Regulation of adipocyte gene expression and differentiation by peroxisome proliferator

Peter Tontonoz,
Dana-Farber
Peroxisome the nuclear specifically

activated receptor y

Erding Hu and Bruce M Spiegelman

and Harvard Medical School, Boston, USA

Cancer

Institute

proliferator hormone

activated receptor (PPAR) y is an orphan member of receptor superfamily and is expressed at high levels Fibroblastic cell Recent data suggest that this factor is a central and differentiation. activators of PPARs, can be induced to differentiate into fat

in adipose tissue. PPARr

regulator of adipocyte gene expression lines that express ectopically cells by a variety of lipids and lipid-like

suggesting that

this protein may function to link adipogenesis with systemic lipid metabolism.

Current Opinion

in Genetics & Development

1995,

5:571-576

Introduction
involves dramatic changes Adipocyte differentiation in cell morphology and gene expression. Although regulation of mRNA stability is likely to contribute to the expression of several adipocyte proteins (e.g. fatty acid synthetase and adipsin [ 1,2]), alteration of the rate of transcription accounts for changes in the levels of the majority of differentiation-linked mRNAs. In recent years, considerable effort has been directed at the identification of c&acting and truw.c-acting factors involved in the regulation of differentiation-dependent adipocyte genes (reviewed in [3,4]). The ultimate goal of these studies has been to characterize transcription factors that function to coordinate and direct the development of the adipose lineage. The discovery of tissue-specific regulatory proteins in tissues such as skeletal muscle [5,6] and erythrocytes [7,8] suggested that analogous proteins might exist in adipocytes. In this review, we will focus on recent advances that suggest that peroxisome proliferator activated receptor (PPAR) y, a tissue-specific orphan member of the nuclear hormone receptor superfamily, functions as a key regulator of adipose cell development. We will also review data that suggest an important functional relationship between PPARr and a second adipose-enriched transcription factor, CCAAT/enhancer-binding protein (C/EBP) CL.

expression. Among the best characterized of these are the members of the UEBP family of basic leucine zipper proteins [c)]. C/EPBa is expressed primarily in tissues with significant lipogenic capacity, including liver, fat, lung and intestine, and is induced during the differentiation of several cultured adipocyte cell lines [lO,ll]. Binding sites for C/EPBa have been located in the promoters of a number of fat cell genes [l&14]. evidence has Over the past five years, considerable accumulated to suggest that C/EPBa plays an important role in the process of adipocyte differentiation (reviewed in [3]). The most direct evidence for such a role has come from the recent work of Freytag et al. [ 15], who found that retroviral expression of C/EPBa can promote adipocyte differentiation in sitar in transfected fibroblast colonies. In addition, Lin and Lane [lh] have recently reported that expression of C/EPBc( from an inducible promoter can stimulate differentiation of 3T3-Ll preadipocytes. C/EPBa is induced relatively late in the time course of differentiation, however, so it is unlikely that this factor initiates the adipocyte program in Go. Furthermore, as C/EPBa is expressed in liver, lung and intestine as well as fat, it cannot by itself account for the strict tissue-specific expression of many adipocyte genes (for a discussion of C/EBPa and metabolic control, see the review in this issue by GJ Darlington, N Wang and RW Hanson [pp 565-5701). Recent work has suggested that two additional C/EBP family members, C/EBPP and C/EBP& also play a role in the regulation of adipocyte gene expression. Like C/EPBa, these factors are expressed in a number of tissues in addition to fat; however, they are induced significantly earlier in the time course of adipocyte differentiation than is C/EPBa [9]. Yeh CI al. [17**] have recently reported that retroviral expression of

Transcriptional expression
A small potential

regulators

of adipocyte

gene

number of proteins have transcriptional regulators

been identified as of adipocyte gene

Abbreviations aP2-adipocyte
PEPCK-phosphoenolpyruvate P2; C/EBP-CCAAT/enhancer-binding carboxykinase; PPAR-peroxisome protein; ETYA-5,8,11 activated ,14-eicosatetraynoic receptor; acid; receptor. proliferator RXR-retinoid-X

