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Differential Expression of Signal Transducers and Activators of Transcription during Human Adipogenesis
Biochemical and Biophysical Research Communications 281, 907–912 (2001) doi:10.1006/bbrc.2001.4460, available online at http://www.idealibrary.com on
Differential Expression of Signal Transducers and Activators of Transcription during Human Adipogenesis Joyce B. Harp,* ,1 Dawn Franklin,† Abenah A. Vanderpuije,* and Jeffrey M. Gimble† *Department of Nutrition, University of North Carolina at Chapel Hill, North Carolina 27599; and †Zen-Bio, Inc., Research Triangle Park, North Carolina
Received January 30, 2001
Signal Transducers and Activators of Transcription (STATs) display unique expression patterns upon induction of differentiation of murine 3T3-L1 preadipocytes into adipocytes. During differentiation, expression of STAT1 and STAT5 increase, while STAT3 and STAT6 remain relatively unchanged. Here, we determined whether human subcutaneous preadipocytes expressed STATs and if the pattern of expression changed during adipogenesis. We found by Western blot analysis that freshly isolated preadipocytes expressed STAT1, STAT3, STAT5, and STAT6, but not STAT2 and STAT4. Induction of preadipocyte differentiation with 1-methyl-3-isobutylxanthine, dexamethasone, insulin, and BRL49653 decreased expression of STAT1, and increased expression of STAT3 and STAT5. STAT6 expression did not change during adipogenesis. Changes in expression of CCAAT/enhancer binding protein  (C/EBP), C/EBP␦, C/EBP␣, and peroxisome proliferator-activated receptor ␥ were similar to murine cell lines. These results suggest that unlike the traditional adipogenic transcription factors, unique differences exist in STAT expression patterns between murine and human adipose cells. © 2001 Academic Press
Key Words: STAT; preadipocytes; adipocytes; adipogenesis; peroxisome proliferator-activated receptor; CCAAT/enhancer binding proteins; and transcription factor.
Much of our understanding of adipogenesis is based on studies in murine-derived embryonic 3T3-L1 cells. 3T3-L1 cells are a clonal subline of committed fibroblast-like cells that replicate in culture until they reach confluence (1, 2). At confluence, the preadipocytes undergo cell-cell contact-inhibited growth arrest. Upon stimulation with 1-methyl-3-isobutylxanthine 1 To whom correspondence should be addressed at Department of Nutrition, University of North Carolina at Chapel Hill, CB# 7400 McGavran-Greenberg Hall, Chapel Hill, NC 27599. Fax: (919) 9667216. E-mail: [email protected].
(MIX), dexamethasone, and high dose insulin (MDI) for 2–3 days, 3T3-L1 preadipocytes undergo several rounds of mitotic clonal expansion, then exit the cell cycle, and begin to express adipocyte-specific genes. Approximately 5 days after differentiation, up to 90% of the cells display the characteristic lipid-filled adipocyte phenotype. The induction of differentiation and subsequent expression of adipocyte-specific genes depend on transcriptional activation and expression of at least two families of transcription factors: CCAAT/ enhancer binding proteins (C/EBP) and peroxisome proliferator-activated receptor ␥2 (PPAR␥2) (3). More recent studies in 3T3-L1 cells have shown that the STAT transcriptional family of proteins is also expressed and regulated during adipogenesis (4, 5). In mammalian cells, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 comprise a group of latent cytoplasmic transcription factors that are activated by cytokines, peptides, and growth factors (6). Activated receptor and nonreceptor tyrosine kinases phosphorylate STATs on critical tyrosine residues in the carboxy-terminal domain. The phosphorylated STATs then homo- or heterodimerize through reciprocal phosphotyrosine-SH2 domain interaction and translocate to the nucleus where they bind to specific DNA regulatory sequences to stimulate transcription of targeted effector genes. Subsequently, STATs become latent upon dephosphorylation and return to the cytoplasm (7). Functionally, STATs play a role in induction of proliferation and differentiation of a variety of hematopoetic and nonhematopoetic cells. In 3T3-L1 cells, expression of STATs change with differentiation (5, 8). Expression of STAT1 and STAT5 increase significantly after the induction of differentiation, while STAT3 and STAT6 expression do not change. Although there is some evidence that this family of transcription factors also regulates preadipocyte proliferation and differentiation (5, 8), precisely how the specific STATs fit into the differentiation program is not known. Because the newly characterized STATs may play a critical role in adipogenesis and lipid me-
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0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Vol. 281, No. 4, 2001
