The Regulation of Fatty Acid Synthase by STAT5A
http://www.100md.com
糖尿病学杂志 2005年第7期
the Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana
ABSTRACT
Growth hormone (GH) diminishes adipose tissue mass in vivo and decreases expression and activity of fatty acid synthase (FAS) in adipocytes. GH and prolactin (PRL) are potent activators of STAT5 and exert adipogenic and antiadipogenic effects in adipocytes. In this study, we demonstrate that GH and PRL decrease the mRNA and protein levels of FAS in 3T3-L1 adipocytes. We present evidence that indicates that FAS is repressed at the level of transcription. In addition, PRL responsiveness was shown to exist between eC1,594 and eC700 of the rat FAS promoter. Moreover, responsiveness to PRL was abolished with mutation of a site at position eC908 to eC893, which we have shown to bind STAT5A in a PRL-dependent manner. Taken together, these data strongly suggest that PRL directly represses expression of FAS in adipocytes through STAT5A binding to the eC908 to eC893 site. Furthermore, our results indicate that STAT5A has an antilipogenic function in adipocytes and may contribute to the regulation of energy balance.
STAT5 proteins were first identified as mammary gland factor, a protein from mouse mammary glands that is bound to the -casein promoter (1). It was subsequently determined that mammary gland factor was two closely related proteins, STAT5A and STAT5B (2), that are expressed in all tissues (3). Transgenic deletion studies in mice indicate that a major function of STAT5 proteins is the regulation of mammary tissue development (4), but multiple lines of evidence suggest a role for STAT5 proteins in the modulation of adipocyte function. During differentiation of 3T3-L1 adipocytes, expression levels of STAT5A and 5B were highly induced (5). Furthermore, growth hormone (GH)-dependent adipogenesis was attenuated by STAT5 antisense oligonucleotides (6), and constitutively active STAT5 can replace the requirement for GH in the adipogenesis of murine 3T3-F442A cells (7). Moreover, ectopic expression of STAT5A conferred adipogenesis in two different nonprecursor cell lines (8). Transgenic deletion of STAT5A, STAT5B, or both STAT5 genes in mice resulted in significantly reduced fat-pad sizes compared with wild-type mice (4). Yet, in primary cultures of adipose tissue from these animals, GH did not stimulate lipolysis as it does in adipocytes from wild-type mice (9). Also, chronic administration of GH in pigs decreases adipose tissue growth through the attenuation of lipogenesis (10,11) and reduces adipocyte conversion in rat primary adipocytes (12). More recent studies suggest that some of the antiadipogenic effects of GH may be mediated by STAT5A. In rat primary preadipocytes, GH-induced STAT5 inhibits aP2 expression (13). In summary, STAT5 proteins appear to have both adipogenic as well as antiadipogenic effects in adipose tissue of various species, which may depend on the developmental stage of the tissue.
PRL is a peptide hormone primarily known for its role in mammary gland development during lactation, but it has been shown to have pleiotropic effects in a variety of tissues (14). PRL activates multiple signal transduction pathways, including mitogen-activated protein kinase (15) and phosphatidylinositol-3 kinase (16); however, the JAK/STAT pathway is the predominant signaling cascade activated by PRL, resulting in the nuclear translocation of STAT5 proteins (3).
The regulation of mammary tissue by PRL is well characterized, but there is also evidence that PRL modulates adipose tissue. PRL-R is expressed in both mouse (17) and human (18) adipose tissue and is induced during adipogenesis of bone marrow stromal cells (19). Furthermore, ectopic expression of the PRL-R in murine NIH-3T3 cells resulted in efficient adipocyte conversion and activation of the aP2 promoter in a PRL-dependent manner (20). Taken together, these observations strongly suggest a role for PRL in the modulation of adipocyte function. Furthermore, the occurrence of obesity has been correlated with hyperprolactinomas in humans (21). In opposition to these adipogenic effects, PRL has been shown to induce lipolysis in rabbits (22) and mouse adipose tissue explants (23). In addition, studies have shown that PRL reduces lipoprotein lipase (LPL) activity in cultured human adipocytes (18) and the activity of LPL and fatty acid synthase (FAS) in adipose tissue of lactating mice (24). Thus, PRL exerts adipogenic and antilipogenic effects on adipose tissue in a variety of species.
FAS is the key enzyme in de novo lipogenesis, catalyzing the reactions for the synthesis of long-chain fatty acids (25). The importance of FAS in adipocyte function is underscored by the inhibition of adipogenesis of 3T3-L1 cells by C75, an allosteric inhibitor of FAS (26). FAS is regulated primarily at the level of transcription and is sensitive to nutritional and hormonal regulation (25). Previous studies have demonstrated that GH, a cytokine that activates STAT5 in adipocytes (27), abrogates the induction of FAS expression by insulin and downregulates basal expression of FAS in 3T3-F442A cells (28). Although insulin-regulated regions of the FAS promoter have been extensively studied, there has not been a conclusive study to characterize a GH-responsive region or to determine the potential role of STATs in the modulation of FAS expression. Since GH and PRL are potent activators of STAT5 (27,20), we hypothesized that STAT5 directly regulates the expression of FAS. In this study, we have demonstrated that GH and PRL treatment of 3T3-L1 adipocytes resulted in decreased FAS mRNA and protein. In addition, we have identified a region within the FAS promoter that is responsive to PRL. This region contains a nonconsensus STAT5 binding site that, when mutated, results in a loss of sensitivity to PRL. Our results clearly demonstrate that STAT5A binds to this nonconsensus sequence and strongly suggest that FAS is a direct target for regulation by STAT5. These data suggest a novel means for regulation of FAS expression in adipocytes and reveal a mechanism by which STAT5 proteins and PRL exert antiadipogenic effects.
RESEARCH DESIGN AND METHODS
Dulbecco’s modified Eagle medium (DMEM) and leukemia inhibitory factor (LIF) were purchased from Invitrogen. Fetal bovine serum was purchased from Atlanta Biologicals, and calf serum was purchased from Biosource. Polyclonal phospho-specific STAT5 (Y694) antibody and FAS antibody were purchased from BD Transduction Laboratories. STAT1 antibody was purchased from Upstate Biotechnology. Porcine growth hormone, ovine PRL (both prepared from pituitaries), and cycloheximide were purchased from Sigma. STAT3 and STAT5A antibodies were purchased from Santa Cruz. [-32P] dCTP was purchased from Perkin-Elmer and Amersham Biosciences. Deoxynucleotide thymine triphosphate, dATP, and dGTP were purchased from Amersham Biosciences. Oligonucleotides were purchased from Integrated DNA Technologies. DNase polymerase I large (Klenow) fragment was purchased from Promega.
Cell culture.
Murine 3T3-L1 preadipocytes were plated and grown to 2 days postconfluence in DMEM containing 10% bovine serum. Medium was changed every 48 h. Cells were induced to differentiate by changing the medium to DMEM containing 10% fetal bovine serum, 0.5 mmol/l 3-isobutyl-methylxanthine, 1 eol/l dexamethasone, and 1.7 eol/l insulin. After 48 h, this medium was replaced with DMEM supplemented with 10% fetal bovine serum, and the cells were maintained in this medium until used for experimentation.
Preparation of whole-cell extracts.
Cell monolayers were rinsed with PBS and then harvested in a nondenaturing buffer containing 10 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1 mmol/l EGTA, 1 mmol/l EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 eol/l phenylmethylsulfonyl fluoride, 1 eol/l pepstatin, 50 trypsin inhibitory mU aprotinin, 10 eol/l leupeptin, and 2 mmol/l sodium vanadate. Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4°C for 15 min. Supernatants containing whole-cell extracts were analyzed for protein content by bicinchoninic acid analysis (Pierce) according to the manufacturer’s instructions.
RNA analysis.
Total RNA was isolated from cell monolayers with Trizol (Invitrogen), according to manufacturer’s instructions with minor modifications. For Northern blot analysis, 15 e total RNA was denatured in formamide and electrophoresed through a formaldehyde/agarose gel. The RNA was transferred to Zeta Probe-GT (Bio-Rad) in a buffer containing 75 mmol/l sodium citrate tribasic, 10 mmol/l NaOH, and 750 mmol/l NaCl. The blots were cross-linked, hybridized, and washed as previously described (29). Probes were labeled by random priming using Klenow fragment and [-32P] dCTP.
Constructs.
The rat FAS promoter eC250 to +65/luciferase construct was generously provided by Dr. Steve Clarke. The rat FAS promoter eC1,594 to +65/luciferase and eC700 to +65/luciferase constructs were generously provided by Dr. Peter Tontonoz. The FAS eC1,594 to +65/luciferase construct was mutated at positions eC901 and eC902 within the STAT5A binding site using the QuikChange Site Directed Mutagenesis Kit, according to manufacturer’s instructions (Stratagene). The following oligonucleotide and corresponding antisense oligonucleotide were used to alter the STAT binding site with the altered bases underlined: GGG AGG GTG AGG GTC AAG GAA ACC AGC AAC TCA GG. Sequence analysis was performed to confirm the presence of the mutated bases using Big Dye Terminator Extension Reaction (ABI Prism). The minimum promoter thymidine kinase (TK) renilla vector was purchased from Promega.
Transfection and luciferase assay.
3T3-L1 preadipocytes were transiently cotransfected with the various FAS promoter constructs and the TK/renilla vector to control for transfection efficiency, as previously described (30), using Polyfect (Qiagen) according to manufacturer’s instructions. Cell lysates were analyzed for firefly and renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega). Relative light units were determined by dividing firefly luciferase activity by renilla luciferase activity. Results are given as ±SD.
Preparation of nuclear and cytosolic extracts.
