 45-60kDa with six tandem EGF-like repeats, which has some similarities to the Drosophila cell-fate determination proteins Notch and Delta [92]. Pref-1 is expressed in preadipocytes but is absent in adipocytes [92] and its constitutive expression inhibits expression of PPARy and C/EBP~ and blocks adipocyte differentiation [93"], suggesting that it functions as a negative regulator of adipocyte differentiation. T h e inhibitory action of Pref-1 can be exerted either in a juxtacrine manner as a transmembrane protein affecting adjacent cells or in a paracrine manner as a soluble inhibitor of adipocyte differentiation, released by cleavage of the membrane-associated form [93"].
Exit from the cell-cycle: a prerequisite for adipocyte differentiation Upon reaching confluence, preadipocytes in culture undergo contact inhibition and growth arrest between G 1 and S cell-cycle stages. After hormonal induction and before becoming quiescent, these cells undergo several rounds of mitotic clonal expansion which are essential to complete differentiation [94]. Cell-cycle arrest, although not sufficient to induce adipocyte differentiation, is at least as important as the induction of adipogenic genes and transcription factors [95"']. The interaction of the retinoblastoma (Rb) gene product and the E2F family of transcription factors is critical in regulating many genes controlling cell cycle and differentiation [96,97",98,99]. Involvement of the Rb protein in adipocyte differentiation was suggested by the fact that either inactivation or absence of the Rb protein, by binding to the SV40 large T antigen or by the use of Rb-/- cells, inhibited adipocyte differentiation [97",98]. Conversely, reintroduction of Rb in Rb-/- cells or h y pophosphorylation of Rb promoted adipogenesis [84,97"]. Furthermore, recent work suggested that the cell-cycle status of preadipocytes during and after clonal expansion correlates with their Rb phosphorylation status; Rb is first hyperphosphorylated during the clonal expansion and later becomes hypophosphorylated in the terminal differentiation phase [95"']. PPARy activation has also been implicated in the inhibition of DNA-binding activity of the E2F/DP-1, which is sufficient to induce growth arrest in certain cells. This inhibitory effect of PPARy activators on E2F
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activity is due to inactivation of PP2A phosphatase, leading to E2F phosphorylation and ultimately resulting in withdrawal from the cell cycle [100••]. Like PPARy, C/EBPo~ has also been shown to arrest cell growth and proliferation [19,101,102]. In contrast to PPARy, however, the antimitogenic effect of C/EBPc~ does not involve either Rb or the tumor suppressor protein p53 [103]. One of the mechanisms by which C/EBPc~ exerts its antimitogenic effects is through stimulation of the p21 (WAF1/Cipl) protein, a cyclin dependent kinase inhibitor [104]. C/EBPc~ not only induces the expression of p21, but also might cooperate to stabilize the p21 protein [104].
cognate response elements in the PPARy gene promoter and trigger high-level expression of PPARy. In addition, ADD-1/SREBP-1 will induce the production of ligands and activators for PPARy through its well established effects on genes involved in lipogenesis. At this time in the differentiation process, expression of RAR is sufficiently reduced to allow RXR to partner preferentially with PPARy. The ligand-activated PPART-RXR heterodimer will then induce exit from the cell cycle and trigger the expression o f adipocyte-specific genes, resulting in increased energy delivery to the adipocyte. C/EBPo~, whose expression is induced only during the later stages of differentiation, will cooperate with PPARy and will furthermore sustain a high level of PPARy in the mature adipocyte, as part of a feedforward loop.
Figure 3
Adipogenic factors and decreased Pref-1
JL C/EBPI3 and C/EBP8
C/EBPc~ ,<
RAR/RXR
ADD1/SREBP1
, PPAR~' ~> RXR
Although many more signaling molecules and transcription factors may have a role in adipocyte differentiation their role is much less well established at present. Our current knowledge of the adipocyte differentiation process may, already, permit us to devise strategies to modulate the activity of these transcriptional regulators by pharmacological and dietary manipulation. This might lead to new therapeutic approaches for disorders such as obesity and cachexia.
