Regulation of Signal Transducers and Activators of Transcription

Biochimica et Biophysica Acta 1452 (1999) 188^196 www.elsevier.com/locate/bba

Regulation of signal transducers and activators of transcription (STATs) by e¡ectors of adipogenesis: coordinate regulation of STATs 1, 5A, and 5B with peroxisome proliferator-activated receptor-Q and C/AAAT enhancer binding protein-K

William C. Stewart a, Ron F. Morrison b, Sherry L. Young a, Jacqueline M. Stephens c;*

a Middle Tennessee State University, Department of Biology, Murfreesboro, TN 37132, USA b Boston University Medical Center, Department of Biochemistry, 80 East Concord St., Boston, MA 02118, USA c Louisiana State University, Department of Biological Sciences, Life Sciences Bldg., Room 508, Baton Rouge, LA 70803, USA

Received 29 July 1999; accepted 16 September 1999

Abstract

We have recently demonstrated that three signal transducers and activators of transcription (STAT) family members are induced during adipocyte differentiation (Stephens et al., J. Biol. Chem. 271 (1996) 10441^10444). Since STATs 1, 5A, and 5B are induced during adipocyte differentiation, we have examined the ability of these proteins to be regulated by components of the differentiation cocktail. In addition, we have examined the effects of potent effectors of differentiation on STAT protein expression during adipogenesis. A negative effector, tumor necrosis factor-K (TNFK), and a positive effector, a thiazolidinedione, were used in these experiments. Our results demonstrate that the expression of STATs 1, 5A, and 5B is not dramatically influenced by individual components of the differentiation cocktail. However, the expression of these three STAT family members tightly correlates with lipid accumulation. Moreover, the expression of STATs 1, 5A, and 5B, but not STATs 3 and 6, are regulated in an identical fashion to both C/AAAT enhancer binding proteins K and peroxisome proliferator-activated receptor-Q by TNFK and a thiazolidinedione. Furthermore, the expression of adipocyte-expressed JAK kinases are unaffected by effectors of differentiation. These findings suggest that three STAT family members may play a role in the regulation of adipocyte gene expression. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: STAT 1; STAT 5A; STAT 5B; Adipocyte; Adipogenesis

1. Introduction 3, 4, 5A, 5B, and 6) which, in response to stimulation of various receptors, mainly those for cytokines, are The STAT (signal transducers and activators of phosphorylated on tyrosine residues which causes transcription) family of mammalian transcription their translocation to the nucleus. Each STAT family factors is comprised of seven proteins (STATs 1, 2, member shows a distinct pattern of activation by cytokines and upon nuclear translocation can regulate the transcription of particular genes [2]. STATs * Corresponding author. Fax: +1-225-388-2597; can be activated by the JAK family of cytosolic ki- E-mail: [email protected] nases or can be activated independently of these ki-