Cell monolayers were rinsed with PBS and then harvested in a nuclear homogenization buffer containing 20 mmol/l Tris (pH 7.4), 10 mmol/l NaCl, 3 mmol/l MgCl2, 1 eol/l dithiothreitol, 1 eol/l phenylmethylsulfonyl fluoride, 1 eol/l pepstatin, 50 trypsin inhibitory mU aprotinin, 10 eol/l leupeptin, and 2 mmol/l sodium vanadate. Igepal CA-630 (Nonidet P-40) was added to a final concentration of 0.15%, and cells were homogenized with 16 strokes in a Dounce homogenizer. The homogenates were centrifuged at 1,500 rpm for 5 min. Supernatants were saved as cytosolic extract, and the nuclear pellets were resuspended in one-half volume of nuclear homogenization buffer and were centrifuged as before. The pellet of intact nuclei was resuspended again in one-half of the original volume of nuclear homogenization buffer and centrifuged again. The majority of the pellet (intact nuclei) was resuspended in an extraction buffer containing 20 mmol/l HEPES (pH 7.9), 420 mmol/l NaCl, 1.5 mmol/l MgCl2, 0.2 mmol/l EDTA, 1 eol/l dithiothreitol, 1 eol/l phenylmethylsulfonyl fluoride, 1 eol/l pepstatin, 50 trypsin inhibitory mU aprotinin, 10 eol/l leupeptin, 2 mmol/l sodium vanadate, and 25% glycerol. Nuclei were extracted for 30 min on ice. The samples were subjected to centrifugation at 10,000 rpm at 4°C for 10 min. Supernatants containing nuclear extracts were analyzed for protein content, using a bicinchoninic acid protein assay kit (Pierce).
Gel electrophoresis and immunoblotting.
Proteins were separated in 6% polyacrylamide (National Diagnostics) gels containing SDS according to Laemmli (31) and transferred to nitrocellulose (Bio-Rad) in 25 mmol/l Tris, 192 mmol/l glycine, and 20% methanol. Following transfer, the membrane was blocked in 4% milk overnight at 4°C. Results were visualized with horseradish peroxidaseeCconjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and enhanced chemiluminescence (Pierce).
Electrophoretic mobility shift analysis.
Double-stranded oligonucleotides were end labeled with [-32P] dCTP using Klenow. Binding reactions were performed with nuclear extracts, according to Ritzenthaler et al. (32). Protein-DNA complexes were resolved and visualized, as previously described (30). For supershift analysis, nuclear extracts were preincubated with 4 e antibody for 1 h at room temperature.
RESULTS
GH is known to reduce adipose tissue in vivo (11) and decrease the expression of FAS in adipocytes (10). GH is also a potent activator of STAT5 in adipocytes (27). Hence, we hypothesized that STAT5 proteins directly modulate FAS expression. Therefore, we examined the regulation of FAS by two STAT5 activators, GH and PRL. As shown in Fig. 1A, we observed that both GH and PRL resulted in a decrease in FAS mRNA in fully differentiated 3T3-L1 adipocytes. GH decreased FAS mRNA by 12 h of treatment, and PRL treatment resulted in decreased expression within 6 h. In an independent experiment, we observed that both GH and PRL also resulted in a decrease in expression of FAS protein levels (Fig. 1B). To demonstrate the specificity of this effect, fully differentiated 3T3-L1 adipocytes were treated with PRL for 8 h with the various doses indicated in Fig. 1C. There was a notable decrease in FAS protein level with 8.8 nmol/l PRL treatment, and the decrease of FAS protein levels by PRL treatment was dose dependent. An acute treatment of 1.3 eol/l PRL was included as a positive control for STAT5 phosphorylation. The level of STAT5A expression was unchanged by GH or PRL (Fig. 1B and C) and is shown to indicate even loading of protein samples.
Clearly, the analysis of mRNA and whole-cell extracts demonstrated that activators of STAT5 decreased expression of FAS protein and mRNA. Yet, it was unclear if the effects of GH and PRL were mediated by affecting FAS transcription and/or protein turnover. To assess whether the effects of PRL on FAS expression could be attributed to changes in the turnover of FAS protein, we examined the loss of FAS in the presence of cycloheximide (5 eol/l) or ethanol, a vehicle control. Whole-cell extracts were collected at various times and used for Western blot analysis. As shown in Fig. 2, either cycloheximide or PRL treatment caused a decrease in FAS protein. In the presence of cycloheximide, a loss of FAS was observed at 8 h, regardless of the presence of PRL. PRL treatment alone decreased the level of FAS within 12 h of treatment. These results indicate that PRL does not affect the turnover of FAS protein in 3T3-L1 adipocytes and suggest that PRL may exert its effects on FAS at the level of transcription.
Our data indicate that PRL directly regulates the expression of FAS. Recent studies by Yin, Clarke, and Etherton (28) have shown that GH abrogates the induction of FAS by insulin and suggested that the eC112 to +65 region of the rat FAS promoter was sensitive to the regulation by GH. Therefore, we hypothesized that a STAT5 recognition element may be present in this region. To address this question, 3T3-L1 preadipocytes were transiently transfected with a luciferase construct containing the FAS promoter fragment of eC250 to +65. After 48 h, cells were stimulated with PRL for the times indicated in Fig. 3Aand were then analyzed for luciferase activity as described in RESEARCH DESIGN AND METHODS. We observed that PRL treatment had no significant effects on the relative luciferase activity (Fig. 3A), clearly demonstrating that the eC250 to +65 region of the FAS promoter is not sensitive to PRL. Therefore, we examined the PRL responsiveness of two additional rat FAS promoter/luciferase constructs that incorporated larger regions, eC1,594 to +65 and eC700 to +65, of the promoter. Transfection of these constructs into 3T3-L1 cells revealed a PRL-responsive region present between eC1,594 and eC700 of the rat FAS promoter. As shown in Fig. 3B, treatment with PRL resulted in a 64% decrease in luciferase activity of the rat FAS promoter (eC1,594 to +65)/luciferase construct. Although the basal level of luciferase activity for the rat FAS promoter (eC700 to +65)/luciferase construct was similar to that of the eC1,594 to +65 construct, PRL had no effect on the level of luciferase activity (Fig. 3B). These data demonstrate that a PRL-sensitive region exists between eC1,594 and eC700 of the FAS promoter and support our hypothesis that FAS is transcriptionally regulated by STAT5 activators.
Since PRL is a potent activator of STAT5, we hypothesized that the eC1,594 to eC700 region of the FAS promoter may contain a STAT5 binding site that conferred PRL responsiveness. Therefore, we examined the rat FAS promoter (GenBank X62889) for the presence of STAT consensus sites (TTCNNNGAA). An examination of the FAS promoter did not result in the identification of any sequences that precisely matched the STAT consensus site. However, as shown in Table 1, we identified four regions that were similar to the consensus sequence. To evaluate these potential STAT5 binding sites, we performed a series of electromobility shift assays (EMSA). For these experiments, nuclear and cytosolic extracts were prepared from 3T3-L1 adipocytes acutely treated with PRL for 15 min. As shown in Fig. 4A, PRL did not induce the binding of nuclear protein complexes to the eC951 to eC933, eC1,226 to eC1,214, or eC4,639 to eC4,623 regions of the FAS promoter. The induction of binding by a PRL-activated protein complex to the rat -casein STAT5 binding site (1) is included as a positive control. However, we did observe PRL-dependent binding by a nuclear protein complex to the eC908 to eC893 region of the FAS promoter (Fig. 4B). To determine the specificity of binding, an EMSA was performed using a mutant version of the eC908 to eC893 oligonucleotide, in which two nucleotides were changed (Table 1). As shown in Fig. 4B, there was no detectable binding to the mutant form of the binding site following PRL stimulation. In addition, binding of the PRL-activated protein complex was successively competed with increasing concentrations of the unlabeled eC908 to eC893 oligonucleotide (Fig. 4C). Further evidence of specificity is shown in Fig. 4D, in which an excess of the eC908 to eC893 oligonucleotide competed away binding induced by PRL treatment (lane 4), whereas the mutant form of the oligonucleotide did not (lane 5). Although we did not detect binding to the eC951 to eC933 site, an excess of this oligonucleotide moderately diminished binding to the radiolabeled eC908 to eC893 probe (lane 6). Yet, there was no appreciable competition of binding by the eC1,226 to eC1,214 oligonucleotide (lane 7). As anticipated, binding was competed away with the STAT5 binding site of the rat -casein promoter. Interestingly, a STAT3 binding site, eC168 to eC148 from the rat 2-macroglobulin promoter (33), also resulted in binding competition. However, binding was not competed with a STAT1 binding site that is present at eC221 to eC207 of the peroxisome proliferatoreCactivated receptor 2 promoter (34).
To determine whether the protein complex binding the eC908 to eC893 contained STAT proteins, we performed supershift analysis using antibodies directed against the STAT proteins expressed in adipocytes. As shown in Fig. 5, the protein complex induced by PRL was fully supershifted with a STAT5A antibody (lane 5) and weakly supershifted with an STAT5B antibody (lane 6). STAT1 and STAT3 antibodies had no effect on the mobility of the complex (lanes 3 and 4). We also investigated the specificity of binding by STAT proteins by comparing the binding induced by LIF (0.5 nmol/l), a cytokine that activates STAT1 and STAT3 but not STAT5 in 3T3-L1 adipocytes (35). LIF stimulated binding to the eC908 to eC893 site by a protein complex that exhibited highly reduced binding affinity and slightly faster mobility than the complex induced by PRL (Fig. 5). Moreover, the LIF-induced complex was supershifted by a STAT1 antibody (lane 8) but not with antibodies for STAT3 or STAT5A (lanes 9 and 10). We used three other STAT3 antibodies capable of supershifting STAT3 complexes and did not observe any STAT3 LIF-stimulated binding to the eC908 to eC893 site (data not shown).
