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Endocrinology Vol. 138, No. 7 2776-2783
Copyright © 1997 by The Endocrine Society

ARTICLES

TNF{alpha}-Mediated Inhibition and Reversal of Adipocyte Differentiation Is Accompanied by Suppressed Expression of PPAR{gamma} without Effects on Pref-1 Expression1

Hong Xing, Jeffrey P. Northrop, J. Russell Grove, Katherine E. Kilpatrick, Jui-Lan Su and Gordon M. Ringold

Affymax Research Institute (H.X., J.P.N., J.R.G., G.M.R.), Santa Clara, California 95051; and Department of Molecular Sciences (K.E.K., J.-L.S.), GlaxoWellcome Research and Development, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Hong Xing, Aurora Biosciences Corp., 11149 North Torrey Pines Road, La Jolla, California 92037. E-mail: XingH{at}aurorabio.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor {alpha} (TNF{alpha}) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNF{alpha} inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPAR{gamma} and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPAR{gamma} occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNF{alpha}-mediated inhibition of adipocyte differentiation, PPAR{gamma} messenger RNA (mRNA) expression stays at low levels. In contrast, TNF{alpha} treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors [such as TNF{alpha}, basic fibroblast growth factor, transforming growth factor ß, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPAR{gamma} mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNF{alpha} to fully differentiated adipocytes results in the rapid disappearance of PPAR{gamma} protein expression and the rapid loss of PPAR{gamma} DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNF{alpha}-generated signals, whereas PPAR{gamma} expression remains sensitive to TNF{alpha} at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPOCYTE differentiation is a complex process involving profound changes in cell morphology and gene expression. Adipocytes are a highly specialized cell type that function to store and release lipid in response to metabolic changes of an organism (reviewed in Ref. 1). These cells are characterized by their unique morphology, an ability to accumulate intracellular lipid, and expression of a set of adipocyte-specific genes (reviewed in Refs. 2, 3, 4, 5, 6, 7). These include genes encoding enzymes involved directly and indirectly in lipid metabolism, such as lipoprotein lipase, fatty acid synthetase, and phosphoenolpyruvate carboxykinase (PEPCK). Several adipocyte-specific genes, such as aP2 [which encodes an intracellular lipid-binding protein (8, 9)] and FSP27 [whose function is unclear (10)], and the well-defined transcription factor CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}) (11, 12, 13), are also expressed. Because C/EBP{alpha} is a widely expressed transcription factor (14), it is unlikely to be the key factor for induction of the adipocyte phenotype. In contrast, both peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (15, 16, 17, 18) [a member of the nuclear receptor superfamily (reviewed in Refs. 19, 20, 21)] and preadipocyte factor 1 (Pref-1) (22, 23) [a novel member of the epidermal growth factor (EGF)-like family with similarity to the Notch/Delta transmembrane proteins involved in cell fate determination (reviewed in Refs. 24, 25)] are highly tissue-specific proteins. PPAR{gamma} is found only in adipose tissue (16, 17, 26), whereas Pref-1 is expressed only in preadipocytes and in adult adrenal tissue (22). The highly restricted tissue distribution and early changes in messenger RNA (mRNA) expression of PPAR{gamma} and Pref-1 suggest that they may play essential roles in the differentiation process. Recently, PPAR{gamma} was identified as a key regulator of at least two adipocyte genes, aP2 and PEPCK (17, 27). PPAR{gamma} serves as a positive regulator of adipocyte differentiation that can cooperate with C/EBP{alpha} in stimulating the adipogenic program (16). Although increased expression of many genes is observed during adipocyte differentiation, few have been identified that are down-regulated during adipogenesis. Pref-1 is abundant in preadipocytes but is not expressed in adipocytes, and constitutive expression of Pref-1 in preadipocytes inhibits adipogenesis (22). Thus, Pref-1 seems to function as a negative regulator of adipocyte differentiation.

In mouse adipogenic cell lines, such as 3T3-L1 and TA1 (28, 29, 30), tumor necrosis factor {alpha} (TNF{alpha}) is capable of inhibiting adipocyte differentiation and of inducing dedifferentiation of fully differentiated adipocytes (31, 32). Addition of TNF{alpha} to adipocytes causes down-regulation of enzymes involved in lipid metabolism and of adipocyte specific genes, such as aP2 and FSP27, and addition of TNF{alpha} to preadipocytes prevents their increased expression (31, 32). Though it has been demonstrated that rapid down-regulation of C/EBP{alpha} (33, 34) and induced expression of c-myc (35) are associated with TNF{alpha}-mediated reversal of adipocyte differentiation, the exact mechanism(s) by which TNF{alpha} affects the adipogenic program remain obscure.