Acknowledgements
Adipocyte-specific target genes (e.g. aP2, LPL, ACS) CurrentOpinionin CellBiology Coordinate regulation of adipogenesis by the proteins Pref-1, RAR, RXR, PPART, C/EBP and ADD-1/SREBP-1. Interactions between these different transcription factors determine the cascade of events during adipocyte differentiation.
Bruce Spiegelman and Michclle Guerre-Millo are gratefully acknowledged for helpful discussion as well as disclosure of unpublished material. We thank the members of the Auwerx laboratory for support and discussion. Grant support to J Auwerx's laboratory by INSERM, Institut Pasteur de Lille, Universit6 de Lille II, Association de Recherche pour le Cancer (ARC 6403), Fondation pour la Recherche Medicale, Region Nord-Pas de Calais, Ligand Pharmaceuticals, Merck Research Laboratories, and Janssen Research Foundation is acknowledged. J Auwerx is a Research Director, and L Fajas is supported by a fellowship of the Janssen Research Foundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest == of outstanding interest
Conclusions Adipose tissue mass is determined to a large extent by adipocyte number, which can increase by replication and differentiation of preadipocytes. In this review we have focused on the role of factors regulating the differentiation process as determinants of adipogenesis. Adipocyte differentiation is influenced both by anti-adipogenic factors, such as T N F ~ , Pref-1, and RA, and by pro-adipogenic factors such as PPARy, the various members of the C/EBP family, ADD-1/SREBP-1, and the Rb protein. Current evidence suggests that PPARy is the master coordinator of the adipocyte differentiation process (Figure 3). Early during the differentiation process, Pref-1 expression decreases and hormonal agents induce the expression of C/EBPI3 and 8. In addition, ADD-1/SREBP-1 becomes activated by proteolytic cleavage as a result of cellular cholesterol depletion resulting from the last phase ofclonal expansion of the preadipocytes. C/EBPI3 and 8 and the activated ADD-1/SREBP-1 will then interact with their
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Transcriptional control of adipogenesis Lluis Fajas, Jean-Charles Fruchart and Johan Auwerx
Adipocyte differentiation is coordinatedly regulated by several transcription factors. C/EBPI3, C/EBP8 and ADD-1/SREBP-1 are active early during the differentiation process and induce the expression and/or activity of the peroxisome proliferator activated receptor-y (PPARy), the pivotal coordinator of the adipocyte differentiation process. Activated PPARy induces exit from the cell cycle and triggers the expression of adipocyte-specific genes, resulting in increased delivery of energy to the cells. C/EBPc~, whose expression coincides with the later stages of differentiation, cooperates with PPAR7 in inducing additional target genes and sustains a high level of PPARy in the mature adipocyte as part of a feedforward loop. Altered activity and/or expression of these transcription factors might underlie the pathogenesis of disorders characterized by increased or decreased adipose tissue depots.
Addresses INSERM U 325, D6partement d'Ath~roscl~rose, Institut Pasteur, 1 Rue Calmette, F-59019 Lille, France Correspondence: Johan Auwerx: e-mail: [email protected] Current Opinion in Cell Biology 1998, 10:165-173 http://biomednet.com/elecref/095506 7401000165
© Current Biology Ltd ISSN 0955-0674 Abbreviations ADD adipose differentiation and determination factor bHLH basic helix-loop-helix C/EBP CCAAT-enhancer-binding protein FAS fatty acid synthase GR glucocorticoid receptor LPL lipoproteinlipase PKB proteinkinase B PKC~] protein kinase Crl PPAR peroxisomeproliferator activated receptor RA retinoic acid RAR RA receptor Rb retinoblastoma RXR retinoicX receptor SREBP sterol regulatory binding protein STAT signaltransducer and activator of transcription TNF tumor necrosis factor WAT white adipose tissue
Introduction White adipose tissue (WAT) is mainly composed of adipocytes, cells that store energy in the form of triglycerides in times of nutritional affluence and release free fatty acids during nutritional deprivation [1]. WAT mass is determined by the balance between energy intake and expenditure and is controlled by genetic neuroendocrine and environmental factors [1]. Perturbances of this steady state can lead to increased or decreased amounts of