Our data clearly demonstrate that the eC908 to eC893 region of the rat FAS promoter binds nuclear PRL-activated STAT5 proteins in vitro. To determine whether this region of the FAS promoter contributed to the regulation of FAS by PRL in living cells, we performed site-specific mutagenesis to alter two basepairs at positions eC902 and eC901 within the rat FAS promoter (eC1,594 to +65)/luciferase construct. We have shown that this mutation abolished binding of PRL-induced proteins to this site (Fig. 4B and D). Transfection of the wild-type and mutant constructs into 3T3-L1 cells revealed that the basal level of luciferase activity was unaffected by mutation of the eC902 and eC901 bp of the FAS promoter (Fig. 6). However, the 60% decrease in luciferase activity induced by PRL for the wild-type construct was eliminated with the mutation. Thus, these data clearly indicate that the eC908 to eC893 site of the FAS promoter is sensitive to PRL and suggest that this site confers the negative regulation of FAS by PRL-activated STAT5A protein complexes.
DISCUSSION
The novel findings in this study include data demonstrating that FAS levels are decreased following stimulation with activators of STAT5 in 3T3-L1 adipocytes, the identification of a PRL-responsive region of the rat FAS promoter, and the characterization of a STAT5 binding site in this region. These results strongly suggest that STAT5A directly represses the expression of FAS in adipocytes. Moreover, our data indicate that STAT5A has an antilipogenic function in murine adipocytes, in addition to an adipogenic role previously described by our laboratory (8) and others (4,6,7).
Previous studies have shown that GH, an activator of STAT5, attenuated the induction of FAS in adipocytes by insulin in 3T3-F442A adipocytes (10) and decreased expression of FAS in vivo in pigs (11). We observed that two activators of STAT5 in adipocytes, GH and PRL, decreased protein and mRNA expression of FAS in 3T3-L1 adipocytes. These results are consistent with the findings that GH in cows (36) and PRL in rats (24) can inhibit the activity of FAS in adipose tissue. Surprisingly, our results revealed that the effects of GH on FAS are transient and reversible, whereas the effects of PRL are more pronounced with time. The differences in the action of these two STAT5 activators may be due to receptor levels or specific signal proteins that have yet to be identified. Alternatively, the differences we observe in GH and PRL effects on FAS in our murine cells may also be affected by different species of hormones we used (sheep and pig) on murine cells. Our results show that PRL did not affect the protein turnover of FAS but indicate that changes in FAS levels are mediated at the transcriptional level. Our experiments are supported by other studies (25,28,37eC48) that demonstrate that FAS can be modulated at the transcriptional level. However, increased turnover of FAS mRNA in 3T3-F442A adipocytes has been demonstrated as one of the means through which GH attenuates the induction of FAS by insulin (10). Nonetheless, our results strongly suggest that the PRL-induced repression of FAS is mediated by an inhibition of transcription.
To elucidate the mechanism of transcriptional regulation by PRL, we investigated the regulation of the FAS promoter. Although a previous study (28) indicated that the eC112 to +65 region of the rat FAS promoter was sensitive to regulation by GH in murine cells, we did not observe PRL-mediated regulation of the rat FAS promoter within this region in murine cells (Fig. 3A). However, our analysis of larger regions of the rat FAS promoter clearly indicated that a PRL-responsive region existed between eC1,594 and eC700 of the rat FAS promoter (Fig. 3B). Yin, Clarke, and Etherton (28) have previously shown that GH attenuated the stimulation by insulin of the rat FAS promoter (eC112 to +65)/luciferase construct, a region of the promoter that does not contain a STAT consensus site. Furthermore, in another study (37), it was demonstrated that staurosporine, an inhibitor of JAK/STAT signaling, did not block the effect of GH on insulin-stimulated FAS expression in murine cells. Thus, it is unlikely that STAT5 proteins mediate the inhibitory effects of GH on insulin regulation of FAS. Yet, in light of our current findings, we postulate that the repression of basal levels of FAS by PRL is directly regulated by STAT5 proteins via binding to a site within the eC1,594 to eC700 region of the rat FAS promoter. We also hypothesize that the delayed effects we observe on FAS mRNA and protein are due to the stability of the FAS mRNA and protein. Our studies indicate that FAS protein is very stable (Fig. 2), and preliminary studies suggest that the FAS mRNA is more stabile than other adipocyte mRNAs (data not shown).
Since PRL is a potent activator of STAT5 (Fig. 1B), we examined the rat FAS promoter for sites that resembled the STAT consensus sequence, TTCNNNGAA. We identified four sites that were similar to the STAT consensus, but our analysis by EMSA indicated that only one site, at position eC908 to eC893, was bound by a PRL-induced nuclear protein complex in a highly specific manner (Fig. 4). Similar results were obtained with GH (data not shown). In our experiments, the PRL-induced protein complex was fully supershifted by a STAT5A antibody (Fig. 5). The functional significance of the eC908 to eC893 site was determined by mutating two nucleotides at positions eC902 and eC901, within the STAT5 binding site of the rat FAS promoter (eC1,594 to +65)/luciferase construct. Mutation of this site completely abrogated the downregulation by PRL that was observed with the wild-type construct (Fig. 6). Taken together, these data strongly suggest that eC908 to eC893 of the rat FAS promoter is a STAT5 binding site, which confers the negative transcriptional regulation of FAS by PRL. These results support our hypothesis that STAT5 directly represses expression of FAS in adipocytes.
The association of increased transcription of FAS in rodent obesity (49) and the inhibition of adipogenesis by an allosteric inhibitor of FAS (26) are highly indicative that regulation of FAS expression and activity in adipocytes is an important control of energy homeostasis. The characterization of a STAT5 binding site in the rat FAS promoter identifies a novel mechanism for repression of FAS expression. The eC908 to eC893 site of the rat FAS promoter is also present in the murine FAS promoter (AL663090) at position eC895 to eC887. Interestingly, the human FAS promoter (AF250144) contains a site at eC1,091 to eC1,083 (TTCGAGGAA) that is different from the sequence identified from the rodent promoters but complements the STAT consensus sequence TTCNNNGAA. Hence, although the precise sequence of the STAT5 binding site is not conserved across species, the binding by STAT5 to these sites in the FAS promoter may be an evolutionarily retained mechanism of regulating FAS expression in adipose tissue.
Our observation that STAT5A, not other adipocyte STATs, preferentially binds to the eC908 to eC893 site (Fig. 5) is consistent with previous studies (50,51) demonstrating that STAT proteins bind similar sequences but exhibit subtle differences in affinity for nucleotides between and beyond the half-sites of the palindrome. This specificity in STAT binding may account for the distinct repertoire of target genes regulated by each STAT protein (51). Our data suggest that the regulation of transcription mediated by the eC908 to eC893 region of the rat FAS promoter is primarily regulated by STAT5A binding.
PRL has been shown to have antilipogenic effects in adipose tissue through inhibition of LPL expression and the repression of FAS (24) and LPL (18) activity. At this time, our studies and others suggest that both PRL and GH repress FAS expression. However, these growth factors appear to have different effects (transient versus sustained) on FAS regulation. These differences may be attributable to expression levels or actions of specific GH and PRL signaling proteins and/or specific cell types. Nonetheless, our results strongly suggest that PRL modulates the expression of FAS in adipocytes through STAT5A and supports a role for STAT5 as a regulator of energy balance in adipocytes. PRL appears to have dual functions, positively and negatively affecting adipocyte gene expression. This hypothesis is supported by another study (13) showing that STAT5 can act as a modulator of GH-induced inhibition of aP2 expression. The association of STAT5 proteins with coactivators, corepressors, and other transcription factors likely affects the ability of STAT5A to have adipogenic and antiadipogenic effects. Recent work from our laboratory has demonstrated that the association between STAT5A and the glucocorticoid receptor is highly regulated during fat cell differentiation (8). Hence, cooperation between STAT5A and glucocorticoid receptor may occur in the modulation of FAS, since glucocorticoids have been demonstrated to affect FAS transcription and activity in adipose tissue (52,53). We are currently investigating the cross talk between these pathways in adipocytes. In summary, we have observed that PRL represses expression of FAS in adipocytes and negatively regulates the rat FAS promoter. Our identification of a STAT5 binding site in the promoter of FAS characterizes a novel mechanism of regulating FAS expression. In summary, we hypothesize that the regulation of FAS by STAT5 is likely an important contribution to the maintenance of energy homeostasis.
ACKNOWLEDGMENTS
This work was supported by grant R01DK52968-05 from the National Institutes of Health to J.M.S.
We thank James E. Baugh and Patricia Arbour-Reily for technical assistance with this project.