0 Current

Biology Ltd ISSN 0959-437X

571

572

Differentiation and gene regulation C/EBPa can promote adipose differentiation of cultured NIH-3T3 cells. Expression of C/EBPG, on the other hand, was observed to promote differentiation of established 3T3-Ll preadipocytes, but not of NIH-3T3 cells. These results differ corn those of Freytag et al. [15**], who failed to observe an effect of retroviral in expression of C/EBPP on adipocyte differentiation similar studies. PPARy was first linked to adipogenic gene expression through analysis of the fat-specific enhancer from the adipocyte P2 (aP2) gene. The key transcriptional regulator of this gene was initially defined as a novel differentiation-dependent nuclear factor, termed ARF6, that binds to two r&acting DNA elements within the enhancer [18]. This same factor was subsequently identified as an important regulator of the adipocyte-specific enhancer from the phosphoenolpyruvate carboxykinase (PEPCK) gene [19]. Target sequences for ARF6 in the aP2 and PEPCK enhancers exhibit homology to a direct repeat of hormone-response elements spaced by one nucleotide; this motif (DR-1) had been previously demonstrated to be the preferred binding site for heterodimers of the retinoid-X receptor (RXR) and PPAR. We have cloned a novel member of the PPAR gene family by PCR amplification of adipocyte cDNA [20]. The encoded receptor was designated PPARy2 because of its homology to the Xenopus PPARy. A different isoform of the mammalian receptor, termed PPARrl, was isolated simultaneously by several groups [21,22,23]. PPARr forms a heterodimeric complex with RXRa, and the DNA-binding and trancriptional activation properties of this complex are identical to those of ARF6. The endogenous ARFh complex has also been purified from a cultured adipocyte cell line by a combination of conventional and sequence-specific DNA affinity chromatography [24]. Chemical sequencing and mass spectral analysis of tryptic peptides deriving fi-om the purified polypeptides definitively identified the ARF6 complex as a heterodimer of RXRa and PPARy. RXRa mRNA is expressed at high levels in a number of tissues, but is most abundant in liver and fat. Expression of PPARr mRNA is strikingly specific for adipose tissue [20**,24,25*]. To date, PPARy is the only transcription factor known to be expressed with adipose specificity. In addition, expression of PPAR? is induced very early in the differentiation of cultured adipocyte cell lines, suggesting that it serves an important function early in the differentiation process. include the steroid, thyroid and retinoid receptors, as well as a growing number of so-called orphan receptors whose endogenous ligands have not been identified. All of these receptors share a common structure, the hallmark features of which are a well conserved DNA-binding domain containing two zinc finger motifs and a divergent ligand-binding/dimerization domain (reviewed in [26]). Peroxisome proliferators are a structurally diverse group of compounds that cause hepatic peroxisomal proliferation and induction of the enzymes of the fatty acid P-oxidation pathway when administered to rodents (reviewed in [27]). Several years ago, Isseman and Green [28] described the cloning of a novel member of the nuclear hormone receptor superfamily, termed PPARa, that could be activated in cells treated with peroxisome proliferators. Three related PPAR genes (a,p and r) were subsequently identified in Xenopus [29]. Analysis of genes induced by peroxisome proliferators identified specific response elements comprising direct repeats of AGGTCA-like motifs spaced by one nucleotide (DR-1 [29-311). The PPARs bind to DNA as heterodimers with the RXRs [32,33]. Three PPAR family members have now been described in mammals: PPARa, PPAR? [20**,21,22**,23], and PPARG (also called NUC-I) [22*,23,34,35]. These mammalian PPARs exhibit distinct patterns of expression: PPARa is expressed predominantly in liver, heart, kidney and brown fat [28], PPARG is expressed at a low level in many tissues [22,35,36], and PPARy is expressed at high levels specifically in adipose tissue [20,25*]. Although the DNA-binding domains of these three receptors are well conserved, their ligand-binding domains are significantly divergent. Accordingly, each mammalian PPAR is more closely related to its Xenoprrs homologue than to the other mammalian receptors. the three receptors have recently been Moreover, shown to be pharmacologically distinct, that is they are differentially activated by the known PPAR activators [22**]. These observations suggest that each PPAR family member serves a distinct physiological function and that each may respond to a different endogenous ligand. Considerable evidence has accumulated to suggest that PPARa plays an important role in the regulation of the peroxisomal and microsomal fatty acid P-oxidation pathways [32]. Acyl CoA oxidase, the enzyme that catalyzes the rate-limiting step in this pathway, has been shown to be a direct transcriptional target of PPARa [29,31]. PPARa is also likely to be responsible for the phenomenon of peroxisome proliferation (as it is preferentially activated by peroxisome proliferators [22**]) and is specifically expressed in those tissues capable of undergoing peroxisomal proliferation. Several groups have observed that PPARa can be activated by physiological concentrations of polyunsaturated fatty acids [37,38]. This has led to the proposal that fatty acids are in fact the endogenous ligands for PPARa and that the physiological role of this receptor is to

Peroxisome

proliferator

activator

receptors: a by lipids

family of nuclear

receptors activated

In vertebrates, the nuclear hormone receptor superfamily serves to link lipophilic extracellular signals directly to cellular gene expression. Members of this superfamily