WAT, such as seen, respectively, in obesity or in the lipodystrophic syndromes [1]. Obesity affects between 25% and 30% of the population in industrialized countries and its frequency is still rising [2,3]. It is an independent risk factor for insulin resistance, non-insulin-dependent diabetes mellitus and coronary artery disease. Conversely, decreased amounts of adipose tissue, such as seen in malnutrition, cachexia and anorexia nervosa are also associated with significant levels of morbidity and mortality. Unlike brown adipose tissue, WAT is not present during embryonic life [4] and the adipocyte lineage derives from an embryonic stem cell precursor which has the capacity to differentiate to mesodermal cell types such as adipocytes and myocytes (reviewed in [5-7]). Alteration in WAT mass can involve alterations in both adipocyte volume and number. Increased adipocyte number is the result of a continuous and highly regulated differentiation process, whereas decreased adipocyte number is the consequence of apoptosis and adipocyte dedifferentiation (for review see [8]). This review will be limited to intrinsic regulatory mechanisms determining adipocyte differentiation, which are coordinated by transcription factors. For more general information on adipose tissue biology and obesity we refer the reader to some recent reviews [2-4,8-12]. Cell culture models, like the 3T3-L1 [13] or ob 1771 [14] immortalized mouse preadipocyte cell lines, have been instrumental in the understanding of adipocyte differentiation. The first step of adipocyte differentiation in these models involves arrest of cell proliferation [15]. At this stage, when appropriately induced with hormonal agents, preadipocytes differentiate into adipocytes, in a process orchestrated by a set of interdependently acting transcription factors, including peroxisome-proliferator-activated receptors (PPARs) [16,17], the CCAAT-enhancerbinding proteins (C/EBPs) [18-20], and the adipocyte differentiation and determination factor (ADD)-I, also known as the sterol regulatory clement binding protein (SREBP)-I [21,22°°]. Together these transcription factors stimulate the expression of adipocyte-specific genes such as those for lipoprotein lipase (LPL), leptin and the fatty acid binding protein, aP2 (reviewed in [23]), thereby leading to the characteristic phenotype of the mature adipocyte (Figure 1). These changes in gene transcription are tightly coordinated with changes in the cell cycle. Efforts to identify the master regulators triggering adipocytc differentiation arc driven by the assumption that controlling or reversing adipocyte differentiation might ultimately lead to a way to cure obesity and associated pathologies.
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Cell regulation
Figure 1
Adipocyte differentiation
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A scheme illustrating the expression of some regulatory proteins and target genes (indicated in italics) during the different stages of adipocyte differentiation.
PPARs, C / E B P s and A D D - 1 / S R E B P - 1 , the three tenors that orchestrate a d i p o g e n e s i s Peroxisome proliferator-activated receptors (PPARs) Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. After dimerization with the retinoic X receptor (RXR) and ligand activation, they control the expression of genes containing specific response elements called PPREs (reviewed in [24-28]). One of the PPARs, PPARy was elegantly shown by Tontonoz et al. to be a key player in adipocyte differentiation [16,17]. Indeed, infection of fibroblast [16] and muscle [29] cells with a retroviral vector expressing PPARy could induce adipocyte differentiation. Initially two different PPARy isoforms, PPARy1 and PPARy2, were identified [30], both derived from the same gene by alternative promoter usage and splicing. PPARy2 is identical to PPARy1 except for 28 additional amino acid at its amino-terminus [30,31°,32-34]. Recently, a third PPARy m R N A , PPARy3, was identified. PPARy3 mRNA produces a protein identical to PPARy1, but is transcribed from an independent promoter region, which could control the regulation of the entire PPARy locus (L Fajas and J Auwerx, unpublished data). So far, little is known about PPARy3 expression, PPARy1 seems ubiquitously expressed, whereas PPARy2 expression is mainly restricted to adipose tissue, suggesting that the PPARy2 form is most important for adipocyte physiology [31°,35]. A recent report that the anaino-terminus of PPARy2 enhances the activity of the ligand-independent transactivation domain, making PPARy2 a better transactivator ([361; L Fajas and