To investigate the mechanisms by which TNF{alpha} and certain growth factors inhibit and reverse adipocyte differentiation, we have studied the expression of two key regulatory proteins, PPAR{gamma} and Pref-1. We show that PPAR{gamma} mRNAs, their corresponding proteins, and DNA-binding activity are rapidly diminished after addition of TNF{alpha} to 3T3-L1 adipocytes. Moreover, PPAR{gamma} expression stays at low levels during TNF{alpha}-mediated inhibition of adipogenesis. Thus, TNF{alpha} is capable of regulating PPAR{gamma} expression in both preadipocytes and adipocytes. In contrast, Pref-1 message fails to reappear during TNF{alpha}-mediated reversal of adipocyte differentiation and is autonomously down-regulated in differentiating adipocytes in the presence of TNF{alpha}. We conclude that the effects of TNF{alpha} on adipogenesis do not involve a change in expression of Pref-1 but do elicit specific changes in PPAR{gamma} expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Vector construction
Part of PPAR{gamma} complementary DNA was obtained from 3T3-L1 adipocyte total RNA by reverse transcription and PCR using the following primers: 5'-GGGGAATTCCGTTATGGGTGAAACTCTGGGA-3' and 5'-CTGGTCGATATCACTGGAGAT-3'. The resulting PCR fragment was digested with EcoR I and EcoR V. A ribovector for PPAR{gamma} was constructed by inserting this PCR fragment into pcDNA3 (Invitrogen Corp., San Diego, CA), digested with EcoR I and EcoR V, and the insert was confirmed by DNA sequencing. A riboprobe template for PPAR{gamma} was generated by StuI digest. Pref-1 complementary DNA was obtained from 3T3-L1 preadipocyte total RNA using two sets of primers: 5'-GATTTGGATCCGAGATGATCGCGACCGGAGCCC-3', 5'-GGTCTTGTCGACGAATCCAGCTG-G-3' and 5'-GATTCGTCGACAAGACCTGCAGCC-3', 5'-GTGTATCTA-GAGAATTCTGTCAATCTTCTCGGGGAAGATG-3'. The resulting PCR fragments were digested with BamHI/SalI and SalI/XbaI, respectively. A ribovector for Pref-1 was created by subcloning of these two fragments into pcDNA3 and linearizing with SmaI. The accuracy of the Pref-1 insert was confirmed by DNA sequencing. Riboprobes for ß-actin and FSP27 have been described previously (36).

Cell culture
3T3-L1 cells (obtained from ATCC, Rockville, MD) were maintained in minimum essential medium Eagle (MEM Earle’s salt medium) (Sigma, St. Louis, MO) supplemented with 10% FCS at 37 C, 5% CO2. All serum used was heat inactivated at 55 C for 30 min. To induce adipocyte differentiation, 3T3-L1 cells were treated at confluence with 1 µM dexamethasone (Dex) and 30 µM indomethacin (Indo) for 6–10 days (8, 37). Alternatively, 2 days after reaching confluence, cells were treated with 1 µM Dex, 0.5 µM 1-methyl-3-isobutyl-xanthine, and 1.7 µM insulin for 2 days, followed by 2 additional days in 1.7 µM insulin, and finally, 2 days in MEM with 10% FCS (38). Typically, at this time, approximately 70% of the cells had differentiated into adipocytes, as judged by cell morphology. No difference in cell morphology or specific gene expression was observed between these two differentiation protocols. TNF{alpha}, basic fibroblast growth factor (bFGF), and transforming growth factor ß (TGFß) were purchased from Collaborative Biomedical Products (Bedford, MA). The phorbol ester tetradecanoyl phorbol-13-acetate (TPA) was from Sigma, and the calcium ionophore ionomycin was from Calbiochem-Behring Corp. (La Jolla, CA). Dex, 1-methyl-3-isobutyl-xanthine, and Indo were from Sigma.