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tabolism, we questioned whether the STATs are also expressed in human adipose cells and whether their expression changes during adipogenesis. We report here that STAT1, STAT3, STAT5A/B, and STAT6 are expressed in freshly isolated preadipocytes. Induction of preadipocyte differentiation into adipocytes leads to a decrease in expression of STAT1, increase in expression of STAT3 and STAT5, and no major change in expression of STAT6. We conclude that during adipogenesis, human cells express STAT5 and STAT6 in a manner similar to 3T3-L1 cells, but display a different pattern of STAT1 and STAT3 expression. Moreover, the differentiation stage of human adipose cell may determine whether the cell is able to respond to ligands that signal through the various STAT pathways. MATERIALS AND METHODS Materials. Monoclonal antibodies to STAT1 (C-terminus), STAT3, and STAT5A/B were from Pharmingen/Transduction Laboratories (Lexington, KY). Polyclonal antibodies to STAT6 (S20) and C/EBP␣ (S61), and monoclonal antibodies to C/EBP (H-7), C/EBP␦ (M17), and PPAR␥ (E8), and protein A/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) detection kit and horseradish peroxidase conjugated secondary antibodies were from Amersham Pharmacia Biotech (Piscataway, NJ). Cell culture. Human subcutaneous preadipocytes isolated from adipose tissue of healthy subjects undergoing liposuction surgery were obtained from Zen Bio, Inc (Research Triangle Park, NC) and cultured as previously described (9). Patient informed consent was obtained and use of the discarded surgical adipose tissue approved by the hospitals’ Institutional Review Boards. Briefly, preadipocytes were plated at 30,000 cells/cm 2 in DMEM/F-10 with 10% FBS supplemented with antibiotics for 16 h to allow attachment. Medium was then changed to DMEM/F-10 supplemented with antibiotics, 3% fetal bovine serum (FBS), 15 mM Hepes (pH 7.4), 33 M biotin, 17 M pantothenate, 100 nM human recombinant insulin, 1 M dexamethasone, 0.25 mM 3-isobutyl-1-methylxanthine, and 1 M BRL49653 (MDIB) to induce differentiation. Cells were maintained in this medium for 3 days, and then switched to medium containing insulin and dexamethasone for the remaining 6 –9 days. Immunoblot analysis. Cells were washed twice in phosphate buffered saline with 1 mM orthovanadate (PBS-V), and then placed immediately in sample buffer (1% NP40, 20 mM Tris–HCL, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% NaN 3, 10 g/ml aprotinin, 1 M pepstatin, 16.4 g/ml leupeptin, 1 mM PMSF, 0.1 mM Na 3VO 4, 2% SDS, 10% glycerol) without DTT or tracking dye. Lysates were heated and protein concentrations determined prior to adding 100 mM DTT and tracking dye. Protein concentrations were determined in cell lysates using the Bio-Rad DC protein determination kit. Bovine serum albumin was used as standard. Samples were heated for 5 min at 95°C, separated by 8 –10% SDS–PAGE, and analyzed by immunoblotting as previously described (10, 11). Immunoblots were developed with the ECL kit. Densitometric analyses of scanned autoradiographs were performed with Scion Image software (Frederick, MD).
RESULTS STAT Expression in Freshly Isolated and Cultured Human Adipose Cells We first examined by Western blot analysis STAT expression in freshly isolated human subcutaneous
FIG. 1. STAT expression in isolated subcutaneous human preadipocytes and adipocytes. Cellular lysates were prepared from freshly isolated preadipocytes and adipocytes. Western blot analysis of 40 g of protein was performed with anti-STAT antibodies. The following dilutions were used: anti-STAT1 1:2500, anti-STAT3 1:2500, anti-STAT5 1:250, and anti-STAT6 1:200. Results are representative of three separate experiments.
preadipocytes and adipocytes that had not been placed in culture dishes or cryopreserved (Fig. 1). Freshly isolated preadipocytes expressed STAT1, STAT3, STAT5, and STAT6. Freshly isolated adipocytes only expressed STAT5 and low levels of STAT3. STAT2 and STAT4 were not expressed in human adipose cells (data not shown). To study adipogenesis in vitro, isolated human preadipocytes were plated at a density to reach confluence within 24 h (see Materials and Methods). Differentiation was induced with MDIB in one-day post-confluent cells (Fig. 2). On day three following induction, cells began to accumulate intracellular lipid droplets. The adipocytes were lipid-laden by day 9. To determine whether cultured human preadipocytes and adipocytes expressed STATs and whether expression of these factors changed during adipogenesis, STAT expression was determined by Western blot analysis in human preadipocytes and adipocytes prior to and 1, 2, 3, 6, and 9 days after the induction of differentiation (Fig. 3). To determine the effect of the culture conditions on STAT expression, unstimulated preadipocytes were also maintained in culture for the same period of time as the MDIB-stimulated preadipocytes. Results show that STAT1 expression declined,
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FIG. 2. Lipid accumulation at different stages of human adipogenesis. Subcutaneous human preadipocytes were induced to differentiate in culture as described under Materials and Methods. Phase-contrast microscopy was performed on, before (day 0), and 1, 2, 3, 6, and 9 days after differentiation.