BBAMCR 14553 28-10-99 W.C. Stewart et al. / Biochimica et Biophysica Acta 1452 (1999) 188^196 189 nases. Since the tissue distribution and function of sion and lipid accumulation during adipogenesis. each STAT is unique, the regulation of tissue-speci¢c Here, we demonstrate that the expression of STATs genes may be a physiological role for these proteins 1, 5A, and 5B tightly correlates with lipid accumu- [3]. Transgenic knockout experiments have revealed lation and the expression of both C/EBPK and PPARQ crucial roles for each known mammalian STAT [2], during adipogenesis. At this time, the function of and cell-speci¢c functions for STAT family members these three STAT family members in fat cells in un- have been identi¢ed. We have recently demonstrated known. However, these studies suggest that STATs that three STAT family members are induced during 1, 5A, and 5B are likely to be important transcrip- the di¡erentiation of 3T3-L1 cells from ¢broblasts to tional regulators of adipocyte development and/or adipocytes. This expression is not an artifact of the maintenance of adipocyte gene expression. cell culture system, as these same proteins are readily detectable in rat adipose cells [1]. The 3T3-L1 cell line di¡erentiates under the con- 2. Materials and methods trolled conditions of cell culture from ¢broblasts, or preadipocytes, into cells with the morphological and 2.1. Materials biochemical properties of adipocytes [4,5] in a process which closely resembles the development of adi- Dulbecco's modi¢ed Eagle's medium (DMEM) pose tissue in vivo. Adipocyte di¡erentiation is a was purchased from Gibco-BRL. Bovine and fetal complex process characterized by dramatic changes bovine serum (FBS) were obtained from Hyclone in cell morphology and gene expression which is and Gibco-BRL, respectively. Englitazone and dar- largely the result of coordinate transcriptional acti- glitazone were generously provided by P¢zer (Gro- vation of adipocyte-speci¢c genes and concomitant ton, CT). TNFK was obtained from Biosource Inter- silencing of preadipocyte genes which are not re- national. STAT antibodies were monoclonal IgGs quired for adipocyte function [6,7]. Regulation of purchased from Transduction Laboratories or poly- this program of gene expression involves two well- clonal IgGs from Quality Controlled Biochemicals or characterized families of transcription factors, the Santa Cruz. Anti-C/EBPK, a rabbit polyclonal, and C/EBPs (C/AAAT enhancer binding proteins) and anti-PPARQ, a mouse monoclonal, were purchased peroxisome proliferator-activated receptor (PPARs). from Santa Cruz. All other reagents were purchased Members of both of these transcription factor fami- from Sigma. lies are induced during adipocyte di¡erentiation. Ec- topic expression of members of both C/EBP and 2.2. Cell culture PPAR family members in non-precursor ¢broblasts has been shown to result in the induction of fat spe- Murine 3T3-L1 preadipocytes were plated and ci¢c genes and accumulation of lipids in cells [8^11]. grown to 2 days postcon£uence in DMEM with Thiazolidinediones have been shown to be positive 10% bovine serum. Medium was changed every 48 e¡ectors of adipocyte di¡erentiation [12] and selec- h. Cells were induced to di¡erentiate by changing the tive ligands for PPARQ [13,14]. Tumor necrosis fac- medium to DMEM containing 10% fetal bovine se- tor-K, TNFK, is a cytokine which was one of the ¢rst rum, 0.5 mM 3-isobutyl-1-methylxanthine, 1 WM identi¢ed inhibitors of adipocyte di¡erentiation [15] dexamethasone, and 1.7 WM insulin. After 48 h, and has recently been shown to inhibit C/EBPK and this medium was replaced with DMEM supple- PPARQ expression [16,17]. mented with 10% FBS, and cells were maintained Thus, in the present study, we have investigated in this medium until utilized for experimentation. the regulation of STAT protein expression by com- In some experiments, TNFK and/or englitazone pounds or proteins which are known e¡ectors of were added at the time of induction of di¡erentia- adipocyte di¡erentiation. We have examined the ef- tion. In other experiments, TNFK was added at var- fects of TNFK, thiazolidinediones, and the compo- ious times following the induction of di¡erentiation nents of the di¡erentiation cocktail on STAT expres- and was replaced with each media change.

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2.3. Preparation of whole cell extracts

Cultured 3T3-L1 cells were rinsed with phosphate- bu¡ered saline (PBS) and then harvested in a non- denaturing bu¡er containing 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EGTA, 1 mM EDTA, 1% Tri- ton X-100, 0.5% Nonidet P-40, 1 WM PMSF, 1 WM pepstatin, 50 trypsin inhibitory milliunits of aproti- nin, 10 WM leupeptin, and 2 mM sodium vanadate. Samples were extracted for 30 min on ice and then centrifuged at 15 000 rpm for 30 min. Supernatants containing whole cell extracts were analyzed for protein content using a BCA kit (Pierce) according to the manufacturer's instructions.