DMEM, Dulbecco’s modified Eagle medium; EMSA, electromobility shift assays; FAS, fatty acid synthase; GH, growth hormone; LIF, leukemia inhibitory factor; LPL, lipoprotein lipase; PRL, prolactin; TK, thymidine kinase
REFERENCES
Schmitt-Ney M, Doppler W, Ball RK, Groner B: Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol11 :3745 eC3755,1991
Wakao H, Gouilleux F, Groner B: Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J13 :2182 eC2191,1994
Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW: Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene285 :1 eC24,2002
Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G, Ihle JN: Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell93 :841 eC850,1998
Stephens JM, Morrison RF, Wu Z, Farmer SR: PPARgamma ligand-dependent induction of STAT1, STAT5A, and STAT5B during adipogenesis. Biochem Biophys Res Commun262 :216 eC222,1999
Yarwood SJ, Sale EM, Sale GJ, Houslay MD, Kilgour E, Anderson NG: Growth hormone-dependent differentiation of 3T3-F442A preadipocytes requires Janus kinase/signal transducer and activator of transcription but not mitogen-activated protein kinase or p70 S6 kinase signaling. J Biol Chem274 :8662 eC8668,1999
Shang CA, Waters MJ: Constitutively active signal transducer and activator of transcription 5 can replace the requirement for growth hormone in adipogenesis of 3T3eCF442A preadipocytes. Mol Endocrinol17 :2494 eC2508,2003
Floyd ZE, Stephens JM: STAT5A promotes adipogenesis in nonprecursor cells and associates with the glucocorticoid receptor during adipocyte differentiation. Diabetes52 :308 eC314,2003
Fain JN, Ihle JH, Bahouth SW: Stimulation of lipolysis but not of leptin release by growth hormone is abolished in adipose tissue from Stat5a and b knockout mice. Biochem Biophys Res Commun263 :201 eC205,1999
Yin D, Clarke SD, Peters JL, Etherton TD: Somatotropin-dependent decrease in fatty acid synthase mRNA abundance in 3T3eCF442A adipocytes is the result of a decrease in both gene transcription and mRNA stability. Biochem J331 :815 eC820,1998
Magri KA, Adamo M, Leroith D, Etherton TD: The inhibition of insulin action and glucose metabolism by porcine growth hormone in porcine adipocytes is not the result of any decrease in insulin binding or insulin receptor kinase activity. Biochem J266 :107 eC113,1990
Wabitsch M, Braun S, Hauner H, Heinze E, Ilondo MM, Shymko R, De Meyts P, Teller WM: Mitogenic and antiadipogenic properties of human growth hormone in differentiating human adipocyte precursor cells in primary culture. Pediatr Res40 :450 eC456,1996
Richter HE, Albrektsen T, Billestrup N: The role of signal transducer and activator of transcription 5 in the inhibitory effects of GH on adipocyte differentiation. J Mol Endocrinol30 :139 eC150,2003
Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA: Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev19 :225 eC268,1998
Avruch J, Zhang XF, Kyriakis JM: Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem Sci19 :279 eC283,1994
Bailey JP, Nieport KM, Herbst MP, Srivastava S, Serra RA, Horseman ND: Prolactin and transforming growth factor-beta signaling exert opposing effects on mammary gland morphogenesis, involution, and the Akt-forkhead pathway. Mol Endocrinol18 :1171 eC1184,2004
Ling C, Hellgren G, Gebre-Medhin M, Dillner K, Wennbo H, Carlsson B, Billig H: Prolactin (PRL) receptor gene expression in mouse adipose tissue: increases during lactation and in PRL-transgenic mice. Endocrinology141 :3564 eC3572,2000
Ling C, Svensson L, Oden B, Weijdegard B, Eden B, Eden S, Billig H: Identification of functional prolactin (PRL) receptor gene expression: PRL inhibits lipoprotein lipase activity in human white adipose tissue. J Clin Endocrinol Metab88 :1804 eC1808,2003
McAveney KM, Gimble JM, Yu-Lee L: Prolactin receptor expression during adipocyte differentiation of bone marrow stroma. Endocrinology137 :5723 eC5726,1996
Nanbu-Wakao R, Fujitani Y, Masuho Y, Muramatu M, Wakao H: Prolactin enhances CCAAT enhancer-binding protein-beta (C/EBP beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) messenger RNA expression and stimulates adipogenic conversion of NIH-3T3 cells. Mol Endocrinol14 :307 eC316,2000
Greenman Y, Tordjman K, Stern N: Increased body weight associated with prolactin secreting pituitary adenomas: weight loss with normalization of prolactin levels. Clin Endocrinol (Oxf )48 :547 eC553,1998
Fortun-Lamothe L, Langin D, Lafontan M: Influence of prolactin on in vivo and in vitro lipolysis in rabbits. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol115 :141 eC147,1996
Fielder PJ, Talamantes F: The lipolytic effects of mouse placental lactogen II, mouse prolactin, and mouse growth hormone on adipose tissue from virgin and pregnant mice. Endocrinology121 :493 eC497,1987
Flint DJ, Clegg RA, Vernon RG: Prolactin and the regulation of adipose-tissue metabolism during lactation in rats. Mol Cell Endocrinol22 :265 eC275,1981
Sul HS, Wang D: Nutritional and hormonal regulation of enzymes in fat synthesis: studies of fatty acid synthase and mitochondrial glycerol-3-phosphate acyltransferase gene transcription. Annu Rev Nutr18 :331 eC351,1998
Liu LH, Wang XK, Hu YD, Kang JL, Wang LL, Li S: Effects of a fatty acid synthase inhibitor on adipocyte differentiation of mouse 3T3eCL1 cells. Acta Pharmacol Sin25 :1052 eC1057,2004
Balhoff JP, Stephens JM: Highly specific and quantitative activation of STATs in 3T3eCL1 adipocytes. Biochem Biophys Res Commun247 :894 eC900,1998
Yin D, Clarke SD, Etherton TD: Transcriptional regulation of fatty acid synthase gene by somatotropin in 3T3eCF442A adipocytes. J Anim Sci79 :2336 eC2345,2001
Stephens JM, Pekala PH: Transcriptional repression of the C/EBP-alpha and GLUT4 genes in 3T3eCL1 adipocytes by tumor necrosis factor-alpha: regulation is coordinate and independent of protein synthesis. J Biol Chem267 :13580 eC13584,1992
Zvonic S, Hogan JC, Arbour-Reily P, Mynatt RL, Stephens JM: Effects of cardiotrophin (CT-1) on adipocytes. J Biol Chem279 :47572 eC47579,2004
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227 :680 eC685,1970
Ritzenthaler JD, Goldstein RH, Fine A, Lichtler A, Rowe DW, Smith BD: Transforming-growth-factor-beta activation elements in the distal promoter regions of the rat alpha 1 type I collagen gene. Biochem J280 :157 eC162,1991
Hattori M, Abraham LJ, Northemann W, Fey GH: Acute-phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2-macroglobulin gene. Proc Natl Acad Sci U S A87 :2364 eC2368,1990
Hogan JC, Stephens JM: The identification and characterization of a STAT 1 binding site in the PPARgamma2 promoter. Biochem Biophys Res Commun287 :484 eC492,2001
Stephens JM, Lumpkin SJ, Fishman JB: Activation of signal transducers and activators of transcription 1 and 3 by leukemia inhibitory factor, oncostatin-M, and interferon-gamma in adipocytes. J Biol Chem273 :31408 eC31416,1998
Lanna DP, Houseknecht KL, Harris DM, Bauman DE: Effect of somatotropin treatment on lipogenesis, lipolysis, and related cellular mechanisms in adipose tissue of lactating cows. J Dairy Sci78 :1703 eC1712,1995
Yin D, Griffin MJ, Etherton TD: Analysis of the signal pathways involved in the regulation of fatty acid synthase gene expression by insulin and somatotropin. J Anim Sci79 :1194 eC1200,2001
Rufo C, Gasperikova D, Clarke SD, Teran-Garcia M, Nakamura MT: Identification of a novel enhancer sequence in the distal promoter of the rat fatty acid synthase gene. Biochem Biophys Res Commun261 :400 eC405,1999
Bennett MK, Lopez JM, Sanchez HB, Osborne TF: Sterol regulation of fatty acid synthase promoter: coordinate feedback regulation of two major lipid pathways. J Biol Chem270 :25578 eC25583,1995
Moon YS, Latasa MJ, Kim KH, Wang D, Sul HS: Two 5'-regions are required for nutritional and insulin regulation of the fatty-acid synthase promoter in transgenic mice. J Biol Chem275 :10121 eC10127,2000
Latasa MJ, Moon YS, Kim KH, Sul HS: Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element. Proc Natl Acad Sci U S A97 :10619 eC10624,2000
Kim JB, Sarraf P, Wright M, Yao KM, Mueller E, Solanes G, Lowell BB, Spiegelman BM: Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest101 :1 eC9,1998
Boizard M, Le Liepvre X, Lemarchand P, Foufelle F, Ferre P, Dugail I: Obesity-related overexpression of fatty-acid synthase gene in adipose tissue involves sterol regulatory element-binding protein transcription factors. J Biol Chem273 :29164 eC29171,1998
Kim S, Dugail I, Standridge M, Claycombe K, Chun J, Moustaid-Moussa N: Angiotensin II-responsive element is the insulin-responsive element in the adipocyte fatty acid synthase gene: role of adipocyte determination and differentiation factor 1/sterol-regulatory-element-binding protein 1c. Biochem J357 :899 eC904,2001
Claycombe KJ, Wang Y, Jones BH, Kim S, Wilkison WO, Zemel MB, Chun J, Moustaid-Moussa N: Transcriptional regulation of the adipocyte fatty acid synthase gene by agouti: interaction with insulin. Physiol Genomics3 :157 eC162,2000
Roder K, Wolf SS, Beck KF, Sickinger S, Schweizer M: NF-Y binds to the inverted CCAAT box, an essential element for cAMP-dependent regulation of the rat fatty acid synthase (FAS) gene. Gene184 :21 eC26,1997
Rolland V, Liepvre XL, Jump DB, Lavau M, Dugail I: A GC-rich region containing Sp1 and Sp1-like binding sites is a crucial regulatory motif for fatty acid synthase gene promoter activity in adipocytes: implication in the overactivity of FAS promoter in obese Zucker rats. J Biol Chem271 :21297 eC21302,1996
Moustaid N, Sakamoto K, Clarke S, Beyer RS, Sul HS: Regulation of fatty acid synthase gene transcription: sequences that confer a positive insulin effect and differentiation-dependent expression in 3T3eCL1 preadipocytes are present in the 332 bp promoter. Biochem J292 :767 eC772,1993
Guichard C, Dugail I, Le L, Liepvre XL, Lavau M: Genetic regulation of fatty acid synthetase expression in adipose tissue: overtranscription of the gene in genetically obese rats. J Lipid Res33 :679 eC687,1992
Soldaini E, John S, Moro S, Bollenbacher J, Schindler U, Leonard WJ: DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites. Mol Cell Biol20 :389 eC401,2000
Ehret GB, Reichenbach P, Schindler U, Horvath CM, Fritz S, Nabholz M, Bucher P: DNA binding specificity of different STAT proteins: comparison of in vitro specificity with natural target sites. J Biol Chem276 :6675 eC6688,2001
Wang Y, Jones Voy B, Urs S, Kim S, Soltani-Bejnood M, Quigley N, Heo YR, Standridge M, Andersen B, Dhar M, Joshi R, Wortman P, Taylor JW, Chun J, Leuze M, Claycombe K, Saxton AM, Moustaid-Moussa N: The human fatty acid synthase gene and de novo lipogenesis are coordinately regulated in human adipose tissue. J Nutr134 :1032 eC1038,2004
Volpe JJ, Marasa JC: Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver. Biochim Biophys Acta380 :454 eC472,1975(Jessica C. Hogan, and Jac)
ABSTRACT
Growth hormone (GH) diminishes adipose tissue mass in vivo and decreases expression and activity of fatty acid synthase (FAS) in adipocytes. GH and prolactin (PRL) are potent activators of STAT5 and exert adipogenic and antiadipogenic effects in adipocytes. In this study, we demonstrate that GH and PRL decrease the mRNA and protein levels of FAS in 3T3-L1 adipocytes. We present evidence that indicates that FAS is repressed at the level of transcription. In addition, PRL responsiveness was shown to exist between eC1,594 and eC700 of the rat FAS promoter. Moreover, responsiveness to PRL was abolished with mutation of a site at position eC908 to eC893, which we have shown to bind STAT5A in a PRL-dependent manner. Taken together, these data strongly suggest that PRL directly represses expression of FAS in adipocytes through STAT5A binding to the eC908 to eC893 site. Furthermore, our results indicate that STAT5A has an antilipogenic function in adipocytes and may contribute to the regulation of energy balance.