J Auwerx, unpublished data), supports the importance of PPARy2 relative to PPARy1 in regulating gene expression T h e transcriptional activity of the PPARy protein can be modulated by the binding of ligands, by posttranslational modifications and by interaction with other nuclear receptors and various cofactors. Thiazolidinedione antidiabetic agents [37] and prostaglandin J2 derivatives [38,39], which are synthetic and natural ligands of PPARy respectively, all facilitate adipogenesis. T h e fact that the insulin-sensitizing thiazolidinediones are potent PPARy activators suggests that this receptor has a key role not only in adipocyte biology but also in glucose homeostasis (reviewed in [9,28]). In addition to regulation by ligands, PPARy activity is also controlled by post-translational modifications [40°°,41,42]. MAP kinase-mediated phosphorylation of PPARy leads to its inactivation, which could underlie the well known inhibitory effects of growth factors and certain cytokines on adipogenesis [40°°,41,42]. However, other authors have not confirmed this observation [43]. Interaction with corepressors and coactivators, which form a bridge between nuclear receptors and the basal transcription machinery and influence chromatin structure, is another important way of modulating their transcriptional activity (for review see [44]). Recently, it was demonstrated that the transcriptional activity of PPARy was enhanced by ligand-dependent interactions of the receptor with the cofactors steroid receptor coactivator-1 (SRC-1) [45 °°] and p300 (L Gelman, L Fajas, J Auwerx, unpublished data). Furthermore,
Transcriptional control of adipogenesis Fajas, Fruchart and Auwerx
Spiegelman's group has demonstrated that interaction with PGC-1, a novel cofactor, could enhance PPARy transcriptional activity specifically in brown adipose tissue (personal communication). Finally, PPARy activity can be modulated by its absolute requirement for RXR in order to bind to DNA (Figure 2). On a DR-1 response element (such as a PPRE), RXR is a nonpermissive inactive partner in heterodimers with RAR, whereas it is a permissive partner in the PPAR-RXR heterodimer. Because the levels of RAR and PPARy change considerably during adipocyte differentiation, a switch in partners by RXR will result in differential recruitment of corepressors and coactivators to the DR-1 element, which is ultimately translated into an alteration of the activity of the gene from repressed to activated (Figure 2) [45°°]. Such a switch might underlie the mechanism by which not only PPARy agonists, but also RXR agonists, exert their effects on adipogenesis and glucose homeostasis. Figure 2
I Coactivator 1
PPAR
DR-1
Active
RXR
Co-repressor
RAR
DR-1
RXR
Inactive CurrentOpinionin CellBiology
PPAR-RXR and RAR-RXR heterodimers respectively activate and repress DR-l-containing promoters. When the RAR-RXR heterodimer is bound to a DR-1 site, RAR blocks the binding of ligands to RXR, keeping the heterodimer in an inactive state due to the binding of corepressors such as N-CoR or SMRT. In contrast, the PPAR-RXR heterodimer configuration permits ligand binding to RXR, allowing for activation of both partners in the PPAR-RXR heterodimer.
Although the mechanisms by which PPAR7 activation drives adipogenesis are not fully elucidated, one particular feature is that it induces the formation of small-sized adipocytes [46]. T h e enhanced adipocyte differentiation most likely involves the regulation of adipocyte-specific gene expression. Indeed, PPARy controls the expression of several crucial adipocyte genes, including those for LPL, acyl coenzyme A synthase, fatty acid synthase (FAS) and phospho-enol pyruvate carboxykinase, which are all involved in coordinating fatty acid uptake and storage (reviewed in [23,28]). Furthermore, PPAR¥ decreases the expression of the adipocyte-derived signaling molecule leptin, which results in an increase in energy intake
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and optimization of energy usage, contributing further to PPARy's adipogenic effect [47-50]. PPARy-mediated activation of adipocyte gene expression and differentiation most probably also underlies its antidiabetic effects. Indeed the low level of expression of PPARy in muscle, the main tissue for glucose metabolism, makes a direct effect of PPARy activators in this tissue unlikely and suggests rather that they modify the production of adipocyte-derived mediators of insulin resistance, such as free fatty acids or the cytokine tumor necrosis factor (TNF) cc PPAR 7 activation will decrease production of TNF(x by adipocytes [51,52], reduce TNF's antidifferentiation effects [52-54] and interfere with its inhibitory effect on insulin signaling [55°']. In addition, because of its tissue-selective effects on genes involved in fatty acid uptake, PPARy activation will induce repartitioning of fatty acids in the body, with enhanced accumulation of fatty acids in adipose tissue at the expense of a relative depletion of muscle fatty acids [56°°]. T h e relative lipid depletion of muscle cells will improve their glucose metabolism and result in an improvement in insulin sensitivity [57].