sixfold, 6 days after the induction of differentiation and was barely detectable on day 9. In unstimulated preadipocytes maintained in culture, STAT1 expression remained constant over the 9-day culture period. Both STAT3 and STAT5 showed trends of increased expression upon induction of differentiation. STAT3 expression increased threefold and 11-fold on days 1 and 6, respectively. STAT5 expression increased 1.5-fold and sixfold, on days 1 and 6, respectively. Levels of STAT3 and STAT5 expression remained low in cells that were not stimulated by MDIB. In contrast to the differentiation-related changes in STAT1, STAT3, and STAT5 expression, STAT6 expression remained constant throughout adipogenesis, 1.5-fold decline. C/EBP and PPAR␥ Expression upon Adipogenesis An association between the C/EBP and STAT families of transcription factors has been shown in other cell systems. Over expression of C/EBP and C/EBP␦ in NIH-3T3 cells leads to an increase in expression of STAT1, STAT5A, and STAT5B upon induction of adipogenesis (8). Conversely, in HepG2 and mammary cells, STAT3 activation induces C/EBP␦ transcription (12, 13). In the next set of studies, we measured C/EBP, C/EBP␦, and C/EBP␣ expression during human adipogenesis. C/EBP showed an increase in expression one day after the induction of differentiation (Fig. 4). This expression was transient, but lasted until day 6 after differentiation. C/EBP␦ expression increased on day 1 and declined on day 3. C/EBP␣ pro-
tein expression increased on day 3 of differentiation and remained high through day 9. PPAR␥ expression was measured using an antibody that detects both ␥1 and ␥2. PPAR␥1 expression increased on day 2 and PPAR␥2 on day 6 after differentiation (Fig. 4). DISCUSSION There are very few studies translating the wellcharacterized events of adipogenesis in mouse 3T3-L1 cells to human adipose cells (14, 15). In this report, we used primary human preadipocytes and adipocytes obtained at liposuction to study patterns of expression of STATs, as well as the C/EBPs and PPAR␥ family of transcription factors during adipogenesis. We found that STAT1, STAT3, STAT5, and STAT6 were expressed in freshly isolated preadipocytes. Only STAT5 and to a lesser extent STAT3 were expressed in freshly isolated adipocytes. When STAT expression was examined during adipogenesis, unique expression patterns were seen. STAT1 decreased, STAT3 and STAT5 increased, and STAT6 remained relatively unchanged. Our results demonstrating the absence of expression of STAT1 in freshly isolated adipocytes, and declining STAT1 expression during human adipogenesis are in sharp contrast to the pattern of increased STAT1 expression during 3T3-L1 adipogenesis (4, 5, 16). This opposite pattern of STAT1 expression suggests that the transcriptional or translational regulators of STAT1 expression differ in murine and human adipose cells.
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expression (8). This suggest that in murine cells, during adipogenesis, transcriptional regulation of STAT1 expression may be driven by PPAR␥/RXR␣. Previous studies in human preadipocytes have demonstrated that the PPAR␥ ligands induce PPAR␥ transcription within 2 to 4 h following initial exposure (19). In our culture conditions, the PPAR␥ ligand BRL49653 was present in the culture medium on days 1–3 and PPAR␥1 protein was expressed on day 2 and PPAR␥2 on day 6. Thus, it appears that, unlike murine cells, in human adipose cells STAT1 expression is not stimulated by PPAR␥ ligands. Moreover, based on our results, it appears that STAT1 expression may be negatively regulated during human adipocyte differentiation. Despite the dissimilarities in STAT1 expression between mouse and human adipose cells, both cell types displayed similar patterns of expression of STAT5 and STAT6. As in 3T3-L1 cells, STAT6 was constitutively expressed throughout adipogenesis. STAT5 expression occurred late, day 6, in differentiation. This suggests that STAT5 may not be an important initiator of human fat cell formation, but may play a role in late stages of adipocyte formation or lipid metabolism. The
FIG. 3. STAT expression during human adipogenesis. Lysates were prepared from cells during progressive stages of preadipocyte differentiation or maintained in culture without induction of adipogenesis. (A) Western blot analysis of 40 g of protein was performed with anti-STAT1, STAT3, STAT5, and STAT6 antibodies. Results are representative of three separate experiments. (B) Densitometric analysis of the Western blots is shown for each STAT and presented as area curves (n ⫽ 3).
The murine STAT1 promoter has been cloned and contains a retinoic acid (RA) response element, which in vivo preferentially interacts with RA receptor  and RXR␣ (17). PPAR␥, which is expressed late in 3T3-L1 differentiation, heterodimerizes with RXR␣ prior to DNA binding (18). In MDI treated NIH-3T3 cells transfected with C/EBP/␦, PPAR␥ ligands enhance STAT1
FIG. 4. Expression of C/EBPs and PPAR␥ during human adipogenesis. Cellular lysates were prepared during progressive stages of preadipocyte differentiation into adipocytes. Western blot analysis of 40 g of protein was performed with anti-C/EBP, C/EBP␦, and PPAR␥ antibodies all at 1:200, except the anti-C/EBP␣ antibody was used at 1:100. Results are representative of two separate experiments.