Total RNA preparation and ribonuclease protection assay
Cells were dissolved in guanidium isothiocyanate-containing solution (Micro RNA Isolation Kit; Stratagene, La Jolla, CA), and total RNA was isolated according to the manufacturer’s protocol. Riboprobes for PPAR{gamma}, Pref-1, FSP27, and ß-actin were generated using a MAXIscript Kit (Ambion, Austin, TX). Briefly, in vitro transcription reactions were set up in 10-µl vols containing 1X transcription buffer; 0.2–0.5 µg template; 500 µM each ATP, CTP, and GTP; 20 µM dithiothreitol (DTT); 25 µCi [{alpha}-32P]UTP (800 Ci/mmol, Amersham, Arlington Heights, IL); and 5 U of T7 or SP6 RNA polymerase. After incubation at room temperature for 30–45 min, DNA template was digested with deoxyribonuclease I at 37 C for 15 min. RNA probes were then separated from unincorporated nucleotides using S-200 HR columns (Pharmacia, Alemeda, CA). Ribonuclease protection assays were performed with RPA II kit (Ambion) and carried out according to manufacturer’s protocol, with minor modifications. Typically, 2 µg of total RNA and 60,000 cpm of each riboprobe were included in each assay. Protected RNA fragments were separated on 5% acrylamide, 8 M urea gels.

Antibody production
Murine monoclonal antibodies to human recombinant PPAR{gamma} (39) were generated from immune lymphocytes isolated from a BALB/c mouse (Charles River, Research Triangle Park, NC). The mouse was immunized a total of four times with recombinant protein-encoding residues 195–475 of the ligand-binding domain of human PPAR{gamma}. At 2-week intervals, 50 and 5 µg of recombinant PPAR{gamma}, emulsified in RIBIs adjuvant (RIBI Immuno. Chem. Research, Inc., Hamilton, MT), were injected ip. The mouse was then immunized with 10 µg ip, then 5 µg iv (recombinant PPAR{gamma} in 200 µl PBS) 96 and 24 h before death. A polyethylene glycol-induced somatic fusion of immune splenocytes was performed using a previously published procedure (40). Monoclonal antibodies P{gamma}M4–17.2 and P{gamma}M2–15.22 produce IgG2a.

Nuclear extract preparation and DNA-binding assay
Nuclear extracts were prepared as described by Schreiber et al. (41). Briefly, two 150-cm2 confluent flasks of cells were harvested and cells resuspended in 1 ml of cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 25 µg/ml aprotinin). Then, 62.5 µl of 10% NP-40 solution was added, and the cell suspension was vortexed vigorously for 10 sec. Nuclei were recovered by centrifugation for 30 sec at 4 C and subsequently resuspended in 150 µl of ice-cold buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT plus protease inhibitors, as described above] and rocked vigorously at 4 C for 30 min. The nuclear lysate was centrifuged at 100,000 rpm for 12 min in Beckman TL-100 ultracentrifuge at 4 C, and the supernatant was aliquoted and stored at -80 C.

The sequences of double-stranded oligonucleotides used were as follows: ARE7s, 5'-TCGAGTGTGAACTCTGATCCAGC-3'; ARE7as, 5'-TCGAGCTGGATCAGAGTTCACAC-3'; Histone octamer 1s, 5'-TCGAGTGTAATATGCAAATCATTTGC-3'; Histone octamer 1as, 5'-TCGAGCAAAATGATTTGCATATTACAC-3'. The DNA fragments were allowed to anneal, labeled with [{gamma}-32P] ATP (>5000 µci/mmol, Amersham), filled in the protruding ends with Klenow fragment, and purified with Bio-Spin 6 chromatography columns (Bio-Rad Laboratories, Richmond, CA) to dispose of unincorporated [{gamma}-32P] ATP and dNTPs.

Electrophoretic mobility shift assay (EMSA) was performed as described by P. Tontonoz et al. (17), with modifications as follows: 5 µg of nuclear extract were incubated with 0.3 µg poly dI-dC for 15 min at 4 C, followed by the addition of 25,000 cpm of 32P-labeled double-stranded oligonucleotide ARE7. The mixture was incubated for 20 min at room temperature and then electrophoresed on a 4% native gel in 0.5 x TBE. When antibody was used, binding reactions were incubated with antibody for 15 min at room temperature before the addition of the probes.