2.4. Gel electrophoresis and immunoblotting

Proteins were separated in 7.5 or 12% polyacryl- amide (acrylamide from National Diagnostics) gels containing sodium dodecyl sulfate (SDS) according to Laemmli [18] and transferred to nitrocellulose (Bio-Rad) in 25 mM Tris, 192 mM glycine, and 20% methanol. Following transfer, the membrane Fig. 1. Regulation of STAT protein expression by components was blocked in 4% milk for 1 h at room temperature. of the di¡erentiation cocktail. (1) Whole cell extracts were pre- Results were visualized with horseradish peroxidase pared 1 week (144 h) after the induction of di¡erentiation. (HRP)-conjugated secondary antibodies (Sigma) and Nine di¡erent di¡erentiation conditions were examined as indicated at the top of the ¢gure. One hundred Wg of each extract enhanced chemiluminescence (Pierce). was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. The STAT antibodies were 2.5. Oil Red O staining monoclonal or polyclonal IgGs. Results were visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies Oil Red O staining was performed following the and enhanced chemiluminescence. (2) Cells cultured in each of procedure described by Green and Kehinde [4]. the nine conditions indicated in panel 1 were analyzed for % adipocyte conversion by examining lipid accumulation by Oil Red O staining. The percent of adipocyte conversion was determined by counting cells with and without Oil Red O stain us- 3. Results ing phase contrast microscopy. The percent of cells containing lipid and the standard deviation was determined for each condi- The respective roles of the components of the nor- tion. This is a representative experiment independently performed three times. mal induction cocktail (MIX, DEX, insulin, and FBS) on the expression of STAT family members and lipid accumulation during adipocyte di¡erentia- whole cell extracts was performed one week follow- tion was examined. We exposed con£uent 3T3-L1 ing treatment with the induction cocktails. Three cells to di¡erentiation cocktails in which di¡erent plates from each condition were exposed to Oil- components of the normal induction cocktail were Red O staining and cells containing lipid droplets systematically omitted. Con£uent monolayers of were counted by visual examination using phase con- 3T3-L1 cells were exposed to the nine di¡erent cock- trast microscopy. Fig. 1A depicts the expression of tails indicated at the top of Fig. 1A for 48 h and then STATs 1, 3, 5A, 5B, and 6 in the nine di¡erent con- the cells from each condition were maintained in 10% ditions. Fig. 1B is a quantitation of Oil red O stain- FBS in DMEM. Oil Red O staining and isolation of ing which was used as an indicator of lipid accumu-

BBAMCR 14553 28-10-99 W.C. Stewart et al. / Biochimica et Biophysica Acta 1452 (1999) 188^196 191 lation. Both the % adipocyte conversion and the has not been fully investigated. Therefore, we exam- standard deviation were determined for each condi- ined the ability of TNFK to inhibit di¡erentiation at tion. Optimal di¡erentiation, as judged by Oil red O various times after the induction of di¡erentiation. In staining, was observed when 3T3-L1 cells were ex- this experiment, 3T3-L1 cells were induced to di¡er- posed to a complete di¡erentiation cocktail contain- entiate and 0.5 nM TNFK was added to the di¡er- ing FBS, MIX, DEX, and insulin and resulted in entiation cocktail at the time of induction of di¡er- 100% adipocyte conversion as has been previously entiation to inhibit adipogenesis. TNFK was also reported [9]. Parallel with the 100% conversion, added at 24, 48 72, 96, or 120 h after the induction STATs 1, 5A and 5B were highly expressed. Subse- of di¡erentiation. Whole cell extracts were isolated quent subtraction of individual components of the from each of the six time points 144 h after the in- cocktail resulted in varying degrees of incomplete duction of di¡erentiation and subjected to Western adipocyte conversion which was accompanied by a blot analysis (Fig. 2B). In addition, Oil-red O stain- comparable reduction in STAT expression. For ex- ing was performed on duplicate plates to examine ample, deletion of MIX from the cocktail reduced lipid accumulation (Fig. 2A). As shown in Fig. 2B, adipocyte conversion to 9% while STAT 1 expression the addition of TNFK to the induction cocktail (time was minimally detectable. A similar trend was ob- 0) resulted in an inhibition of STATs 1, 5A, and 5B served with STAT 5A and STAT 5B expression. As expression. When TNFK was added 24 h after the previously reported [9], cocktails containing MIX, initiation of di¡erentiation, we detected a small, but DEX, insulin, or FBS alone failed to result in signi¢- notable, increase in the expression of STATs 1, 5A, cant lipid accumulation (0^24%) as indicated in Fig. and 5B. However, when TNFK was added 48 h after 1B. In these conditions, we have demonstrated that the initiation of di¡erentiation there was a notable MIX, DEX, insulin, or FBS alone also failed to increase in STATs 1, 5A, and 5B expression (Fig. 2B) cause the induction of immunodetectable levels of which corresponded with the presence of lipid (Fig. STATs 1, 5A, and 5B expression. Neither STATs 3 2A, plate C). There was no immunodetectable di¡er- or 6 expression was e¡ected by the nine di¡erent ences in STAT expression from cells that received conditions which demonstrates the speci¢city of these TNFK treatment at 72, 96, or 120 h after the induc- results on STATs 1, 5A, and 5B expression. As tion of di¡erentiation. Furthermore, the addition of shown in Fig. 1A, the expression of STATs 1, 5A, TNFK at these times had no inhibitory e¡ects on and 5B did not appear to be directly regulated by adipocyte di¡erentiation as judged by Oil Red O individual components of the di¡erentiation cocktail, staining (Plates D^F). These results clearly demon- but the expression of these three STAT family mem- strate that the expression of STATs 1, 5A, and 5B bers tightly correlated with the presence of lipid correlate with adipocyte di¡erentiation and suggests droplets. However, a combination of di¡erentiation that the expression of these STATs is not directly e¡ectors may serve to a¡ect multiple regulatory fac- regulated by TNFK. In addition, our data shows tors such that, when they are combined, they can that there is a narrow window of time in which directly regulate STAT expression. Although the re- TNFK can alter the di¡erentiation program. Once sults varied slightly in three independent experi- this window has passed, di¡erentiation proceeds ments, the expression of STATs 1, 5A, and 5B al- even in the presence of TNFK. ways correlated with lipid accumulation and did not In addition to examining an inhibitor of di¡eren- appear to be exclusively regulated by components of tiation, we examined the e¡ects of englitazone on the di¡erentiation cocktail. It should also be noted STAT expression. Thiazolidinediones, such as engli- that STATs 1, 5A, and 5B are present in 3T3-L1 tazone, are enhancers of adipogenesis and speci¢c preadipocytes (data not shown), but their expression ligands for PPARQ [12^14]. Recent studies have is not readily immunodetectable when compared to also demonstrated that the inhibitory e¡ects of their induction following adipocyte di¡erentiation. TNFK on adipocyte di¡erentiation can be overcome The inhibitory e¡ects of TNFK on adipocyte dif- by the presence of a thiazolidinedione [19]. There- ferentiation are well described [15], yet a time course fore, we examined the regulation of STAT protein of TNFK addition on the inhibition of di¡erentiation expression under conditions of englitazone and/or