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role of STAT5 in lipid metabolism is supported by recent studies in COS-1 cells transfected with growth hormone receptors, JAKs, STAT5, and peroxisome proliferator reporter constructs, which demonstrate that STAT5B mediates growth hormone induced inhibition of PPAR␣ activity (20). PPAR␣ is important in -oxidation. One would expect to find that STAT5mediated inhibition of PPAR␣ activity in adipose or liver cells would increase lipid accumulation. There is some evidence in whole animals that STAT5 affects adipose tissue. Transgenic mice with a null mutation in the STAT5A/B gene have disproportionate decreases in fat pad size (21). However, no studies were reported in these mice to determine whether the decrease in adiposity was related to a change in energy balance (21). PPAR␥ ligands also enhance STAT5 expression in MDI treated C/EBP/␦-NIH-3T3 cells (8). Thus, it is possible that either the BRL compound or the process of adipogenesis led to the increase in STAT5 expression in our human adipose cells. Our data showing increased STAT3 expression early in adipocyte formation, and before lipid accumulation, suggests that STAT3 may be important in the induction of adipogenesis. In HepG2 and mammary cells, C/EBP␦ gene transcription is mediated by STAT3 activation (12, 13). In these human cells, STAT3 was expressed in preadipocytes, and this expression increased early after the induction of differentiation. C/EBP␦ expression also increased one day after the induction of differentiation. Thus, this temporal relationship, in expression of STAT3 and C/EBP␦, although close, suggests that it is possible that STAT3 regulates human adipogenesis through induction of expression of C/EBP␦, and subsequent expression of C/EBP␣. C/EBP␦ and C/EBP␣ are transcriptional regulators of murine adipogenesis (22). In human cells, C/EBP␣ was expressed late in adipogenesis similarly to 3T3-L1 cells. Because thiazolidindiones are widely used to treat patients with type 2 diabetes mellitus, it is clinically important to sort out the effect of PPAR␥ agonists on STAT1 and STAT 5 expression in human adipose cells. Others have shown that PPAR␥ agonists both activate and induce the expression of PPAR␥ mRNA (19). However, previous studies suggest that protein expression of PPAR␥2 has been difficult to detect in human adipose cells (23). In this study, we show by Western blot analysis that PPAR␥2 is expressed on day 6 of differentiation, albeit at levels lower than in 3T3-L1 cells where comparable amounts of protein are assessed. In addition, PPAR␥2 expression is lower than PPAR␥1. In our culture system, BRL49653 was present in the culture medium on days 1-3 of differentiation. Thus, these data suggests that the adipogenic process rather than the BRL agonist alone induces PPAR␥2 expression. In conclusion, human adipose cells express several members of the STAT family of transcription factors. The
patterns of STAT expression change with differentiation and, with respect to STAT1 and STAT3, differ from patterns of expression in murine adipose cells. Future studies may determine the role of STATs in the regulation of human adipogenesis and lipid metabolism. ACKNOWLEDGMENTS This work was supported by the US Public Health Service Grant DK53398 to J.B.H. and the Clinical Nutrition Research Center at UNC-Chapel Hill Grant DK56350. We kindly thank all of the patients and health care providers involved in providing the adipose tissue used in these studies. We also thank the scientific staff of Zen-Bio, Inc. and the members of the Harp laboratory for critical comments and suggestions during the course of this study.
REFERENCES 1. Green, H., and Kehinde, O. (1974) Sublines of mouse 3T3 cells that accumulate lipid. Cell 1, 113–116. 2. Green, H., and Kehinde, O. (1979) Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J. Cell. Physiol. 101, 169 –172. 3. MacDougald, O., and Lane, M. D. (1995) Transcriptional regulation of gene expression during adipocyte differentiation. Annu. Rev. Biochem. 64, 345–373. 4. Stephens, J., Morrison, R. F., and Pilch, P. F. (1996) The expression and regulation of STATs during 3T3-L1 adipocyte differentiation. J. Biol. Chem. 271, 10441–10444. 5. Deng, J., Hua, K., Lesser, S. S., and Harp, J. B. (2000) Activation of STAT3 during proliferative phases of 3T3-L1 adipogenesis. Endocrinology 141, 2370 –2376. 6. Darnell, J. E., Jr. (1997) STATs and gene regulation. Science 277, 1630 –1635. 7. Haspel, R., Salditt-Georgieff, M., and Darnell, J. E., Jr. (1996) The rapid inactivation of nuclear tyrosine phosphorylated Stat1 depends on a protein tyrosine phosphatase. EMBO J. 15, 6262– 6268. 8. Stephens, J., Morrison, R. F., Wu, Z., and Farmer, S. R. (1999) PPAR gamma ligand-dependent induction of STAT1, STAT5A, and STAT5B during adipogenesis. Biochem. Biophys. Res. Commun. 262, 216 –222. 9. Zhou, L., Halvorsen, Y-D, Cryan, E. V., Pelton, P. D., Burris, T. P., and Demarest, K. T. (1999) Analysis of the pattern of gene expression during human adipogenesis by DNA microarray. Biotech. Techniques 13, 513–517. 10. Marrero, M. B., Schieffer, B., Bing, L., Sun, J., Harp, J. B., and Ling, B. N. (1997) Role of Janus Kinase/Signal Transducer and Activator of Transcription and mitogen-activated protein kinase cascade in angiotensin II- and platelet-derived growth factorinduced vascular smooth muscle cell proliferation. J. Biol. Chem. 272, 24684 –24690. 11. Laemmli, U. K. (1970) Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. 12. Yamada, T., Tobita, K., Osada, S., Nishihara, T., and Imagawa, M. (1997) CCAAT/enhancer -binding protein delta gene expression is mediated by APRF/STAT3. J. Biochem. 121, 731–738. 13. Hutt, J., O’Rourke, J. P., and DeWille, J. (2000) Signal transducer activator of transcription 3 activates CCAAT enhancerbinding protein ␦ gene transcription in G 0 growth-arrested mouse mammary epithelial cells and in involuting mouse mammary gland. J. Biol. Chem. 275, 29123–29131. 14. Hauner, H., Rohrig, K., Spelleken, M., Liu, L. S., and Ecke, J.