Immunoblotting
Nuclear extracts (20 µg) were added to SDS-PAGE loading solution, boiled for 8–10 min, and electrophoresed on a denaturing gel consisting of a 3.8% acrylamide stacking gel and a 12% acrylamide separating gel, according to Laemmli (42). Proteins were transferred to Immobilon-P transfer membrane (Millipore, Bedford, MA) and blots subsequently treated with 5% nonfat milk in TBS [50 mM Tris-HCl (pH 7.4), 150 mM NaCl], containing 0.2% Tween 20, for 20 min at room temperature. Blocked membranes were incubated with anti-PPAR{gamma} monoclonal antibody, P{gamma}M 2–15.22 at 4 C overnight, followed by three washes with TBS containing 0.05% tween. Membranes were then incubated for 1 h with antimouse IgG, conjugated with horseradish peroxidase, and PPAR{gamma} proteins visualized with the enhanced chemiluminescence system (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR{gamma} and Pref-1 expression during adipocyte differentiation of 3T3-L1 cells
Confluent 3T3-L1 cells differentiate into mature adipocytes when cultured in 10% FCS in the presence of both Dex and Indo. To examine the time course of changes in PPAR{gamma} and Pref-1 gene expression, total cellular RNA was prepared at various stages during the differentiation process, and PPAR{gamma} and Pref-1 messages were examined by ribonuclease protection assay (Fig. 1Go). In preadipocytes, PPAR{gamma} mRNA was just detectable and remained at low levels as cells reached confluence (Fig. 1AGo). In the presence of FCS, Dex, and Indo, PPAR{gamma} mRNA was induced at a very early stage in the differentiation process (within 2 days after cells reached confluence, day zero). Maximal induction of PPAR{gamma} message was observed by day 6 (Fig. 1AGo). Pref-1 mRNA expression level also was altered early during adipocyte differentiation, decreasing just 1 day after confluence and remaining at low levels during the entire time course (Fig. 1BGo). These changes in expression of PPAR{gamma} and Pref-1 occurred well before accumulation of intracellular lipid droplets (data not shown). The induction of PPAR{gamma} mRNA and decrease of Pref-1 mRNA expression precede the induction of adipocyte-specific message FSP27 (3 days post confluence, Fig. 1BGo) and expression of transcription factor C/EBP{alpha} on day 3 (16).



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  		Figure 1. Expression of PPAR{gamma} and Pref-1 during differentiation of 3T3-L1 cells to adipocytes. Confluent 3T3-L1 cells (day zero) were switched into fresh differentiation medium (MEM containing 10% FCS, 1 µM Dex, and 30 µM Indo) and subsequently refed with fresh medium every 3–4 days. Total RNA was isolated on the indicated days. Lane Preadipo, Subconfluent preadipocytes. Ribonuclease protection assays were performed using 2 µg of total RNA with ß-actin as an internal control. A, Time course of PPAR{gamma} induction; B, time course of expression of Pref-1 and FSP27 using the same RNA samples as in panel A.

 
Regulation of PPAR{gamma} and Pref-1 message during TNF{alpha}-mediated inhibition of adipocyte differentiation
Because PPAR{gamma} and Pref-1 act as positive and negative regulators of adipogenesis, respectively, determining the effect of TNF{alpha} treatment on their expression is important to understand TNF{alpha}-mediated inhibition of adipocyte differentiation. TNF{alpha}, at various concentrations, was added to confluent cells in the presence of Dex and Indo, and cells were cultured in differentiation medium for 10 days. In the absence of TNF{alpha}, cells differentiated into adipocytes, and PPAR{gamma} message increased (Fig. 2AGo, compare nontreated preadipocytes and adipocytes). With TNF{alpha} present in the differentiation media, PPAR{gamma} message remained at low levels, even with the lowest dose of TNF{alpha} used (Fig. 2AGo). As expected, levels of Pref-1 message decreased when cells differentiated into adipocytes (Fig. 2BGo). Surprisingly, Pref-1 mRNA decreased significantly, even when differentiation was blocked by the presence of a wide range of TNF{alpha} concentrations (Fig. 2BGo). In this experiment, TNF{alpha}-mediated inhibition of adipocyte differentiation was judged both by inhibition of adipocyte-specific marker FSP27 expression (Fig. 2BGo) and by morphological resemblance to preadipocytes (data not shown).



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  		Figure 2. Dose-dependent inhibition of 3T3-L1 adipocyte differentiation by TNF{alpha}. Confluent cells (day zero) were cultured in differentiation medium, as in Fig. 1Go, without [lane 2, Adipo. NT (not treated)] or with (lanes 3–6, TNF) the indicated amounts of TNF{alpha}. Every 2 days, cells were refed with fresh differentiation medium plus TNF{alpha} and harvested on day 10. Ribonuclease protection assays were performed as in Fig. 1Go. Lane Preadipo, Subconfluent preadipocytes; A, PPAR{gamma} expression; B, Pref-1 and FSP27 expression.