Regulation of adipocyte gene expression and differentiation by PPARr Tontonoz,

Hu and Spiegelman

573

safeguard against systemic lipid overload. Direct binding of fatty acids to a PPAR has not yet been demonstrated, however. PPAR6 is expressed in a wide range of tissues and is expressed early during mammalian development [22**]. At present, the physiological role of this PPAR family member is a mystery. PPARG is activated significantly by polyunsaturated fatty acids such as linoleic acid, but only weakly by peroxisome proliferators such as clofibrate [22**]. Grimaldi and colleagues [35-l have recently provided evidence to suggest that PPARG can mediate fatty acid induction of certain adipocyte genes, including aP2, when overexpressed in cultured cells. As PPARG is widely expressed, however, it is likely to function in many tissues.

results strongly suggest that the physiological role of PPARr is to regulate adipocyte gene expression and differentiation in response to one or more endogenous lipid activators. Interestingly, the murine PPARs appear to differ in their ability to stimulate adipocyte gene expression and differentiation. PPARa activates expression of the aP2 gene in response to 5,8,11,14-eicosatetraynoic acid (ETYA), but, unlike PPARy, does not stimulate overt differentiation [36**]. There are two plausible explanations for the observed difference in activity between these two receptors. The first possibility is that they may in fact be functionally distinct, with each receptor being competent to activate a discrete set of target genes. As the DNA-binding domains of PPARr and PPARa recognize a similar sequence motif (DR-I), this implies that the two receptors interact differently, either with the transcriptional machinery at the promoter or with other enhancer-binding factors. The second possibility for the observed difference in differentiation-promoting activity is that PPARa is not maximally activated in fibroblasts by ETYA. Although the aP2 gene is effectively induced under these conditions, differentiation may simply require more PPAR activity. Thus, it is formally possible that a more potent activator would enable PPARa to functionally substitute for PPARr and stimulate differentiation. As reviewed above, considerable evidence has accumulated to suggest that both PPARr and C/EPBa function as important regulators of adipocyte gene expression and differentiation. When PPARr and C/EPBa are coexpressed in fibroblasts, they act synergistically to induce adipogenesis [36**]. This suggests that the endogenous differentiation program involves the combined action of both proteins. Although other transcription factors are likely to play a role in the differentiation process, expression of PPARy and C/EPBa is, in some contexts, sufficient to define the adipocyte phenotype. The simplest explanation for the observed synergy between PPARr and C/EPBa in promoting adipogenesis is that both are required for the activation of certain critical genes. In contrast to genes expressed specifically in cell types such as liver and muscle, the transcriptional regulatory sequences of relatively few adipocyte-specific genes have been carefully studied. In fact, PEPCK and aP2 are the only genes for which regulatory sequences sufficient to direct adipose-specific expression in ho have been characterized [43-451. In both of these genes, PPARr binds to an upstream adipose-specific enhancer region and C/EPBa binds to sites within the proximal promoter. It remains to be determined whether similar regulatory arrangements are utilized by other adipocyte genes. The temporal expression of these two factors suggests that they act sequentially and that some genes may be controlled principally by one or the other. PPARy2, which is induced very early, may be important for the activation of certain early genes and may serve to commit the primitive mesodermal

PPARy

and the adipocyte

developmental

program Prior to the cloning of murine PPARy, the known regulatory targets of the PPARs were primarily confined to enzymes involved in hepatic peroxisomal and microsomal fatty acid oxidation. It is now clear, however, that the regulatory targets of the PPARs are not limited to genes involved in lipid oxidation. A number of adipocyte genes, including aP2 and PEPCK, also contain DR-l-like repeats. In fat, these sites are primary targets for the PPARyRXRa heterodimer. aP2 is the major lipid-binding protein in adipose cells, and PEPCK catalyzes the rate-limiting step in glyceroneogenesis. These proteins are important for triglyceride synthesis and storage, rather than lipid catabolism. The phenomenon of peroxisomal proliferation occurs in brown adipose tissue, but not in white adipose tissue [32,39]. Accordingly, PPARa is expressed in brown fat, but not in white fat [28]. PPARy, like aP2 and PEPCK, is expressed in both tissues [20**]. It had been recognized for many years that clofibrate and related hypolipidemic drugs could potentiate the differentiation of cultured preadipocyte cell lines such as 3T3-Ll and Ob-17 [40,41]. More recently, Chawla and Lazar [42**] have shown that a number of peroxisome proliferators can cause differentiation of 3T3-Ll preadipocytes in the absence of other inducing agents. These observations, together with the tissue-specificity of PPARr expression and the ability of this factor to activate adipocyte-specific enhancers in non-adipose cells, suggested that PPARr functions to regulate the process of adipocyte differentiation per sc. We demonstrated recently that retroviral expression of PPARy is, in fact, sufficient to convert a number of uncommitted fibroblast cell lines into bona jidc differentiation-competent preadipocytes [36]. PPAR activators, including naturally occurring polyunsaturated Betty acids, promote the differentiation of PPARexpressing cells in a dose-dependent manner. These

574

Differentiation and gene regulation

cell to the adipose lineage. C/EPBa, which is induced later in the differentiation process, probably functions to coordinate expression of late genes and may serve to trigger growth arrest, terminal differentiation and/or lipid accumulation [46-481.