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(1998) Development of insulin-responsive glucose uptake and GLUT4 expression in differentiating human adipocyte precursor cells. Int. J. Obes. Relat. Metab. Disord. 22, 448 – 453. Hube, F., Lee, Y. M., Rohrig, K., and Hauner, H. (1999) The phosphodiesterase inhibitor IBMX suppresses TNF-alpha expression in human adipocyte precursor cells: A possible explanation for its adipogenic effect. Horm. Metab. Res. 31, 359 –362. Stephens, J., Lumpkin, S. J., and Fishman, J. B. (1998) Activation of Signal Transducers and Activators of Transcription 1 and 3 by leukemia inhibitory factor, oncostatin-M, and interfeuron-␥ in adipocytes. J. Biol. Chem. 273, 31408 –31416. Weihua, X., Kolla, V., and Kalvakolanu, D. V. (1997) Modulation of interferon action by retinoids: Induction of murine STAT1 gene expression by retinoic acid. J. Biol. Chem. 272, 9742–9748. Tontonoz, P., Graves, R. A., Budavari, A. I., ErdjumentBromage, H., Lui, M., Hu, E., Tempst, P., and Spiegelman, B. M. (1994) Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR gamma and RXR alpha. Nucleic Acids Res. 22, 5628 –5634. Saladin, R., Fajas, L., Dana, S., Halvorsen, Y-D., Auwerx, J., and
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Briggs, M. (1999) Differential regulation of peroxisome proliferator activated receptor gamma1 (PPARgamma1) and PPARgamma2 messenger RNA expression in the early stages of adipogenesis. Cell Growth Differ. 10, 43– 48. Zhou, Y., and Waxman, D. J. (1999) Cross-talk between Janus Kinase-Signal Transducer and Activator of Trancription (JAKSTAT) and peroxisome proliferator-activated receptor-␣ (PPAR␣) signaling pathways. J. Biol. Chem. 274, 2672–2681. Teglund, S., McKay, C., Schuetz, E., van Deursen, J. M., Stravopodis, D., Wang, D., Brown, M., Bodner, S., Grosveld, G., and Ihle, J. N. (1998) STAT5 knockout. Cell 93, 841– 850. Darlington, G., Ross, S. E., and MacDougald, O. A. (1998) The role of C/EBP genes in adipocyte differentiation. J. Biol. Chem. 273, 30057–30060. Adams, M., Montague, C. T., Prins, J. B., Holder, J. C., Smith, S. A., Sanders, L., Digby, J. E., Sewter, C. P., Lazar, M. A., Chatterjee, V. K. K., and O’Rahilly. S. (1997) Activators of peroxisome proliferator- activated receptor have depot-specific effects on human preadipocyte differentiation. J. Clin. Invest. 100, 3149 –3153.
Differential Expression of Signal Transducers and Activators of Transcription during Human Adipogenesis Joyce B. Harp,* ,1 Dawn Franklin,† Abenah A. Vanderpuije,* and Jeffrey M. Gimble† *Department of Nutrition, University of North Carolina at Chapel Hill, North Carolina 27599; and †Zen-Bio, Inc., Research Triangle Park, North Carolina
Received January 30, 2001
Signal Transducers and Activators of Transcription (STATs) display unique expression patterns upon induction of differentiation of murine 3T3-L1 preadipocytes into adipocytes. During differentiation, expression of STAT1 and STAT5 increase, while STAT3 and STAT6 remain relatively unchanged. Here, we determined whether human subcutaneous preadipocytes expressed STATs and if the pattern of expression changed during adipogenesis. We found by Western blot analysis that freshly isolated preadipocytes expressed STAT1, STAT3, STAT5, and STAT6, but not STAT2 and STAT4. Induction of preadipocyte differentiation with 1-methyl-3-isobutylxanthine, dexamethasone, insulin, and BRL49653 decreased expression of STAT1, and increased expression of STAT3 and STAT5. STAT6 expression did not change during adipogenesis. Changes in expression of CCAAT/enhancer binding protein  (C/EBP), C/EBP␦, C/EBP␣, and peroxisome proliferator-activated receptor ␥ were similar to murine cell lines. These results suggest that unlike the traditional adipogenic transcription factors, unique differences exist in STAT expression patterns between murine and human adipose cells. © 2001 Academic Press
Key Words: STAT; preadipocytes; adipocytes; adipogenesis; peroxisome proliferator-activated receptor; CCAAT/enhancer binding proteins; and transcription factor.
Much of our understanding of adipogenesis is based on studies in murine-derived embryonic 3T3-L1 cells. 3T3-L1 cells are a clonal subline of committed fibroblast-like cells that replicate in culture until they reach confluence (1, 2). At confluence, the preadipocytes undergo cell-cell contact-inhibited growth arrest. Upon stimulation with 1-methyl-3-isobutylxanthine 1 To whom correspondence should be addressed at Department of Nutrition, University of North Carolina at Chapel Hill, CB# 7400 McGavran-Greenberg Hall, Chapel Hill, NC 27599. Fax: (919) 9667216. E-mail: [email protected].