STAT5 proteins were first identified as mammary gland factor, a protein from mouse mammary glands that is bound to the -casein promoter (1). It was subsequently determined that mammary gland factor was two closely related proteins, STAT5A and STAT5B (2), that are expressed in all tissues (3). Transgenic deletion studies in mice indicate that a major function of STAT5 proteins is the regulation of mammary tissue development (4), but multiple lines of evidence suggest a role for STAT5 proteins in the modulation of adipocyte function. During differentiation of 3T3-L1 adipocytes, expression levels of STAT5A and 5B were highly induced (5). Furthermore, growth hormone (GH)-dependent adipogenesis was attenuated by STAT5 antisense oligonucleotides (6), and constitutively active STAT5 can replace the requirement for GH in the adipogenesis of murine 3T3-F442A cells (7). Moreover, ectopic expression of STAT5A conferred adipogenesis in two different nonprecursor cell lines (8). Transgenic deletion of STAT5A, STAT5B, or both STAT5 genes in mice resulted in significantly reduced fat-pad sizes compared with wild-type mice (4). Yet, in primary cultures of adipose tissue from these animals, GH did not stimulate lipolysis as it does in adipocytes from wild-type mice (9). Also, chronic administration of GH in pigs decreases adipose tissue growth through the attenuation of lipogenesis (10,11) and reduces adipocyte conversion in rat primary adipocytes (12). More recent studies suggest that some of the antiadipogenic effects of GH may be mediated by STAT5A. In rat primary preadipocytes, GH-induced STAT5 inhibits aP2 expression (13). In summary, STAT5 proteins appear to have both adipogenic as well as antiadipogenic effects in adipose tissue of various species, which may depend on the developmental stage of the tissue.
PRL is a peptide hormone primarily known for its role in mammary gland development during lactation, but it has been shown to have pleiotropic effects in a variety of tissues (14). PRL activates multiple signal transduction pathways, including mitogen-activated protein kinase (15) and phosphatidylinositol-3 kinase (16); however, the JAK/STAT pathway is the predominant signaling cascade activated by PRL, resulting in the nuclear translocation of STAT5 proteins (3).
The regulation of mammary tissue by PRL is well characterized, but there is also evidence that PRL modulates adipose tissue. PRL-R is expressed in both mouse (17) and human (18) adipose tissue and is induced during adipogenesis of bone marrow stromal cells (19). Furthermore, ectopic expression of the PRL-R in murine NIH-3T3 cells resulted in efficient adipocyte conversion and activation of the aP2 promoter in a PRL-dependent manner (20). Taken together, these observations strongly suggest a role for PRL in the modulation of adipocyte function. Furthermore, the occurrence of obesity has been correlated with hyperprolactinomas in humans (21). In opposition to these adipogenic effects, PRL has been shown to induce lipolysis in rabbits (22) and mouse adipose tissue explants (23). In addition, studies have shown that PRL reduces lipoprotein lipase (LPL) activity in cultured human adipocytes (18) and the activity of LPL and fatty acid synthase (FAS) in adipose tissue of lactating mice (24). Thus, PRL exerts adipogenic and antilipogenic effects on adipose tissue in a variety of species.
FAS is the key enzyme in de novo lipogenesis, catalyzing the reactions for the synthesis of long-chain fatty acids (25). The importance of FAS in adipocyte function is underscored by the inhibition of adipogenesis of 3T3-L1 cells by C75, an allosteric inhibitor of FAS (26). FAS is regulated primarily at the level of transcription and is sensitive to nutritional and hormonal regulation (25). Previous studies have demonstrated that GH, a cytokine that activates STAT5 in adipocytes (27), abrogates the induction of FAS expression by insulin and downregulates basal expression of FAS in 3T3-F442A cells (28). Although insulin-regulated regions of the FAS promoter have been extensively studied, there has not been a conclusive study to characterize a GH-responsive region or to determine the potential role of STATs in the modulation of FAS expression. Since GH and PRL are potent activators of STAT5 (27,20), we hypothesized that STAT5 directly regulates the expression of FAS. In this study, we have demonstrated that GH and PRL treatment of 3T3-L1 adipocytes resulted in decreased FAS mRNA and protein. In addition, we have identified a region within the FAS promoter that is responsive to PRL. This region contains a nonconsensus STAT5 binding site that, when mutated, results in a loss of sensitivity to PRL. Our results clearly demonstrate that STAT5A binds to this nonconsensus sequence and strongly suggest that FAS is a direct target for regulation by STAT5. These data suggest a novel means for regulation of FAS expression in adipocytes and reveal a mechanism by which STAT5 proteins and PRL exert antiadipogenic effects.
RESEARCH DESIGN AND METHODS
Dulbecco’s modified Eagle medium (DMEM) and leukemia inhibitory factor (LIF) were purchased from Invitrogen. Fetal bovine serum was purchased from Atlanta Biologicals, and calf serum was purchased from Biosource. Polyclonal phospho-specific STAT5 (Y694) antibody and FAS antibody were purchased from BD Transduction Laboratories. STAT1 antibody was purchased from Upstate Biotechnology. Porcine growth hormone, ovine PRL (both prepared from pituitaries), and cycloheximide were purchased from Sigma. STAT3 and STAT5A antibodies were purchased from Santa Cruz. [-32P] dCTP was purchased from Perkin-Elmer and Amersham Biosciences. Deoxynucleotide thymine triphosphate, dATP, and dGTP were purchased from Amersham Biosciences. Oligonucleotides were purchased from Integrated DNA Technologies. DNase polymerase I large (Klenow) fragment was purchased from Promega.
Cell culture.
Murine 3T3-L1 preadipocytes were plated and grown to 2 days postconfluence in DMEM containing 10% bovine serum. Medium was changed every 48 h. Cells were induced to differentiate by changing the medium to DMEM containing 10% fetal bovine serum, 0.5 mmol/l 3-isobutyl-methylxanthine, 1 eol/l dexamethasone, and 1.7 eol/l insulin. After 48 h, this medium was replaced with DMEM supplemented with 10% fetal bovine serum, and the cells were maintained in this medium until used for experimentation.
Preparation of whole-cell extracts.
Cell monolayers were rinsed with PBS and then harvested in a nondenaturing buffer containing 10 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1 mmol/l EGTA, 1 mmol/l EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 eol/l phenylmethylsulfonyl fluoride, 1 eol/l pepstatin, 50 trypsin inhibitory mU aprotinin, 10 eol/l leupeptin, and 2 mmol/l sodium vanadate. Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4°C for 15 min. Supernatants containing whole-cell extracts were analyzed for protein content by bicinchoninic acid analysis (Pierce) according to the manufacturer’s instructions.
RNA analysis.
Total RNA was isolated from cell monolayers with Trizol (Invitrogen), according to manufacturer’s instructions with minor modifications. For Northern blot analysis, 15 e total RNA was denatured in formamide and electrophoresed through a formaldehyde/agarose gel. The RNA was transferred to Zeta Probe-GT (Bio-Rad) in a buffer containing 75 mmol/l sodium citrate tribasic, 10 mmol/l NaOH, and 750 mmol/l NaCl. The blots were cross-linked, hybridized, and washed as previously described (29). Probes were labeled by random priming using Klenow fragment and [-32P] dCTP.
Constructs.
The rat FAS promoter eC250 to +65/luciferase construct was generously provided by Dr. Steve Clarke. The rat FAS promoter eC1,594 to +65/luciferase and eC700 to +65/luciferase constructs were generously provided by Dr. Peter Tontonoz. The FAS eC1,594 to +65/luciferase construct was mutated at positions eC901 and eC902 within the STAT5A binding site using the QuikChange Site Directed Mutagenesis Kit, according to manufacturer’s instructions (Stratagene). The following oligonucleotide and corresponding antisense oligonucleotide were used to alter the STAT binding site with the altered bases underlined: GGG AGG GTG AGG GTC AAG GAA ACC AGC AAC TCA GG. Sequence analysis was performed to confirm the presence of the mutated bases using Big Dye Terminator Extension Reaction (ABI Prism). The minimum promoter thymidine kinase (TK) renilla vector was purchased from Promega.
Transfection and luciferase assay.
3T3-L1 preadipocytes were transiently cotransfected with the various FAS promoter constructs and the TK/renilla vector to control for transfection efficiency, as previously described (30), using Polyfect (Qiagen) according to manufacturer’s instructions. Cell lysates were analyzed for firefly and renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega). Relative light units were determined by dividing firefly luciferase activity by renilla luciferase activity. Results are given as ±SD.
Preparation of nuclear and cytosolic extracts.