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Fig. 2. Analysis of lipid accumulation and STAT protein expression during a time course of TNFK addition during 3T3-L1 adipocyte di¡erentiation. Oil Red O staining and isolation of whole cell extracts was performed on 3T3-L1 cells 1 week (144 h) following the induction of di¡erentiation. Cells were induced to di¡erentiate at 2 days postcon£uence and TNFK was added at the initiation of di¡erentiation (time 0) and at various times following the induction of di¡erentiation. After 48 h, the induction medium was replaced with DMEM and 10% FBS. Once cells were exposed to TNFK, the culture was maintained in the presence of 0.5 nM TNFK and was freshly added with each medium change. (A) Cells cultured in each of the six conditions were analyzed for adipocyte conversion by examining lipid accumulation stained with Oil Red O. Cells on Plate A were induced to di¡erentiate with the standard induction cocktail. Cells on Plate B were induced to di¡erentiate with the standard induction, which was supplemented with 0.5 nM TNFK at 24 h after the induction of di¡erentiation. Cells on Plates C, D, E, and F were treated with TNFK at 48, 72, 96, and 120 h after the induction of di¡erentiation, respectively. (B) Whole cell extracts were isolated for each condition indicated above and were analyzed for STAT protein expression. One hundred Wgof each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. Samples were processed and results were visualized as described in the legend to Fig. 1. This is a representative experiment independently performed three times. 6

zone in the induction cocktail, or with both TNFK and englitazone in the induction cocktail. Following the 48-h treatment with the di¡erent induction cocktails, each condition was maintained in FBS and TNFK and/or englitazone treatment. Cells exposed to the normal di¡erentiation cocktail resulted in 100% di¡erentiation (Fig. 3A, Plate A) as indicated by Oil Red O staining. The presence of TNFK in the induction cocktail resulted in a substantial inhibition of lipid accumulation (Fig. 3A, Plate B). The presence of englitazone during adipocyte di¡erentiation resulted in accelerated lipid accumulation at earlier time points (data not shown), but this treatment resulted in the same end point of 100% di¡erentiation (Fig. 3A, Plate C). When both TNFK and englitazone were added to the induction cocktail, the cells di¡erentiated normally and Oil Red O staining re- sembled the control group (Fig. 3A, Plate D). In the same experiment, we also isolated whole cell TNFK treatment during adipogenesis. In this experi- extracts 1 week after the induction of di¡erentiation ment, 3T3-L1 cells were induced to di¡erentiate to examine the expression of STATs, C/EBPK, and under normal conditions (control), with 0.5 nM PPARQ (Fig. 3B). Normal di¡erentiation conditions TNFK in the induction cocktail, with 2 WM englita- resulted in an abundant induction of STAT 1, STAT