(MIX), dexamethasone, and high dose insulin (MDI) for 2–3 days, 3T3-L1 preadipocytes undergo several rounds of mitotic clonal expansion, then exit the cell cycle, and begin to express adipocyte-specific genes. Approximately 5 days after differentiation, up to 90% of the cells display the characteristic lipid-filled adipocyte phenotype. The induction of differentiation and subsequent expression of adipocyte-specific genes depend on transcriptional activation and expression of at least two families of transcription factors: CCAAT/ enhancer binding proteins (C/EBP) and peroxisome proliferator-activated receptor ␥2 (PPAR␥2) (3). More recent studies in 3T3-L1 cells have shown that the STAT transcriptional family of proteins is also expressed and regulated during adipogenesis (4, 5). In mammalian cells, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 comprise a group of latent cytoplasmic transcription factors that are activated by cytokines, peptides, and growth factors (6). Activated receptor and nonreceptor tyrosine kinases phosphorylate STATs on critical tyrosine residues in the carboxy-terminal domain. The phosphorylated STATs then homo- or heterodimerize through reciprocal phosphotyrosine-SH2 domain interaction and translocate to the nucleus where they bind to specific DNA regulatory sequences to stimulate transcription of targeted effector genes. Subsequently, STATs become latent upon dephosphorylation and return to the cytoplasm (7). Functionally, STATs play a role in induction of proliferation and differentiation of a variety of hematopoetic and nonhematopoetic cells. In 3T3-L1 cells, expression of STATs change with differentiation (5, 8). Expression of STAT1 and STAT5 increase significantly after the induction of differentiation, while STAT3 and STAT6 expression do not change. Although there is some evidence that this family of transcription factors also regulates preadipocyte proliferation and differentiation (5, 8), precisely how the specific STATs fit into the differentiation program is not known. Because the newly characterized STATs may play a critical role in adipogenesis and lipid me-
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0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Vol. 281, No. 4, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tabolism, we questioned whether the STATs are also expressed in human adipose cells and whether their expression changes during adipogenesis. We report here that STAT1, STAT3, STAT5A/B, and STAT6 are expressed in freshly isolated preadipocytes. Induction of preadipocyte differentiation into adipocytes leads to a decrease in expression of STAT1, increase in expression of STAT3 and STAT5, and no major change in expression of STAT6. We conclude that during adipogenesis, human cells express STAT5 and STAT6 in a manner similar to 3T3-L1 cells, but display a different pattern of STAT1 and STAT3 expression. Moreover, the differentiation stage of human adipose cell may determine whether the cell is able to respond to ligands that signal through the various STAT pathways. MATERIALS AND METHODS Materials. Monoclonal antibodies to STAT1 (C-terminus), STAT3, and STAT5A/B were from Pharmingen/Transduction Laboratories (Lexington, KY). Polyclonal antibodies to STAT6 (S20) and C/EBP␣ (S61), and monoclonal antibodies to C/EBP (H-7), C/EBP␦ (M17), and PPAR␥ (E8), and protein A/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) detection kit and horseradish peroxidase conjugated secondary antibodies were from Amersham Pharmacia Biotech (Piscataway, NJ). Cell culture. Human subcutaneous preadipocytes isolated from adipose tissue of healthy subjects undergoing liposuction surgery were obtained from Zen Bio, Inc (Research Triangle Park, NC) and cultured as previously described (9). Patient informed consent was obtained and use of the discarded surgical adipose tissue approved by the hospitals’ Institutional Review Boards. Briefly, preadipocytes were plated at 30,000 cells/cm 2 in DMEM/F-10 with 10% FBS supplemented with antibiotics for 16 h to allow attachment. Medium was then changed to DMEM/F-10 supplemented with antibiotics, 3% fetal bovine serum (FBS), 15 mM Hepes (pH 7.4), 33 M biotin, 17 M pantothenate, 100 nM human recombinant insulin, 1 M dexamethasone, 0.25 mM 3-isobutyl-1-methylxanthine, and 1 M BRL49653 (MDIB) to induce differentiation. Cells were maintained in this medium for 3 days, and then switched to medium containing insulin and dexamethasone for the remaining 6 –9 days. Immunoblot analysis. Cells were washed twice in phosphate buffered saline with 1 mM orthovanadate (PBS-V), and then placed immediately in sample buffer (1% NP40, 20 mM Tris–HCL, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% NaN 3, 10 g/ml aprotinin, 1 M pepstatin, 16.4 g/ml leupeptin, 1 mM PMSF, 0.1 mM Na 3VO 4, 2% SDS, 10% glycerol) without DTT or tracking dye. Lysates were heated and protein concentrations determined prior to adding 100 mM DTT and tracking dye. Protein concentrations were determined in cell lysates using the Bio-Rad DC protein determination kit. Bovine serum albumin was used as standard. Samples were heated for 5 min at 95°C, separated by 8 –10% SDS–PAGE, and analyzed by immunoblotting as previously described (10, 11). Immunoblots were developed with the ECL kit. Densitometric analyses of scanned autoradiographs were performed with Scion Image software (Frederick, MD).