The C/EBP family of transcription factors Three members of the C/EBP family of transcription factors--C/EBPct, 13 and ~i- - although not specific for the adipocyte lineage, are expressed at defined times during adipogenesis, and each has a specific regulatory role. Adipogenic hormones such as glucocorticoids, cyclic AMP (cAMP) and insulin induce a transient increase in the expression of C/EBP13 and 8 early in adipocyte differentiation [58-60]. C/EBPI3, in synergy with C/EBP~5, then induces PPARy expression in the preadipocyte [61,62°], subsequently triggering full-blown adipocyte differentiation. Supporting this idea is the fact that there are at least two binding sites for C/EBP in the human PPARy2 promoter [30,31°].
In contrast to the early effects of C/EBPI3 and 8 on PPARy expression and adipocyte differentiation, C/EBPct seems to play an important part in the later stages of differentiation by sustaining high levels of PPARy expression and by maintaining the differentiated adipocyte phenotype. Several lines of evidence support an important role for C/EBP(x in adipocyte differentiation. First, temporal activation of C/EBPc~ expression occurs immediately before the coordinate expression of a group of adipocyte-specific genes, suggesting its involvement in their regulation [18]. Second, antisense C/EBP(x RNA can inhibit adipocyte differentiation [63]. Third, premature induction or overexpression of C/EBPct triggers adipocyte differentiation [20,63]. And finally, adipocytes from mice in which the C/EBP(x gene was disrupted by homologous recombination failed to accumulate lipids in adipose tissue [64].
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The ADD-1/SREBP-1 family of transcription factors ADD-1 and SREBP-1, which are rodent and human homologs, were independently cloned and characterized as transcription factors implicated, respectively, in the control of adipocyte differentiation [21] and in cholesterol control of gene expression [65]. T h e protein is synthesized as a membrane-bound precursor localized in the nuclear envelope and the endoplasmic reticulum. Upon sterol depletion the precursor is proteolytically cleaved to generate a soluble amino-terminal fragment, which contains an acidic activation domain and basic helix-loop-helix (bHLH) region mediating protein dimerization and DNA binding (reviewed in [66]). This mature form translocates to the nucleus and has a dual DNA-binding specificity. It can bind either to a cis element containing variations on the core sequence C A N N T G (where N represents any of the four nucleotides), also referred to as the E box, which is the b H L H consensus response element, or to a sequence motif identified as the sterol regulatory element (SRE). T h e expression of ADD-1/SREBP-1, is induced during differentiation of adipocytes, where it activates transcription of target genes involved in both cholesterol metabolism and fatty acid metabolism [22°']. Direct evidence of the adipogenic effect of ADD1/SREBP-1 was provided by its capacity to induce fat accumulation and adipocyte differentiation when overexpressed in fibroblasts with a retroviral vector [22°°]. The adipogenic effect of ADD-1/SREBP-1 is enhanced by PPARy and its activators [22°°]. Furthermore, overexpressing SREBP-1 in the liver of transgenic animals resulted in a massive fat accumulation in the liver subsequent to the induction of genes involved in lipid uptake and biosynthesis [67]. Part of the adipogenic effect of ADD-1/SREBP-1 might involve direct activation of the expression of PPARy through interaction with a response element in the PPARy3 promoter (L Fajas and J Auwerx, unpublished data). In addition, SREBP-1/ADD-1 controls the expression of genes involved in lipid metabolism, such as those for L P L [22"°,67], acetyl coenzyme A carboxylase [68], and FAS [22°*,67,69], which could lead to the generation of natural activators and ligands for PPARy. O t h e r factors i n v o l v e d in a d i p o g e n e s i s In addition to the proteins discussed above, recent preliminary evidence implicates other factors in adipogenesis (reviewed in [23]). For all of these factors, however, further studies are required to identify the molecular pathways in which they operate. Signal transducers and activators of transcription T h e signal transducer and activator of trascription (STAT) family of transcription factors consists of six members that are phosphorylated in response to various cytokines and peptide hormones (reviewed in [70,71]). After phosphorylation and translocation to the nucleus, STATs regulate the transcription of specific target genes. Recently it has