Cell monolayers were rinsed with PBS and then harvested in a nuclear homogenization buffer containing 20 mmol/l Tris (pH 7.4), 10 mmol/l NaCl, 3 mmol/l MgCl2, 1 eol/l dithiothreitol, 1 eol/l phenylmethylsulfonyl fluoride, 1 eol/l pepstatin, 50 trypsin inhibitory mU aprotinin, 10 eol/l leupeptin, and 2 mmol/l sodium vanadate. Igepal CA-630 (Nonidet P-40) was added to a final concentration of 0.15%, and cells were homogenized with 16 strokes in a Dounce homogenizer. The homogenates were centrifuged at 1,500 rpm for 5 min. Supernatants were saved as cytosolic extract, and the nuclear pellets were resuspended in one-half volume of nuclear homogenization buffer and were centrifuged as before. The pellet of intact nuclei was resuspended again in one-half of the original volume of nuclear homogenization buffer and centrifuged again. The majority of the pellet (intact nuclei) was resuspended in an extraction buffer containing 20 mmol/l HEPES (pH 7.9), 420 mmol/l NaCl, 1.5 mmol/l MgCl2, 0.2 mmol/l EDTA, 1 eol/l dithiothreitol, 1 eol/l phenylmethylsulfonyl fluoride, 1 eol/l pepstatin, 50 trypsin inhibitory mU aprotinin, 10 eol/l leupeptin, 2 mmol/l sodium vanadate, and 25% glycerol. Nuclei were extracted for 30 min on ice. The samples were subjected to centrifugation at 10,000 rpm at 4°C for 10 min. Supernatants containing nuclear extracts were analyzed for protein content, using a bicinchoninic acid protein assay kit (Pierce).
Gel electrophoresis and immunoblotting.
Proteins were separated in 6% polyacrylamide (National Diagnostics) gels containing SDS according to Laemmli (31) and transferred to nitrocellulose (Bio-Rad) in 25 mmol/l Tris, 192 mmol/l glycine, and 20% methanol. Following transfer, the membrane was blocked in 4% milk overnight at 4°C. Results were visualized with horseradish peroxidaseeCconjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and enhanced chemiluminescence (Pierce).
Electrophoretic mobility shift analysis.
Double-stranded oligonucleotides were end labeled with [-32P] dCTP using Klenow. Binding reactions were performed with nuclear extracts, according to Ritzenthaler et al. (32). Protein-DNA complexes were resolved and visualized, as previously described (30). For supershift analysis, nuclear extracts were preincubated with 4 e antibody for 1 h at room temperature.
RESULTS
GH is known to reduce adipose tissue in vivo (11) and decrease the expression of FAS in adipocytes (10). GH is also a potent activator of STAT5 in adipocytes (27). Hence, we hypothesized that STAT5 proteins directly modulate FAS expression. Therefore, we examined the regulation of FAS by two STAT5 activators, GH and PRL. As shown in Fig. 1A, we observed that both GH and PRL resulted in a decrease in FAS mRNA in fully differentiated 3T3-L1 adipocytes. GH decreased FAS mRNA by 12 h of treatment, and PRL treatment resulted in decreased expression within 6 h. In an independent experiment, we observed that both GH and PRL also resulted in a decrease in expression of FAS protein levels (Fig. 1B). To demonstrate the specificity of this effect, fully differentiated 3T3-L1 adipocytes were treated with PRL for 8 h with the various doses indicated in Fig. 1C. There was a notable decrease in FAS protein level with 8.8 nmol/l PRL treatment, and the decrease of FAS protein levels by PRL treatment was dose dependent. An acute treatment of 1.3 eol/l PRL was included as a positive control for STAT5 phosphorylation. The level of STAT5A expression was unchanged by GH or PRL (Fig. 1B and C) and is shown to indicate even loading of protein samples.
Clearly, the analysis of mRNA and whole-cell extracts demonstrated that activators of STAT5 decreased expression of FAS protein and mRNA. Yet, it was unclear if the effects of GH and PRL were mediated by affecting FAS transcription and/or protein turnover. To assess whether the effects of PRL on FAS expression could be attributed to changes in the turnover of FAS protein, we examined the loss of FAS in the presence of cycloheximide (5 eol/l) or ethanol, a vehicle control. Whole-cell extracts were collected at various times and used for Western blot analysis. As shown in Fig. 2, either cycloheximide or PRL treatment caused a decrease in FAS protein. In the presence of cycloheximide, a loss of FAS was observed at 8 h, regardless of the presence of PRL. PRL treatment alone decreased the level of FAS within 12 h of treatment. These results indicate that PRL does not affect the turnover of FAS protein in 3T3-L1 adipocytes and suggest that PRL may exert its effects on FAS at the level of transcription.
Our data indicate that PRL directly regulates the expression of FAS. Recent studies by Yin, Clarke, and Etherton (28) have shown that GH abrogates the induction of FAS by insulin and suggested that the eC112 to +65 region of the rat FAS promoter was sensitive to the regulation by GH. Therefore, we hypothesized that a STAT5 recognition element may be present in this region. To address this question, 3T3-L1 preadipocytes were transiently transfected with a luciferase construct containing the FAS promoter fragment of eC250 to +65. After 48 h, cells were stimulated with PRL for the times indicated in Fig. 3Aand were then analyzed for luciferase activity as described in RESEARCH DESIGN AND METHODS. We observed that PRL treatment had no significant effects on the relative luciferase activity (Fig. 3A), clearly demonstrating that the eC250 to +65 region of the FAS promoter is not sensitive to PRL. Therefore, we examined the PRL responsiveness of two additional rat FAS promoter/luciferase constructs that incorporated larger regions, eC1,594 to +65 and eC700 to +65, of the promoter. Transfection of these constructs into 3T3-L1 cells revealed a PRL-responsive region present between eC1,594 and eC700 of the rat FAS promoter. As shown in Fig. 3B, treatment with PRL resulted in a 64% decrease in luciferase activity of the rat FAS promoter (eC1,594 to +65)/luciferase construct. Although the basal level of luciferase activity for the rat FAS promoter (eC700 to +65)/luciferase construct was similar to that of the eC1,594 to +65 construct, PRL had no effect on the level of luciferase activity (Fig. 3B). These data demonstrate that a PRL-sensitive region exists between eC1,594 and eC700 of the FAS promoter and support our hypothesis that FAS is transcriptionally regulated by STAT5 activators.
Since PRL is a potent activator of STAT5, we hypothesized that the eC1,594 to eC700 region of the FAS promoter may contain a STAT5 binding site that conferred PRL responsiveness. Therefore, we examined the rat FAS promoter (GenBank X62889) for the presence of STAT consensus sites (TTCNNNGAA). An examination of the FAS promoter did not result in the identification of any sequences that precisely matched the STAT consensus site. However, as shown in Table 1, we identified four regions that were similar to the consensus sequence. To evaluate these potential STAT5 binding sites, we performed a series of electromobility shift assays (EMSA). For these experiments, nuclear and cytosolic extracts were prepared from 3T3-L1 adipocytes acutely treated with PRL for 15 min. As shown in Fig. 4A, PRL did not induce the binding of nuclear protein complexes to the eC951 to eC933, eC1,226 to eC1,214, or eC4,639 to eC4,623 regions of the FAS promoter. The induction of binding by a PRL-activated protein complex to the rat -casein STAT5 binding site (1) is included as a positive control. However, we did observe PRL-dependent binding by a nuclear protein complex to the eC908 to eC893 region of the FAS promoter (Fig. 4B). To determine the specificity of binding, an EMSA was performed using a mutant version of the eC908 to eC893 oligonucleotide, in which two nucleotides were changed (Table 1). As shown in Fig. 4B, there was no detectable binding to the mutant form of the binding site following PRL stimulation. In addition, binding of the PRL-activated protein complex was successively competed with increasing concentrations of the unlabeled eC908 to eC893 oligonucleotide (Fig. 4C). Further evidence of specificity is shown in Fig. 4D, in which an excess of the eC908 to eC893 oligonucleotide competed away binding induced by PRL treatment (lane 4), whereas the mutant form of the oligonucleotide did not (lane 5). Although we did not detect binding to the eC951 to eC933 site, an excess of this oligonucleotide moderately diminished binding to the radiolabeled eC908 to eC893 probe (lane 6). Yet, there was no appreciable competition of binding by the eC1,226 to eC1,214 oligonucleotide (lane 7). As anticipated, binding was competed away with the STAT5 binding site of the rat -casein promoter. Interestingly, a STAT3 binding site, eC168 to eC148 from the rat 2-macroglobulin promoter (33), also resulted in binding competition. However, binding was not competed with a STAT1 binding site that is present at eC221 to eC207 of the peroxisome proliferatoreCactivated receptor 2 promoter (34).
To determine whether the protein complex binding the eC908 to eC893 contained STAT proteins, we performed supershift analysis using antibodies directed against the STAT proteins expressed in adipocytes. As shown in Fig. 5, the protein complex induced by PRL was fully supershifted with a STAT5A antibody (lane 5) and weakly supershifted with an STAT5B antibody (lane 6). STAT1 and STAT3 antibodies had no effect on the mobility of the complex (lanes 3 and 4). We also investigated the specificity of binding by STAT proteins by comparing the binding induced by LIF (0.5 nmol/l), a cytokine that activates STAT1 and STAT3 but not STAT5 in 3T3-L1 adipocytes (35). LIF stimulated binding to the eC908 to eC893 site by a protein complex that exhibited highly reduced binding affinity and slightly faster mobility than the complex induced by PRL (Fig. 5). Moreover, the LIF-induced complex was supershifted by a STAT1 antibody (lane 8) but not with antibodies for STAT3 or STAT5A (lanes 9 and 10). We used three other STAT3 antibodies capable of supershifting STAT3 complexes and did not observe any STAT3 LIF-stimulated binding to the eC908 to eC893 site (data not shown).
Our data clearly demonstrate that the eC908 to eC893 region of the rat FAS promoter binds nuclear PRL-activated STAT5 proteins in vitro. To determine whether this region of the FAS promoter contributed to the regulation of FAS by PRL in living cells, we performed site-specific mutagenesis to alter two basepairs at positions eC902 and eC901 within the rat FAS promoter (eC1,594 to +65)/luciferase construct. We have shown that this mutation abolished binding of PRL-induced proteins to this site (Fig. 4B and D). Transfection of the wild-type and mutant constructs into 3T3-L1 cells revealed that the basal level of luciferase activity was unaffected by mutation of the eC902 and eC901 bp of the FAS promoter (Fig. 6). However, the 60% decrease in luciferase activity induced by PRL for the wild-type construct was eliminated with the mutation. Thus, these data clearly indicate that the eC908 to eC893 site of the FAS promoter is sensitive to PRL and suggest that this site confers the negative regulation of FAS by PRL-activated STAT5A protein complexes.