 
Regulation of PPAR{gamma} and Pref-1 during TNF{alpha}-mediated reversal of adipocyte differentiation
Addition of TNF{alpha} to differentiated adipocytes causes down-regulation of adipocyte specific genes (31, 32, 35), as well as decreased expression of regulatory factors such as C/EBP{alpha} (33, 34), thus substantially reverting these cells to a state resembling the preadipocyte phenotype. To determine whether TNF{alpha} has an effect on the expression pattern of PPAR{gamma} and Pref-1 in adipocytes, fully differentiated 3T3-L1 cells were transferred to fresh media, containing TNF{alpha}, and analyzed after various time intervals. TNF noticeably reduced the PPAR{gamma} steady-state mRNA level between 0.5 and 1 h after its addition; and after 6 h of TNF{alpha} treatment, no PPAR{gamma} mRNA was detectable (Fig. 3Go, A and E). FSP27 mRNA decreased more slowly, reaching a minimal level after 9.5 h of TNF{alpha} treatment (Fig. 3Go, B and E; 35). In contrast, Pref-1 mRNA was not up-regulated during TNF{alpha}-mediated reversal of adipocyte differentiation for up to 3 days of treatment (Fig. 3Go, C and F). The reversal of adipocyte differentiation during this time course was judged both by the loss of adipocyte specific marker FSP27 expression (Fig. 3Go, D and E) and by morphological resemblance to preadipocytes (data not shown).



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  		Figure 3. Time course of reversal of 3T3-L1 adipocyte differentiation by TNF{alpha}. A and B, Confluent cells were cultured in differentiation media for 10 days. At this time, cells were either harvested for control adipocytes (NT, lane 2) or treated with 25 ng/ml TNF{alpha} for the indicated number of hours (lanes 3–7) before total RNA was isolated. Ribonuclease protection assays were performed as in Fig. 1Go. PPAR{gamma}, Pref-1, and FSP27-specific bands are indicated in panels A and B, respectively. C and D, Cells were cultured in differentiation media for 5 days post confluence and then either harvested for total RNA isolation or refed with fresh medium containing 50 ng/ml TNF{alpha}, and total RNA isolated on the indicated days. Pref-1 and FSP27-specific bands are indicated in panels C and D, respectively. Preadipo. NT, Nontreated preadipocytes; E, autoradiograms in A and B were quantitated by scanning densitometry; Pref-1 (black bars), PPAR{gamma} (grey bars) and FSP27 (light grey bars) signal intensities were normalized to ß-actin. The amount of Pref-1 mRNA seen with control preadipocytes (Preadipo. NT) was defined as 100, whereas both the amount of PPAR{gamma} and FSP27 mRNAs seen with control adipocytes (Adipo. NT) was defined as 100. F, Autoradiograms in C and D were quantitated by scanning densitometry. Pref-1 (black bars) and FSP27 (grey bars) signal intensities were normalized to ß-actin. The amount of Pref-1 mRNA seen with control preadipocytes (Preadipo. NT) was defined as 100, whereas the amount of FSP27 seen with control adipocytes (Adipo. NT) was defined as 100.

 
Down-regulation of PPAR{gamma}, but not reexpression of Pref-1, is associated with TGFß, bFGF, and phorbol ester plus calcium ionophore-mediated reversal of adipocyte differentiation
Treatment of fully differentiated 3T3-L1 adipocytes with TGFß, bFGF, or the phorbol ester, TPA, plus ionomycin induces reversal of the adipocyte phenotype (37). To investigate whether these agents also are capable of modulating PPAR{gamma} or Pref-1 expression, adipocytes were treated with TGFß, bFGF, or TPA plus ionomycin, and total cellular RNA was analyzed. PPAR{gamma} expression decreased significantly upon treatment with TGFß, bFGF, or TPA plus ionomycin (Fig. 4Go, A and C). In contrast, Pref-1 message remained low during these treatments (Fig. 4Go, B and C). These results suggest that down-regulation of PPAR{gamma} may be one mechanism by which these agents reverse the adipocyte phenotype. It seems that increased expression of the negative regulator, Pref-1, may not be involved in their action.