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Fig. 3. Coordinate regulation of C/EBPK, PPARQ, STAT 1, STAT 5A, and STAT 5B by TNFK and englitazone. Oil Red O staining and isolation of whole cell extracts was performed on 3T3-L1 cells 1 week (144 h) following the induction of di¡erentiation. Cells were induced to di¡erentiate at 2 days postcon£u- ence and TNFK and/or englitazone was added to the induction cocktail. After 48 h, the induction medium was replaced with DMEM and 10% FBS and was supplemented with TNFK and/ or englitazone in the treatment groups. (A) Cells cultured in each of the four di¡erentiation conditions were analyzed for adipocyte conversion by examining lipid accumulation by Oil Red O staining. Cells on Plate A were induced with the standard induction cocktail as described in Section 2. Cells on Plate B were induced to di¡erentiate with the standard induction cocktail in the presence of 0.5 nM TNFK. Cells on Plate C were induced to di¡erentiate with the standard induction cocktail in the presence of 2 WM englitazone. Cells on Plate D were induced to di¡erentiate with the standard induction cocktail in the presence of TNFK and englitazone. (B) Undi¡erentiated 3T3-L1 cells (preadipocytes) and cells cultured in each of the four conditions indicated above (Plates A, B, C, and D) were examined for STAT protein expression. One hundred Wgof each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. Samples were processed and results were visualized as described in the legend to Fig. 1. This is a representative experiment independently performed three times. 6

5A, STAT 5B, C/EBPK, and PPARQ when compared to preadipocyte extracts. TNFK-treated cells dis- played a substantial reduction in the expression of STAT 1, STAT 5A, STAT 5B, C/EBPK, and PPARQ while STATs 3 and 6 were completely una¡ected (Fig. 3B, Lane B). Cells which were treated with both TNFK and englitazone resulted in normal adipocyte conversion and an abundant expression of STAT 1, STAT 5A, STAT 5B, C/EBPK, and PPARQ (Fig. 3B, Lane D). As previously reported, thiazolidinediones can block the inhibitory e¡ects of TNFK on adipocyte di¡erentiation [19] and our current study has demonstrated that this treatment is also accompanied by an induction of STATs 1, 5A, and 5B expression. Similar results were observed in the 3T3-F442A cells and with the use of other thiazolidinediones, including darglitazone and pioglitazone (data not shown). We have previously shown that of the four JAK kinases (JAK 1, JAK 2, JAK 3, and Tyk 2), only JAK 1 and JAK 2 are expressed in 3T3-L1 cultured adipocytes and native rat adipocytes (unpublished observation). As shown in the bottom two panels