been shown that three members of the STAT family, STAT-1, -3 and -5 are strongly induced during adipocyte differentiation in a manner similar to C/EBPc~ and PPARy [72°]. Furthermore, inhibition of adipogenesis by TNFc~ correlates with the repression of STATs in 3T3-L1 cells [72°]. Which signaling molecules and receptors are involved in the activations of these STATs is currently unknown. Adipose tissue, however, contains several receptors of the cytokine receptor superfamily, such as the growth hormone receptor and the prolactin receptor, both of which are induced during differentiation [73,74] and which have been shown to control the expression of adipocyte genes such as LPL [75,76]. Altogether, these data suggest that cytokines and peptide hormones, through their effects on STAT-mediated gene expression could have a significant role in the development and/or maintenance of the adipose phenotype. HMGI-C
T h e chromosomal protein HMGI-C belongs to the high mobility group family of architectural DNA-binding proteins that are abundant, heterogeneous nonhistone components of chromatin [77]. Although they do not possess transcriptional activity by themselves, they change the conformation of DNA and may hence influence transcription. Support for the involvement of HMGI-C in adipogenesis is provided by the observation that the mouse mutant pygmy, characterized by its small size and disproportionally reduced body fat content, was found to be a null allele of the HMGI-C gene [78]. Further evidence comes from the finding that in certain lipomas gene rearrangements were found in which the HMGI-C DNA-binding domain was fused to either a LIM domain or an acidic transactivation domain [79,80]. In the first case, the LIM domain of the translocation partner could recruit transcriptional activators to the DNA site, whereas in the second case, the fusion of the HMGI-C protein to an acidic transactivation domain could turn the chimeric protein into a powerful transcriptional activator. Thus, the lack or reduced expression of HMGI-C could predispose to leaness, whereas the juxtaposition of its DNA-binding motif to transcriptional regulatory domains could promote adipogenesis. Glucocorticoid receptor Activated glucocorticoid receptor (GR) is thought to have multiple pro-adipogenic effects, several of them enhancing PPARy activity. First, activated GR will increase the expression of early adipogenic genes, including those for the C/EBPs [58-60], which in their turn will switch on the PPARy gene [62°]. It has also been suggested that GR might directly stimulate the expression of PPARy [62°], although further studies are required to elucidate whether there is a functional binding site for GR in the PPAR7 promoter. In addition, glucocorticoids are known to activate phospholipase A2, hence stimulating the production of prostanoid ligands of PPARy [81].
Transcriptionalcontrol of adipogenesis Fajas, Fruchart and Auwerx
Protein kinase B and protein kinase Crl Protein kinase B (PKB or Rac), a serine/threonine kinase which is activated by insulin, has recently been shown to be involved in adipogenesis. As differentiation of 3T3-L1 preadipose cells into adipocytes is dependent on insulin it was postulated that PKB was responsible for transducing the insulin signal to downstream proteins. Experiments in which an ectopic and constitutively activated form of PKB was expressed in 3T3-L1 preadipocytes showed that these cells spontaneously differentiated into adipocytes, pointing to a crucial role of this kinase in differentiation [82]. PKB might achieve this adipogenic effect by activation of pT0 $6 kinases which in previous studies were shown to be the target of rapamycin-mediated inhibition of adipogenesis [83]. Another protein kinase with a potential role in adipocyte differentiation is protein kinase Clq. Overexpression of PKCrl in quiescent NIH-3T3 cells induces hypophosphorylation of the retinoblastoma protein leading to adipocyte differentiation [84]. As the studies with both PKB and PKCrl involved ectopic expression of the kinase, more work is required to provide conclusive evidence for a role of the endogeneous kinases in adipocyte differentiation.