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  		Figure 4. PPAR{gamma} and Pref-1 expression in adipocytes treated with TGFß, bFGF, or TPA plus ionomycin. 3T3-L1 cells were differentiated as in Fig. 3CGo and D and either harvested as control adipocytes, no treatment (adipo. NT) or refed with fresh MEM containing 10% FCS plus either 1 µg/ml TGFß (TGF), 50 ng/ml bFGF (bFGF), or 100 nM TPA plus 1 µM ionomycin (TPA + Iono) for 2 days before total RNAs were isolated. A and B, Ribonuclease protection showing expression of PPAR{gamma}, Pref-1, and FSP27, respectively; C, autoradiograms were quantitated by scanning densitometry; both PPAR{gamma} (black bars) and Pref-1 (grey bars) signal intensities were normalized to ß-actin. The amount of PPAR{gamma} mRNA seen with control adipocytes (Adipo. NT) was defined as 100, whereas the amount of Pref-1 mRNA seen with control preadipocytes (Preadipo. NT) was defined as 100. Results from two independent experiments are shown; bars represent SE.

 
TNF{alpha}-induced adipocyte dedifferentiation leads to a reversal of the differentiation-induced profile of PPAR{gamma} proteins and their DNA-binding activity
The loss of PPAR{gamma} protein also was monitored during TNF{alpha}-induced adipocyte dedifferentiation by analyzing nuclear protein extracts prepared at the various times after TNF{alpha} addition to 3T3-L1 adipocytes, using EMSA and immunoblot assays. As shown in Fig. 5AGo, no detectable PPAR{gamma} proteins are present in preadipocytes (lane Preadipo.). Upon differentiation, both PPAR{gamma}1 (top) and PPAR{gamma}2 (bottom) are induced (lane Adipo.). Addition of TNF{alpha} into 3T3-L1 adipocytes causes rapid loss of both PPAR{gamma} proteins, reaching a level comparable with that of preadipocytes within 1 h (Fig. 5AGo).



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  		Figure 5. Time course of TNF action on down-regulation PPAR{gamma} proteins and their DNA-binding activity. A, 3T3-L1 cells were treated essentially the same as described in Fig. 3Go. Nuclear extracts were prepared from 3T3-L1 cells before reaching confluence (Preadipo.), 8 days after confluence, when cells were differentiated into adipocytes in the presence of Dex and Indo (Adipo.), or adipocytes treated with 25 ng/ml of TNF{alpha} for 0.5, 1, 2, 6, 11.5, and 24.5 h (TNF{alpha}, 0.5–24.5). Western immunoblot assays were carried out with 20 µg of nuclear extract and electrophoresed in a 12% SDS polyacrylamide gel. PPAR{gamma} proteins were detected as bands of 56 and 52 kDa, caused by the presence of both PPAR{gamma}1 and PPAR{gamma}2 in 3T3-L1 adipocytes. B, Electrophoretic mobility shift assay (EMSA) using the same nuclear extract as described in (A). Double-stranded 32P-labeled PPAR{gamma}-specific ARE7 oligonucleotide (left panel) and histone octamer sequence (a recognition sequence for ubiquitous transcription factor Oct) (right panel) were used as probes. The shifted bands in the presence of ARE7 are labeled as ARF6, a heterodimers of PPAR{gamma} and RXR{alpha}. Free probe is labeled as FP. C, EMSA of 3T3-L1 adipocyte nuclear extract (NE), performed as described in (B), except plus (ARE7 or Oct) or minus (-) of a 40-fold molar excess of cold oligonucleotides before addition of 32P-labeled ARE7 oligonucleotide. D, EMSA of 3T3-L1 NE in the absence (-) and presence (+) of mouse monoclonal antibody against PPAR{gamma}, P{gamma}M 4–17.22. Both double stranded oligonucleotides ARE7 and Oct were used as probes. Supershifted ARF6 complex is indicated by the arrowhead.