PPARy: a molecular metabolism

link between

systemic lipid,

and adipocyte

development?

Whereas the volume of lipid an individual adipocyte can accumulate is finite, the capacity of adipose tissue to expand in response to overfeeding is virtually without limit. Significant expansion of adipose tissue mass, therefore, necessitates the differentiation of new adipocytes from precursor cells [49,50]. Such differentiation can occur at any time throughout the life of an adult organism and must obviously be linked to nutritional status. As a lipid-activated transcription factor, PPARr represents a potential molecular link between systemic lipid metabolism and adipocyte differentiation. In vivo, a chronic hyperlipidemic state might be expected to provide adipose precursor cells with an increased level of potential PPAR activators or their metabolic precursors. Once taken up by the cells, these activators could influence differentiation directly through interaction with PPARy. Alternatively, PPARr activators may be generated intracellularly from lipid precursors in response to other extracellular signals. More research will be required to distinguish between these possibilities. A number of important issues remain to be addressed. Foremost among these is the identity of the endogenous ligand(s) for PPARy. Although polyunsaturated fatty acids and a number of structurally diverse synthetic compounds have been shown to activate PPARs in experimental systems, none of these has been shown to bind to a receptor directly. It is likely that many or all of the known PPAR activators actually function indirectly, by leading to an increase in the cellular levels of the true ligand(s). The observation that the three known PPARs (a, y and 6) have divergent ligand-binding domains and are pharmacologically distinct implies that each may bind a different endogenous ligand [22**]. PPARr activity appears to be required for the maintenance of aP2 and PEPCK gene expression in mature adipocytes, suggesting that the endogenous PPAR? activator is present at significant levels in these cells. Cultured adipocytes could, therefore, represent the ideal source from which to isolate a PPARy ligand. The characterization of PPARy, a potentially central regulator of adipocyte development, could open the door for novel approaches to the treatment of obesity and other disorders of adipose tissue. As a ligand-dependent

transcription factor, PPARy represents an ideal target for intervention. A number of therapeutic agents in clinical use today are directed at nuclear hormone receptors. These include drugs such as the estrogen receptor antagonist tamoxifen (used to treat breast cancer) and the progesterone receptor antagonist RU-486 (the abortion pill). If PPARy is actually required for the development of adipose cells, it may be possible to limit adipocyte differentiation in humans using specific antagonists of PPARy. It remains possible, of course, that in the absence of PPARy, other PPAR family members might be able to complement the defect and allow adipogenesis to proceed. Targeted disruption of the PPARy locus in mice should claritjr this issue.

Whether PPARy antagonists would be effective as anti-obesity agents may depend on whether there are adequate feedback mechanisms in the energy balance system. The possibility that agenesis of adipose tissue may lead to pathological disorders such as hyperlipidemia, fatty liver, pancreatitis or diabetes must also be considered. The recent cloning and characterization of the murine obese locus has provided the most direct evidence for the existence of a feedback loop from adipose tissue to satiety centers in the hypothalamus [51]. If caloric intake is not reduced, excess lipid would have to be shunted to energy-wasting pathways, such as brown adipose tissue thermogenesis, in order to avert hyperlipidemia.

Conclusions It is now clear that members of both the PPAR and C/EBP families of transcriptional regulators play important roles in the development of adipose cells. Under certain conditions, PPARy, C/EPBa and C/EBPB can each stimulate adipogenesis when expressed individually in fibroblastic cells. In addition, PPARy and C/EPBa stimulate adipogenesis synergistically when co-expressed. It should now be possible to define the structural characteristics of these factors that are important for their adipogenic activity and to define the interactions of these proteins with co-activators and/or the core transcriptional machinery. It will also be important to understand the mechanisms by which adipocyte differentiation is coupled to the cessation of cell growth. At some level, these factors must interact, either directly or indirectly, with the cell cycle machinery. Finally, as PPARy is likely to function as a ligand-activated transcription factor, it should be possible to develop specific agonists and antagonists of this receptor. Such molecules might someday allow the regulation of adipogenesis and lipid metabolism in the setting of human metabolic disease.

Regulation of adipocyte gene expression and differentiation by PPARr Tontonoz, Hu and Spiegelman

575

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38.

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1 Tontonoz,

Hu

and 13M Spiegelman,

Institute, Author E-mail:

44 Uinney

Street,

Boston,

I>ana-Farber Cancer Massachusetts 021 15, USA.

for correspondence: UM Spiegelman. Uruce_Spiegehnan@dfci.harvard.edu