DISCUSSION
The novel findings in this study include data demonstrating that FAS levels are decreased following stimulation with activators of STAT5 in 3T3-L1 adipocytes, the identification of a PRL-responsive region of the rat FAS promoter, and the characterization of a STAT5 binding site in this region. These results strongly suggest that STAT5A directly represses the expression of FAS in adipocytes. Moreover, our data indicate that STAT5A has an antilipogenic function in murine adipocytes, in addition to an adipogenic role previously described by our laboratory (8) and others (4,6,7).
Previous studies have shown that GH, an activator of STAT5, attenuated the induction of FAS in adipocytes by insulin in 3T3-F442A adipocytes (10) and decreased expression of FAS in vivo in pigs (11). We observed that two activators of STAT5 in adipocytes, GH and PRL, decreased protein and mRNA expression of FAS in 3T3-L1 adipocytes. These results are consistent with the findings that GH in cows (36) and PRL in rats (24) can inhibit the activity of FAS in adipose tissue. Surprisingly, our results revealed that the effects of GH on FAS are transient and reversible, whereas the effects of PRL are more pronounced with time. The differences in the action of these two STAT5 activators may be due to receptor levels or specific signal proteins that have yet to be identified. Alternatively, the differences we observe in GH and PRL effects on FAS in our murine cells may also be affected by different species of hormones we used (sheep and pig) on murine cells. Our results show that PRL did not affect the protein turnover of FAS but indicate that changes in FAS levels are mediated at the transcriptional level. Our experiments are supported by other studies (25,28,37eC48) that demonstrate that FAS can be modulated at the transcriptional level. However, increased turnover of FAS mRNA in 3T3-F442A adipocytes has been demonstrated as one of the means through which GH attenuates the induction of FAS by insulin (10). Nonetheless, our results strongly suggest that the PRL-induced repression of FAS is mediated by an inhibition of transcription.
To elucidate the mechanism of transcriptional regulation by PRL, we investigated the regulation of the FAS promoter. Although a previous study (28) indicated that the eC112 to +65 region of the rat FAS promoter was sensitive to regulation by GH in murine cells, we did not observe PRL-mediated regulation of the rat FAS promoter within this region in murine cells (Fig. 3A). However, our analysis of larger regions of the rat FAS promoter clearly indicated that a PRL-responsive region existed between eC1,594 and eC700 of the rat FAS promoter (Fig. 3B). Yin, Clarke, and Etherton (28) have previously shown that GH attenuated the stimulation by insulin of the rat FAS promoter (eC112 to +65)/luciferase construct, a region of the promoter that does not contain a STAT consensus site. Furthermore, in another study (37), it was demonstrated that staurosporine, an inhibitor of JAK/STAT signaling, did not block the effect of GH on insulin-stimulated FAS expression in murine cells. Thus, it is unlikely that STAT5 proteins mediate the inhibitory effects of GH on insulin regulation of FAS. Yet, in light of our current findings, we postulate that the repression of basal levels of FAS by PRL is directly regulated by STAT5 proteins via binding to a site within the eC1,594 to eC700 region of the rat FAS promoter. We also hypothesize that the delayed effects we observe on FAS mRNA and protein are due to the stability of the FAS mRNA and protein. Our studies indicate that FAS protein is very stable (Fig. 2), and preliminary studies suggest that the FAS mRNA is more stabile than other adipocyte mRNAs (data not shown).
Since PRL is a potent activator of STAT5 (Fig. 1B), we examined the rat FAS promoter for sites that resembled the STAT consensus sequence, TTCNNNGAA. We identified four sites that were similar to the STAT consensus, but our analysis by EMSA indicated that only one site, at position eC908 to eC893, was bound by a PRL-induced nuclear protein complex in a highly specific manner (Fig. 4). Similar results were obtained with GH (data not shown). In our experiments, the PRL-induced protein complex was fully supershifted by a STAT5A antibody (Fig. 5). The functional significance of the eC908 to eC893 site was determined by mutating two nucleotides at positions eC902 and eC901, within the STAT5 binding site of the rat FAS promoter (eC1,594 to +65)/luciferase construct. Mutation of this site completely abrogated the downregulation by PRL that was observed with the wild-type construct (Fig. 6). Taken together, these data strongly suggest that eC908 to eC893 of the rat FAS promoter is a STAT5 binding site, which confers the negative transcriptional regulation of FAS by PRL. These results support our hypothesis that STAT5 directly represses expression of FAS in adipocytes.
The association of increased transcription of FAS in rodent obesity (49) and the inhibition of adipogenesis by an allosteric inhibitor of FAS (26) are highly indicative that regulation of FAS expression and activity in adipocytes is an important control of energy homeostasis. The characterization of a STAT5 binding site in the rat FAS promoter identifies a novel mechanism for repression of FAS expression. The eC908 to eC893 site of the rat FAS promoter is also present in the murine FAS promoter (AL663090) at position eC895 to eC887. Interestingly, the human FAS promoter (AF250144) contains a site at eC1,091 to eC1,083 (TTCGAGGAA) that is different from the sequence identified from the rodent promoters but complements the STAT consensus sequence TTCNNNGAA. Hence, although the precise sequence of the STAT5 binding site is not conserved across species, the binding by STAT5 to these sites in the FAS promoter may be an evolutionarily retained mechanism of regulating FAS expression in adipose tissue.
Our observation that STAT5A, not other adipocyte STATs, preferentially binds to the eC908 to eC893 site (Fig. 5) is consistent with previous studies (50,51) demonstrating that STAT proteins bind similar sequences but exhibit subtle differences in affinity for nucleotides between and beyond the half-sites of the palindrome. This specificity in STAT binding may account for the distinct repertoire of target genes regulated by each STAT protein (51). Our data suggest that the regulation of transcription mediated by the eC908 to eC893 region of the rat FAS promoter is primarily regulated by STAT5A binding.
PRL has been shown to have antilipogenic effects in adipose tissue through inhibition of LPL expression and the repression of FAS (24) and LPL (18) activity. At this time, our studies and others suggest that both PRL and GH repress FAS expression. However, these growth factors appear to have different effects (transient versus sustained) on FAS regulation. These differences may be attributable to expression levels or actions of specific GH and PRL signaling proteins and/or specific cell types. Nonetheless, our results strongly suggest that PRL modulates the expression of FAS in adipocytes through STAT5A and supports a role for STAT5 as a regulator of energy balance in adipocytes. PRL appears to have dual functions, positively and negatively affecting adipocyte gene expression. This hypothesis is supported by another study (13) showing that STAT5 can act as a modulator of GH-induced inhibition of aP2 expression. The association of STAT5 proteins with coactivators, corepressors, and other transcription factors likely affects the ability of STAT5A to have adipogenic and antiadipogenic effects. Recent work from our laboratory has demonstrated that the association between STAT5A and the glucocorticoid receptor is highly regulated during fat cell differentiation (8). Hence, cooperation between STAT5A and glucocorticoid receptor may occur in the modulation of FAS, since glucocorticoids have been demonstrated to affect FAS transcription and activity in adipose tissue (52,53). We are currently investigating the cross talk between these pathways in adipocytes. In summary, we have observed that PRL represses expression of FAS in adipocytes and negatively regulates the rat FAS promoter. Our identification of a STAT5 binding site in the promoter of FAS characterizes a novel mechanism of regulating FAS expression. In summary, we hypothesize that the regulation of FAS by STAT5 is likely an important contribution to the maintenance of energy homeostasis.
ACKNOWLEDGMENTS
This work was supported by grant R01DK52968-05 from the National Institutes of Health to J.M.S.
We thank James E. Baugh and Patricia Arbour-Reily for technical assistance with this project.