RESULTS STAT Expression in Freshly Isolated and Cultured Human Adipose Cells We first examined by Western blot analysis STAT expression in freshly isolated human subcutaneous
FIG. 1. STAT expression in isolated subcutaneous human preadipocytes and adipocytes. Cellular lysates were prepared from freshly isolated preadipocytes and adipocytes. Western blot analysis of 40 g of protein was performed with anti-STAT antibodies. The following dilutions were used: anti-STAT1 1:2500, anti-STAT3 1:2500, anti-STAT5 1:250, and anti-STAT6 1:200. Results are representative of three separate experiments.
preadipocytes and adipocytes that had not been placed in culture dishes or cryopreserved (Fig. 1). Freshly isolated preadipocytes expressed STAT1, STAT3, STAT5, and STAT6. Freshly isolated adipocytes only expressed STAT5 and low levels of STAT3. STAT2 and STAT4 were not expressed in human adipose cells (data not shown). To study adipogenesis in vitro, isolated human preadipocytes were plated at a density to reach confluence within 24 h (see Materials and Methods). Differentiation was induced with MDIB in one-day post-confluent cells (Fig. 2). On day three following induction, cells began to accumulate intracellular lipid droplets. The adipocytes were lipid-laden by day 9. To determine whether cultured human preadipocytes and adipocytes expressed STATs and whether expression of these factors changed during adipogenesis, STAT expression was determined by Western blot analysis in human preadipocytes and adipocytes prior to and 1, 2, 3, 6, and 9 days after the induction of differentiation (Fig. 3). To determine the effect of the culture conditions on STAT expression, unstimulated preadipocytes were also maintained in culture for the same period of time as the MDIB-stimulated preadipocytes. Results show that STAT1 expression declined,
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FIG. 2. Lipid accumulation at different stages of human adipogenesis. Subcutaneous human preadipocytes were induced to differentiate in culture as described under Materials and Methods. Phase-contrast microscopy was performed on, before (day 0), and 1, 2, 3, 6, and 9 days after differentiation.
sixfold, 6 days after the induction of differentiation and was barely detectable on day 9. In unstimulated preadipocytes maintained in culture, STAT1 expression remained constant over the 9-day culture period. Both STAT3 and STAT5 showed trends of increased expression upon induction of differentiation. STAT3 expression increased threefold and 11-fold on days 1 and 6, respectively. STAT5 expression increased 1.5-fold and sixfold, on days 1 and 6, respectively. Levels of STAT3 and STAT5 expression remained low in cells that were not stimulated by MDIB. In contrast to the differentiation-related changes in STAT1, STAT3, and STAT5 expression, STAT6 expression remained constant throughout adipogenesis, 1.5-fold decline. C/EBP and PPAR␥ Expression upon Adipogenesis An association between the C/EBP and STAT families of transcription factors has been shown in other cell systems. Over expression of C/EBP and C/EBP␦ in NIH-3T3 cells leads to an increase in expression of STAT1, STAT5A, and STAT5B upon induction of adipogenesis (8). Conversely, in HepG2 and mammary cells, STAT3 activation induces C/EBP␦ transcription (12, 13). In the next set of studies, we measured C/EBP, C/EBP␦, and C/EBP␣ expression during human adipogenesis. C/EBP showed an increase in expression one day after the induction of differentiation (Fig. 4). This expression was transient, but lasted until day 6 after differentiation. C/EBP␦ expression increased on day 1 and declined on day 3. C/EBP␣ pro-
tein expression increased on day 3 of differentiation and remained high through day 9. PPAR␥ expression was measured using an antibody that detects both ␥1 and ␥2. PPAR␥1 expression increased on day 2 and PPAR␥2 on day 6 after differentiation (Fig. 4). DISCUSSION There are very few studies translating the wellcharacterized events of adipogenesis in mouse 3T3-L1 cells to human adipose cells (14, 15). In this report, we used primary human preadipocytes and adipocytes obtained at liposuction to study patterns of expression of STATs, as well as the C/EBPs and PPAR␥ family of transcription factors during adipogenesis. We found that STAT1, STAT3, STAT5, and STAT6 were expressed in freshly isolated preadipocytes. Only STAT5 and to a lesser extent STAT3 were expressed in freshly isolated adipocytes. When STAT expression was examined during adipogenesis, unique expression patterns were seen. STAT1 decreased, STAT3 and STAT5 increased, and STAT6 remained relatively unchanged. Our results demonstrating the absence of expression of STAT1 in freshly isolated adipocytes, and declining STAT1 expression during human adipogenesis are in sharp contrast to the pattern of increased STAT1 expression during 3T3-L1 adipogenesis (4, 5, 16). This opposite pattern of STAT1 expression suggests that the transcriptional or translational regulators of STAT1 expression differ in murine and human adipose cells.