Inhibition of adipogenesis: TNF(~, retinoic acid and Pref-1 In contrast to those factors already discussed, which all seem to enhance adipogenesis, knowledge is accumulating about factors which are either involved in the reversal and/or inhibition of this process. TNFc~ is a potent inhibitor of adipocyte differentiation and exposure of 3T3-L1 adipocytes to TNFo~ results in lipid depletion and a complete reversal of adipocyte differentiation [52]. One important way in which TNFc~ exerts its anti-adipogenic action is by downregulation of the expression of adipogenic factors such as C/EBPc~ [85,86] and PPARy [52,87]. Interestingly, several other polypeptides, such as tumor growth factor-13 (TGFI3) and basic fibroblast growth factor (bFGF), as well as calcium ionophores and agents that activated protein kinase C are also capable of reversing adipocyte differentiation through decreased expression of adipogenic transcription factors [87,88]. How these agents mediate this decrease in expression is unclear but further studies are required to investigate the role of NF-KB, AP1, and other transcription factors in the inhibition and reversal of adipocyte differentiation. Retinoic acid (RA), is another molecule capable of blocking early steps of adipocyte differentiation. T h e effects of RA are mediated by the liganded RA receptor (RAR) and downregulation of RARy1 expression during adipocyte differentiation correlates well with the loss of responsiveness to RA [89]. Ectopic expression of RAR extends the RA-responsive period [89]. One mechanism by which RA might block adipogenesis is by interfering with C/EBPl3-mediated transcription, which is only essential in the early stages of adipogenesis [90"]. This would
169
explain why RA is less effective later in the adipocyte differentiation program. In addition, RA can inhibit PPARy action by displacement of the active PPARy-RXR heterodimer from its DR-1 response element. RA favors the formation of the inactive RAR-RXR heterodimer over the PPAR-RXR dimer [45"'] (see also Figure 2). Alternatively, RA has been shown to induce the expression of the repressive orphan receptor COUP-TF [91], which also competes for binding to a DR-1. Another factor with anti-adipogenic activity is Pref-1, a transmembrane protein of 45-60kDa with six tandem EGF-like repeats, which has some similarities to the Drosophila cell-fate determination proteins Notch and Delta [92]. Pref-1 is expressed in preadipocytes but is absent in adipocytes [92] and its constitutive expression inhibits expression of PPARy and C/EBP~ and blocks adipocyte differentiation [93"], suggesting that it functions as a negative regulator of adipocyte differentiation. T h e inhibitory action of Pref-1 can be exerted either in a juxtacrine manner as a transmembrane protein affecting adjacent cells or in a paracrine manner as a soluble inhibitor of adipocyte differentiation, released by cleavage of the membrane-associated form [93"].
Exit from the cell-cycle: a prerequisite for adipocyte differentiation Upon reaching confluence, preadipocytes in culture undergo contact inhibition and growth arrest between G 1 and S cell-cycle stages. After hormonal induction and before becoming quiescent, these cells undergo several rounds of mitotic clonal expansion which are essential to complete differentiation [94]. Cell-cycle arrest, although not sufficient to induce adipocyte differentiation, is at least as important as the induction of adipogenic genes and transcription factors [95"']. The interaction of the retinoblastoma (Rb) gene product and the E2F family of transcription factors is critical in regulating many genes controlling cell cycle and differentiation [96,97",98,99]. Involvement of the Rb protein in adipocyte differentiation was suggested by the fact that either inactivation or absence of the Rb protein, by binding to the SV40 large T antigen or by the use of Rb-/- cells, inhibited adipocyte differentiation [97",98]. Conversely, reintroduction of Rb in Rb-/- cells or h y pophosphorylation of Rb promoted adipogenesis [84,97"]. Furthermore, recent work suggested that the cell-cycle status of preadipocytes during and after clonal expansion correlates with their Rb phosphorylation status; Rb is first hyperphosphorylated during the clonal expansion and later becomes hypophosphorylated in the terminal differentiation phase [95"']. PPARy activation has also been implicated in the inhibition of DNA-binding activity of the E2F/DP-1, which is sufficient to induce growth arrest in certain cells. This inhibitory effect of PPARy activators on E2F
1 70
Cell regulation
activity is due to inactivation of PP2A phosphatase, leading to E2F phosphorylation and ultimately resulting in withdrawal from the cell cycle [100••]. Like PPARy, C/EBPo~ has also been shown to arrest cell growth and proliferation [19,101,102]. In contrast to PPARy, however, the antimitogenic effect of C/EBPc~ does not involve either Rb or the tumor suppressor protein p53 [103]. One of the mechanisms by which C/EBPc~ exerts its antimitogenic effects is through stimulation of the p21 (WAF1/Cipl) protein, a cyclin dependent kinase inhibitor [104]. C/EBPc~ not only induces the expression of p21, but also might cooperate to stabilize the p21 protein [104].