BBAMCR 14553 28-10-99 194 W.C. Stewart et al. / Biochimica et Biophysica Acta 1452 (1999) 188^196 of Fig. 3B, the expression of JAKs 1 and 2 was of induction of STATs 1, 5A, and 5B expression. completely una¡ected by e¡ectors of adipocyte dif- These results suggest that these STATs are not di- ferentiation. However, we have clearly demonstrated rectly inhibited by TNFK during adipogenesis. Alter- coordinate regulation of three STAT family members natively, a direct e¡ect of TNFK could be reduced with C/EBPK and PPARQ expression. In a manner following the initiation of di¡erentiation due to a identical to both C/EBPK and PPARQ, the expression change in the cellular pro¢le or a reduction in of STATs 1, 5A, and 5B tightly correlates with lipid TNFK sensitivity. Regardless, the expression of accumulation. STATs 1, 5A, and 5B is tightly associated with the fat-laden phenotype. One hypothesis which has developed from these 4. Discussion studies and previous studies is that a potential function of STAT 1 is the regulation of genes involved Members of the STAT family of transcription fac- with insulin sensitivity. We have shown that STAT 1 tors have been well documented to regulate gene ex- protein expression is markedly decreased in TNFK pression following their activation by cytokines and induced insulin resistance in fully di¡erentiated fat other stimuli [2]. Transgenic knockout experiments cells [1]. While glucose metabolism is an important have revealed crucial roles for each known mamma- function of the mature adipocyte, it is unlikely that lian STAT [2] and several studies have de¢ned tissue STAT 1 is essential for the morphological fat-laden speci¢c functions for STAT family members [3]. This phenotype. Observations of STAT 1 de¢cient mice is supported by numerous reports that demonstrate have led others to report that the only critical func- that particular STATs are activated di¡erently in re- tion of STAT 1 is to regulate a set of genes which sponse to growth factors and cytokines depending collectively provide innate immunity. Although these upon the cell type. We have recently shown that mice have no overt developmental abnormalities (i.e., three STAT family members are induced during the irregular fat pads), the regulation of gene expression di¡erentiation of 3T3-L1 cells from ¢broblasts to in the adipocytes of these animals has not been in- adipocytes. In our current experiments, we have vestigated. In contrast to STAT 1, the contributions clearly demonstrated that these three STATs are of STATs 5A and 5B to adipocyte morphology ap- not exclusively regulated by individual e¡ectors of pear to be much greater. Transgenic mice lacking di¡erentiation, but their expression tightly correlates either STAT 5A or 5B have signi¢cantly smaller fat with lipid accumulation and the fat-laden phenotype. pads and transgenic animals lacking both STATs 5A Moreover, this regulation is restricted to particular and 5B have fat pads only one-¢fth the size of wild- proteins in the JAK-STAT signaling pathway. The type animals [21]. Moreover, antisense STAT 5A has highly conserved regulation of STATs 1, 5A, and 5B been shown to inhibit the di¡erentiation of 3T3- by e¡ectors of adipogenesis suggests that the expres- F442A preadipocytes [22]. Collectively, these studies sion of these transcription factors are an important support the notion that these three STAT family event leading to the functionally mature adipocyte. members may be important regulators of gene ex- Currently, the inhibitory e¡ects of TNFK on adi- pression mediating adipocyte function and that pogenesis are well documented, but the mechanisms STAT 5A and STAT 5B are likely to play essential involved are poorly understood. In our current ex- regulatory roles during di¡erentiation. periments, we have shown that TNFK must be Like C/EBPK and PPARQ, the protein expression present within the ¢rst 24 h to have a substantial of STATs 1, 5A, and 5B are highly induced during e¡ect on the inhibition of lipid accumulation. This adipocyte di¡erentiation. Yet, C/EBPK and PPARQ time period corresponds with mitotic clonal expan- have a much more limited tissue distribution than sion and the regulation of retinoblastoma proteins by STAT proteins. We have recently demonstrated TNFK which may contribute to the inhibitory e¡ects that the STAT family members can be rapidly acti- of this cytokine on fat cell di¡erentiation [20]. None- vated in cultured and native rat adipocytes by several theless, we have shown that the inhibitory e¡ect of growth factors, suggesting that these proteins medi- TNFK on lipid accumulation coincides with the lack ate cytokine induced gene regulation [23,24]. Unlike