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expression (8). This suggest that in murine cells, during adipogenesis, transcriptional regulation of STAT1 expression may be driven by PPAR␥/RXR␣. Previous studies in human preadipocytes have demonstrated that the PPAR␥ ligands induce PPAR␥ transcription within 2 to 4 h following initial exposure (19). In our culture conditions, the PPAR␥ ligand BRL49653 was present in the culture medium on days 1–3 and PPAR␥1 protein was expressed on day 2 and PPAR␥2 on day 6. Thus, it appears that, unlike murine cells, in human adipose cells STAT1 expression is not stimulated by PPAR␥ ligands. Moreover, based on our results, it appears that STAT1 expression may be negatively regulated during human adipocyte differentiation. Despite the dissimilarities in STAT1 expression between mouse and human adipose cells, both cell types displayed similar patterns of expression of STAT5 and STAT6. As in 3T3-L1 cells, STAT6 was constitutively expressed throughout adipogenesis. STAT5 expression occurred late, day 6, in differentiation. This suggests that STAT5 may not be an important initiator of human fat cell formation, but may play a role in late stages of adipocyte formation or lipid metabolism. The
FIG. 3. STAT expression during human adipogenesis. Lysates were prepared from cells during progressive stages of preadipocyte differentiation or maintained in culture without induction of adipogenesis. (A) Western blot analysis of 40 g of protein was performed with anti-STAT1, STAT3, STAT5, and STAT6 antibodies. Results are representative of three separate experiments. (B) Densitometric analysis of the Western blots is shown for each STAT and presented as area curves (n ⫽ 3).
The murine STAT1 promoter has been cloned and contains a retinoic acid (RA) response element, which in vivo preferentially interacts with RA receptor  and RXR␣ (17). PPAR␥, which is expressed late in 3T3-L1 differentiation, heterodimerizes with RXR␣ prior to DNA binding (18). In MDI treated NIH-3T3 cells transfected with C/EBP/␦, PPAR␥ ligands enhance STAT1
FIG. 4. Expression of C/EBPs and PPAR␥ during human adipogenesis. Cellular lysates were prepared during progressive stages of preadipocyte differentiation into adipocytes. Western blot analysis of 40 g of protein was performed with anti-C/EBP, C/EBP␦, and PPAR␥ antibodies all at 1:200, except the anti-C/EBP␣ antibody was used at 1:100. Results are representative of two separate experiments.
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role of STAT5 in lipid metabolism is supported by recent studies in COS-1 cells transfected with growth hormone receptors, JAKs, STAT5, and peroxisome proliferator reporter constructs, which demonstrate that STAT5B mediates growth hormone induced inhibition of PPAR␣ activity (20). PPAR␣ is important in -oxidation. One would expect to find that STAT5mediated inhibition of PPAR␣ activity in adipose or liver cells would increase lipid accumulation. There is some evidence in whole animals that STAT5 affects adipose tissue. Transgenic mice with a null mutation in the STAT5A/B gene have disproportionate decreases in fat pad size (21). However, no studies were reported in these mice to determine whether the decrease in adiposity was related to a change in energy balance (21). PPAR␥ ligands also enhance STAT5 expression in MDI treated C/EBP/␦-NIH-3T3 cells (8). Thus, it is possible that either the BRL compound or the process of adipogenesis led to the increase in STAT5 expression in our human adipose cells. Our data showing increased STAT3 expression early in adipocyte formation, and before lipid accumulation, suggests that STAT3 may be important in the induction of adipogenesis. In HepG2 and mammary cells, C/EBP␦ gene transcription is mediated by STAT3 activation (12, 13). In these human cells, STAT3 was expressed in preadipocytes, and this expression increased early after the induction of differentiation. C/EBP␦ expression also increased one day after the induction of differentiation. Thus, this temporal relationship, in expression of STAT3 and C/EBP␦, although close, suggests that it is possible that STAT3 regulates human adipogenesis through induction of expression of C/EBP␦, and subsequent expression of C/EBP␣. C/EBP␦ and C/EBP␣ are transcriptional regulators of murine adipogenesis (22). In human cells, C/EBP␣ was expressed late in adipogenesis similarly to 3T3-L1 cells. Because thiazolidindiones are widely used to treat patients with type 2 diabetes mellitus, it is clinically important to sort out the effect of PPAR␥ agonists on STAT1 and STAT 5 expression in human adipose cells. Others have shown that PPAR␥ agonists both activate and induce the expression of PPAR␥ mRNA (19). However, previous studies suggest that protein expression of PPAR␥2 has been difficult to detect in human adipose cells (23). In this study, we show by Western blot analysis that PPAR␥2 is expressed on day 6 of differentiation, albeit at levels lower than in 3T3-L1 cells where comparable amounts of protein are assessed. In addition, PPAR␥2 expression is lower than PPAR␥1. In our culture system, BRL49653 was present in the culture medium on days 1-3 of differentiation. Thus, these data suggests that the adipogenic process rather than the BRL agonist alone induces PPAR␥2 expression. In conclusion, human adipose cells express several members of the STAT family of transcription factors. The
patterns of STAT expression change with differentiation and, with respect to STAT1 and STAT3, differ from patterns of expression in murine adipose cells. Future studies may determine the role of STATs in the regulation of human adipogenesis and lipid metabolism. ACKNOWLEDGMENTS This work was supported by the US Public Health Service Grant DK53398 to J.B.H. and the Clinical Nutrition Research Center at UNC-Chapel Hill Grant DK56350. We kindly thank all of the patients and health care providers involved in providing the adipose tissue used in these studies. We also thank the scientific staff of Zen-Bio, Inc. and the members of the Harp laboratory for critical comments and suggestions during the course of this study.
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