DMEM, Dulbecco’s modified Eagle medium; EMSA, electromobility shift assays; FAS, fatty acid synthase; GH, growth hormone; LIF, leukemia inhibitory factor; LPL, lipoprotein lipase; PRL, prolactin; TK, thymidine kinase
REFERENCES
Schmitt-Ney M, Doppler W, Ball RK, Groner B: Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol11 :3745 eC3755,1991
Wakao H, Gouilleux F, Groner B: Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J13 :2182 eC2191,1994
Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW: Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene285 :1 eC24,2002
Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G, Ihle JN: Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell93 :841 eC850,1998
Stephens JM, Morrison RF, Wu Z, Farmer SR: PPARgamma ligand-dependent induction of STAT1, STAT5A, and STAT5B during adipogenesis. Biochem Biophys Res Commun262 :216 eC222,1999
Yarwood SJ, Sale EM, Sale GJ, Houslay MD, Kilgour E, Anderson NG: Growth hormone-dependent differentiation of 3T3-F442A preadipocytes requires Janus kinase/signal transducer and activator of transcription but not mitogen-activated protein kinase or p70 S6 kinase signaling. J Biol Chem274 :8662 eC8668,1999
Shang CA, Waters MJ: Constitutively active signal transducer and activator of transcription 5 can replace the requirement for growth hormone in adipogenesis of 3T3eCF442A preadipocytes. Mol Endocrinol17 :2494 eC2508,2003
Floyd ZE, Stephens JM: STAT5A promotes adipogenesis in nonprecursor cells and associates with the glucocorticoid receptor during adipocyte differentiation. Diabetes52 :308 eC314,2003
Fain JN, Ihle JH, Bahouth SW: Stimulation of lipolysis but not of leptin release by growth hormone is abolished in adipose tissue from Stat5a and b knockout mice. Biochem Biophys Res Commun263 :201 eC205,1999
Yin D, Clarke SD, Peters JL, Etherton TD: Somatotropin-dependent decrease in fatty acid synthase mRNA abundance in 3T3eCF442A adipocytes is the result of a decrease in both gene transcription and mRNA stability. Biochem J331 :815 eC820,1998
Magri KA, Adamo M, Leroith D, Etherton TD: The inhibition of insulin action and glucose metabolism by porcine growth hormone in porcine adipocytes is not the result of any decrease in insulin binding or insulin receptor kinase activity. Biochem J266 :107 eC113,1990
Wabitsch M, Braun S, Hauner H, Heinze E, Ilondo MM, Shymko R, De Meyts P, Teller WM: Mitogenic and antiadipogenic properties of human growth hormone in differentiating human adipocyte precursor cells in primary culture. Pediatr Res40 :450 eC456,1996
Richter HE, Albrektsen T, Billestrup N: The role of signal transducer and activator of transcription 5 in the inhibitory effects of GH on adipocyte differentiation. J Mol Endocrinol30 :139 eC150,2003
Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA: Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev19 :225 eC268,1998
Avruch J, Zhang XF, Kyriakis JM: Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem Sci19 :279 eC283,1994
Bailey JP, Nieport KM, Herbst MP, Srivastava S, Serra RA, Horseman ND: Prolactin and transforming growth factor-beta signaling exert opposing effects on mammary gland morphogenesis, involution, and the Akt-forkhead pathway. Mol Endocrinol18 :1171 eC1184,2004
Ling C, Hellgren G, Gebre-Medhin M, Dillner K, Wennbo H, Carlsson B, Billig H: Prolactin (PRL) receptor gene expression in mouse adipose tissue: increases during lactation and in PRL-transgenic mice. Endocrinology141 :3564 eC3572,2000
Ling C, Svensson L, Oden B, Weijdegard B, Eden B, Eden S, Billig H: Identification of functional prolactin (PRL) receptor gene expression: PRL inhibits lipoprotein lipase activity in human white adipose tissue. J Clin Endocrinol Metab88 :1804 eC1808,2003
McAveney KM, Gimble JM, Yu-Lee L: Prolactin receptor expression during adipocyte differentiation of bone marrow stroma. Endocrinology137 :5723 eC5726,1996
Nanbu-Wakao R, Fujitani Y, Masuho Y, Muramatu M, Wakao H: Prolactin enhances CCAAT enhancer-binding protein-beta (C/EBP beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) messenger RNA expression and stimulates adipogenic conversion of NIH-3T3 cells. Mol Endocrinol14 :307 eC316,2000
Greenman Y, Tordjman K, Stern N: Increased body weight associated with prolactin secreting pituitary adenomas: weight loss with normalization of prolactin levels. Clin Endocrinol (Oxf )48 :547 eC553,1998
Fortun-Lamothe L, Langin D, Lafontan M: Influence of prolactin on in vivo and in vitro lipolysis in rabbits. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol115 :141 eC147,1996
Fielder PJ, Talamantes F: The lipolytic effects of mouse placental lactogen II, mouse prolactin, and mouse growth hormone on adipose tissue from virgin and pregnant mice. Endocrinology121 :493 eC497,1987
Flint DJ, Clegg RA, Vernon RG: Prolactin and the regulation of adipose-tissue metabolism during lactation in rats. Mol Cell Endocrinol22 :265 eC275,1981
Sul HS, Wang D: Nutritional and hormonal regulation of enzymes in fat synthesis: studies of fatty acid synthase and mitochondrial glycerol-3-phosphate acyltransferase gene transcription. Annu Rev Nutr18 :331 eC351,1998
Liu LH, Wang XK, Hu YD, Kang JL, Wang LL, Li S: Effects of a fatty acid synthase inhibitor on adipocyte differentiation of mouse 3T3eCL1 cells. Acta Pharmacol Sin25 :1052 eC1057,2004
Balhoff JP, Stephens JM: Highly specific and quantitative activation of STATs in 3T3eCL1 adipocytes. Biochem Biophys Res Commun247 :894 eC900,1998
Yin D, Clarke SD, Etherton TD: Transcriptional regulation of fatty acid synthase gene by somatotropin in 3T3eCF442A adipocytes. J Anim Sci79 :2336 eC2345,2001
Stephens JM, Pekala PH: Transcriptional repression of the C/EBP-alpha and GLUT4 genes in 3T3eCL1 adipocytes by tumor necrosis factor-alpha: regulation is coordinate and independent of protein synthesis. J Biol Chem267 :13580 eC13584,1992
Zvonic S, Hogan JC, Arbour-Reily P, Mynatt RL, Stephens JM: Effects of cardiotrophin (CT-1) on adipocytes. J Biol Chem279 :47572 eC47579,2004
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227 :680 eC685,1970
Ritzenthaler JD, Goldstein RH, Fine A, Lichtler A, Rowe DW, Smith BD: Transforming-growth-factor-beta activation elements in the distal promoter regions of the rat alpha 1 type I collagen gene. Biochem J280 :157 eC162,1991
Hattori M, Abraham LJ, Northemann W, Fey GH: Acute-phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2-macroglobulin gene. Proc Natl Acad Sci U S A87 :2364 eC2368,1990
Hogan JC, Stephens JM: The identification and characterization of a STAT 1 binding site in the PPARgamma2 promoter. Biochem Biophys Res Commun287 :484 eC492,2001
Stephens JM, Lumpkin SJ, Fishman JB: Activation of signal transducers and activators of transcription 1 and 3 by leukemia inhibitory factor, oncostatin-M, and interferon-gamma in adipocytes. J Biol Chem273 :31408 eC31416,1998
Lanna DP, Houseknecht KL, Harris DM, Bauman DE: Effect of somatotropin treatment on lipogenesis, lipolysis, and related cellular mechanisms in adipose tissue of lactating cows. J Dairy Sci78 :1703 eC1712,1995
Yin D, Griffin MJ, Etherton TD: Analysis of the signal pathways involved in the regulation of fatty acid synthase gene expression by insulin and somatotropin. J Anim Sci79 :1194 eC1200,2001
Rufo C, Gasperikova D, Clarke SD, Teran-Garcia M, Nakamura MT: Identification of a novel enhancer sequence in the distal promoter of the rat fatty acid synthase gene. Biochem Biophys Res Commun261 :400 eC405,1999
Bennett MK, Lopez JM, Sanchez HB, Osborne TF: Sterol regulation of fatty acid synthase promoter: coordinate feedback regulation of two major lipid pathways. J Biol Chem270 :25578 eC25583,1995
Moon YS, Latasa MJ, Kim KH, Wang D, Sul HS: Two 5'-regions are required for nutritional and insulin regulation of the fatty-acid synthase promoter in transgenic mice. J Biol Chem275 :10121 eC10127,2000
Latasa MJ, Moon YS, Kim KH, Sul HS: Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element. Proc Natl Acad Sci U S A97 :10619 eC10624,2000
Kim JB, Sarraf P, Wright M, Yao KM, Mueller E, Solanes G, Lowell BB, Spiegelman BM: Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest101 :1 eC9,1998
Boizard M, Le Liepvre X, Lemarchand P, Foufelle F, Ferre P, Dugail I: Obesity-related overexpression of fatty-acid synthase gene in adipose tissue involves sterol regulatory element-binding protein transcription factors. J Biol Chem273 :29164 eC29171,1998
Kim S, Dugail I, Standridge M, Claycombe K, Chun J, Moustaid-Moussa N: Angiotensin II-responsive element is the insulin-responsive element in the adipocyte fatty acid synthase gene: role of adipocyte determination and differentiation factor 1/sterol-regulatory-element-binding protein 1c. Biochem J357 :899 eC904,2001
Claycombe KJ, Wang Y, Jones BH, Kim S, Wilkison WO, Zemel MB, Chun J, Moustaid-Moussa N: Transcriptional regulation of the adipocyte fatty acid synthase gene by agouti: interaction with insulin. Physiol Genomics3 :157 eC162,2000
Roder K, Wolf SS, Beck KF, Sickinger S, Schweizer M: NF-Y binds to the inverted CCAAT box, an essential element for cAMP-dependent regulation of the rat fatty acid synthase (FAS) gene. Gene184 :21 eC26,1997
Rolland V, Liepvre XL, Jump DB, Lavau M, Dugail I: A GC-rich region containing Sp1 and Sp1-like binding sites is a crucial regulatory motif for fatty acid synthase gene promoter activity in adipocytes: implication in the overactivity of FAS promoter in obese Zucker rats. J Biol Chem271 :21297 eC21302,1996
Moustaid N, Sakamoto K, Clarke S, Beyer RS, Sul HS: Regulation of fatty acid synthase gene transcription: sequences that confer a positive insulin effect and differentiation-dependent expression in 3T3eCL1 preadipocytes are present in the 332 bp promoter. Biochem J292 :767 eC772,1993
Guichard C, Dugail I, Le L, Liepvre XL, Lavau M: Genetic regulation of fatty acid synthetase expression in adipose tissue: overtranscription of the gene in genetically obese rats. J Lipid Res33 :679 eC687,1992
Soldaini E, John S, Moro S, Bollenbacher J, Schindler U, Leonard WJ: DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites. Mol Cell Biol20 :389 eC401,2000
Ehret GB, Reichenbach P, Schindler U, Horvath CM, Fritz S, Nabholz M, Bucher P: DNA binding specificity of different STAT proteins: comparison of in vitro specificity with natural target sites. J Biol Chem276 :6675 eC6688,2001
Wang Y, Jones Voy B, Urs S, Kim S, Soltani-Bejnood M, Quigley N, Heo YR, Standridge M, Andersen B, Dhar M, Joshi R, Wortman P, Taylor JW, Chun J, Leuze M, Claycombe K, Saxton AM, Moustaid-Moussa N: The human fatty acid synthase gene and de novo lipogenesis are coordinately regulated in human adipose tissue. J Nutr134 :1032 eC1038,2004
Volpe JJ, Marasa JC: Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver. Biochim Biophys Acta380 :454 eC472,1975(Jessica C. Hogan, and Jac)