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C/EBPK and PPARQ, STAT proteins are signaling [4] H. Green, O. Kehinde, Sublines of mouse 3T3 cells that molecules which can activate or repress transcription. accumulate lipid, Cell 1 (1974) 113^116. However, the speci¢c function of these proteins in [5] H. Green, O. Kehinde, An established cell line and its di¡erentiation in culture II. Factors a¡ecting the adipose conver- adipocytes is unknown. Activated STAT proteins sion, Cell 5 (1975) 19^27. may be responsible for the regulation of housekeep- [6] P. Cornelius, O.A. MacDougald, M.D. Lane, Regulation of ing genes, yet our results suggest that STATs 1, 5A, adipocyte development, Annu. Rev. Nutr. 14 (1994) 99^129. and 5B may be involved in the regulation of adipo- [7] C.M. Smas, H.S. Sul, Control of adipocyte di¡erentiation, cyte expressed genes which code for proteins that Biochem. J. 309 (1995) 697^710. [8] P. Tontonoz, E. Hu, B.M. Spiegelman, Stimulation of adipo- confer insulin sensitivity and/or the ability to accu- genesis in ¢broblasts by PPARQ2, a lipid-activated transcrip- mulate lipid. These studies clearly demonstrate that tion factor, Cell 79 (1994) 1147^1156. three STAT family members are regulated in a sim- [9] W. Yeh, Z. Cao, M. Classon, S.L. McKnight, Cascade reg- ilar fashion to both C/EBPK and PPARQ. Whereas, ulation of terminal adipocyte di¡erentiation by three mem- the expression of STATs 3 and 6 and adipocyte-ex- bers of the C/EBP family of leucine zipper proteins, Genes pressed JAK kinases are not regulated by e¡ectors of Dev. 9 (1995) 168^181. [10] F.T. Lin, M.D. Lane, CCAAT/enhancer binding protein K is adipogenesis. These results suggests that STATs 1, su¤cient to initiate the 3T3-L1 adipocyte program, Proc. 5A, and 5B play a role in the regulation of adipocyte Natl. Acad. Sci. USA 91 (1994) 8757^8761. gene expression and our current e¡orts are focused [11] S.O. Freytag, D.L. Paielli, J.D. Gilbert, Ectopic expression on the identi¢cation of adipocyte genes which are of the CCAAT/enhancer-binding protein K promotes the regulated by STAT proteins. Although, the expres- adipogenic program in a variety of mouse ¢broblastic cells, Genes Dev. 8 (1994) 1654^1663. sion of STATs 3 and 6 does not change during di¡er- [12] R.F. Kletzien, L.A. Foellmi, P.K. Harris, B.M. Wyse, S.D. entiation, the activation, or tyrosine phosphoryla- Clarke, Adipocyte fatty acid-binding protein: regulation of tion, of these transcription factors may play a role gene expression in vivo and in vitro by an insulin-sensitizing during adipogenesis. Determining the function of agent, Mol. Pharmacol. 4 (1992) 558^562. STATs during adipogenesis and deciphering the [13] B.M. Forman, P. Tontonoz, J. Chen, R.P. Brun, B.M. Spie- 12;14 complex interaction of these and other adipogenic gelman, R.M. Evans, 15-Deoxy-v -prostaglandin J2 is a ligand for the adipocyte determination factor PPARQ, Cell transcription factors will provide a better under- 83 (1995) 803^812. standing of the molecular processes of adipocyte dif- [14] J.M. Lehmann, L.B. Moore, T.A. Smith-Oliver, W.O. Wil- ferentiation. kison, T.M. Willson, S.A. Kliewer, An antidiabetic thiazolidinedione is a high a¤nity ligand for Peroxisome Prolifera- tor-Activated Receptor Q (PPAR Q), J. Biol. Chem. 270 (1995) 12953^12956. Acknowledgements [15] F.M. Torti, S.V. Torti, J.W. Larrick, G.M. Ringold, Mod- ulation of adipocyte di¡erentiation by tumor necrosis factor This work was supported by National Institutes of and transforming growth factor beta, J. Cell Biol. 108 (1989) Health Grant R29DK52968-01 and a Career Devel- 1105^1113. opment Award from the American Diabetes Associ- [16] J.M. Stephens, P.H. Pekala, Transcriptional repression of ation to J.M.S. the GLUT4 and C/EBP genes in 3T3-L1 adipocytes by tumor necrosis factor-K, J. Biol. Chem. 266 (1991) 21839^ 21845. [17] M.R. Hill, M.D. Young, C.M. McCurdy, J.M. Gimble, De- creased expression of murine PPARgamma in adipose tissue References during endotoxemia, Endocrinology 138 (1997) 3073^3076. [18] U.K. Laemmli, Cleavage of structural proteins during the [1] J.M. Stephens, R.F. Morrison, P.F. Pilch, The expression assembly of the head of bacteriophage T4, Nature 227 and regulation of STATs during 3T3-L1 adipocyte di¡eren- (1970) 680^685. tiation, J. Biol. Chem. 271 (1996) 10441^10444. [19] D. Szalkowski, S. White-Carrington, J. Berger, B. Zhang, [2] J.E. Darnell Jr., STATs Gene Regul. Sci. 277 (1997) 1630^ Antidiabetic thiazolidinediones block the inhibitory e¡ect 1635. of tumor necrosis factor-alpha on di¡erentiation, insulin- [3] C. Schindler, J.E. Darnell, Transcriptional responses to po- stimulated glucose uptake, and gene expression in 3T3-L1 lypeptide ligands: The JAK STAT pathway, Annu. Rev. cells, Endocrinology 136 (1995) 1474^1481. Biochem. 64 (1995) 621^651. [20] R.E. Lyle, V.M. Richon, R.E. McGehee Jr., TNFK disrupts

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