cognate response elements in the PPARy gene promoter and trigger high-level expression of PPARy. In addition, ADD-1/SREBP-1 will induce the production of ligands and activators for PPARy through its well established effects on genes involved in lipogenesis. At this time in the differentiation process, expression of RAR is sufficiently reduced to allow RXR to partner preferentially with PPARy. The ligand-activated PPART-RXR heterodimer will then induce exit from the cell cycle and trigger the expression o f adipocyte-specific genes, resulting in increased energy delivery to the adipocyte. C/EBPo~, whose expression is induced only during the later stages of differentiation, will cooperate with PPARy and will furthermore sustain a high level of PPARy in the mature adipocyte, as part of a feedforward loop.
Figure 3
Adipogenic factors and decreased Pref-1
JL C/EBPI3 and C/EBP8
C/EBPc~ ,<
RAR/RXR
ADD1/SREBP1
, PPAR~' ~> RXR
Although many more signaling molecules and transcription factors may have a role in adipocyte differentiation their role is much less well established at present. Our current knowledge of the adipocyte differentiation process may, already, permit us to devise strategies to modulate the activity of these transcriptional regulators by pharmacological and dietary manipulation. This might lead to new therapeutic approaches for disorders such as obesity and cachexia.
Acknowledgements
Adipocyte-specific target genes (e.g. aP2, LPL, ACS) CurrentOpinionin CellBiology Coordinate regulation of adipogenesis by the proteins Pref-1, RAR, RXR, PPART, C/EBP and ADD-1/SREBP-1. Interactions between these different transcription factors determine the cascade of events during adipocyte differentiation.
Bruce Spiegelman and Michclle Guerre-Millo are gratefully acknowledged for helpful discussion as well as disclosure of unpublished material. We thank the members of the Auwerx laboratory for support and discussion. Grant support to J Auwerx's laboratory by INSERM, Institut Pasteur de Lille, Universit6 de Lille II, Association de Recherche pour le Cancer (ARC 6403), Fondation pour la Recherche Medicale, Region Nord-Pas de Calais, Ligand Pharmaceuticals, Merck Research Laboratories, and Janssen Research Foundation is acknowledged. J Auwerx is a Research Director, and L Fajas is supported by a fellowship of the Janssen Research Foundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest == of outstanding interest
Conclusions Adipose tissue mass is determined to a large extent by adipocyte number, which can increase by replication and differentiation of preadipocytes. In this review we have focused on the role of factors regulating the differentiation process as determinants of adipogenesis. Adipocyte differentiation is influenced both by anti-adipogenic factors, such as T N F ~ , Pref-1, and RA, and by pro-adipogenic factors such as PPARy, the various members of the C/EBP family, ADD-1/SREBP-1, and the Rb protein. Current evidence suggests that PPARy is the master coordinator of the adipocyte differentiation process (Figure 3). Early during the differentiation process, Pref-1 expression decreases and hormonal agents induce the expression of C/EBPI3 and 8. In addition, ADD-1/SREBP-1 becomes activated by proteolytic cleavage as a result of cellular cholesterol depletion resulting from the last phase ofclonal expansion of the preadipocytes. C/EBPI3 and 8 and the activated ADD-1/SREBP-1 will then interact with their
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