 
To determine how PPAR{gamma} DNA-binding activity is altered during TNF{alpha}-induced reversal of adipocyte differentiation, these same nuclear extracts were examined for PPAR{gamma} DNA-binding activity (Fig. 5BGo, left panel). We used the ARE7-binding site in the aP2 enhancer, previously shown to specifically bind PPAR{gamma}-RXR{alpha} heterodimers, also called ARF6 (18), as probe. Only a small amount of DNA-binding activity is detected in nuclear extracts from preadipocytes. Upon differentiation, an increase in ARE7 DNA-binding activity is seen as expected (Fig. 5BGo). Treatment of adipocytes with TNF{alpha}, rapidly results in decreased PPAR{gamma}-RXR{alpha} DNA-binding activity, reaching a level similar to that observed in preadipocytes within 1 h (Fig. 5BGo). As an internal control, the DNA-binding activity of Oct-1 remains relatively constant during of TNF{alpha} treatment (Fig. 5BGo, right panel). The specificity of the observed DNA-binding activity was determined using specific and nonspecific DNA-binding sequence as competitors (Fig. 5CGo). ARF6 DNA-binding activity is competed by unlabeled ARE7 oligonucleotide, but not by a histone octamer sequence (Fig. 5CGo). Evidence of participation of PPAR{gamma} in the ARE7-binding complex was obtained by preincubation of 3T3-L1 adipocyte nuclear extracts with monoclonal antibody P{gamma}M 4–7.22 raised against the ligand-binding domain of PPAR{gamma}. Addition of this antibody results in a supershift of the ARE7-PPAR{gamma}-RXR complexes, indicating that PPAR{gamma} is present in the complexes (Fig. 5DGo). The antibody specificity was confirmed by its failure to supershift Oct-1 complexes. The rapid disappearance of the PPAR{gamma}, as a result of TNF{alpha} application, strongly suggests that TNF{alpha} reversal of the adipose phenotype may, in part, be caused by the rapid loss of this adipocyte-specific transcription factor.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Confluent 3T3-L1 cells initiate an adipogenic program under the influence of serum factors and inducing agents, such as glucocorticoid hormones and Indo. We have observed that the expression of PPAR{gamma} and Pref-1 changes during early stages of adipocyte differentiation. This data is in general agreement with that of others (16, 26). We show that the down-regulation of Pref-1 mRNA expression and induction of PPAR{gamma} mRNA occur 1 day and 2 days, respectively, after cells reach confluence (Fig. 1Go). These changes not only precede the expression of adipocyte specific genes, but also precede the induction of C/EBP{alpha} (1), which has been shown to play an important regulatory role in adipogenesis (12, 13, 43, 44). Whereas glucocorticoid hormones may exert their effects by inducing C/EBP{alpha} mRNA expression during adipogenesis (45), the specific actions of Indo have not previously been defined in this system. Indo, at similar concentrations to those required for induction of differentiation, recently has been found to activate PPAR{gamma}-dependent transcription (H. Xing, unpublished observations; J. Lehmann, GlaxoWellcome, personal communication), which may explain why Indo enhances conversion to adipocytes. The highly restricted tissue distribution and early changes in mRNA expression of PPAR{gamma} and Pref-1 suggest that they play an important role in the adipocyte differentiation process.

Pref-1 is a member of a small group of proteins (identified to date) whose expression decreases during adipocyte differentiation. Although Pref-1 expression is rapidly down-regulated during the early stages of adipocyte differentiation, it may be required for initiation of the differentiation process in a permissive and/or instructive manner. The absence of Pref-1 seems to be essential for differentiation (22). Although it remains a formal possibility that Pref-1 participates in some early inductive signals, this seems unlikely, given recent observation that Pref-1 expression is exceedingly low in TA1 preadipocytes that differentiate normally (H. Xing, unpublished observations).

The presence of EGF-like repeats in the extracellular domain of Pref-1 suggests that it may regulate adipogenesis through protein-protein interactions. Studies with Notch and Delta demonstrate that the EGF-like repeats within these proteins govern cell-cell interactions and, consequently, cell fate decisions (24, 25). Recently, the mammalian protein Jagged has been identified as an activating ligand for Notch and, when expressed in myoblasts, prevents muscle cell differentiation (46). Whether Pref-1 acts as a receptor for a Jagged-like protein in preadipocytes or as a ligand for an as yet unidentified adipocyte receptor remains to be determined.

When present in cultures of confluent adipogenic 3T3-L1 or TA1 cells, TNF{alpha} inhibits their differentiation to adipocytes. TNF{alpha}-treated cells fail to express adipocyte-specific genes and to accumulate intracellular lipid droplets (Fig. 2BGo and Refs. 31, 32). We have found that TNF{alpha}-mediated inhibition of adipocyte differentiation is not associated with continued high expression of Pref-1. Rather, Pref-1 expression decreases in a manner indistinguishable from that of nontreated cells. Thus, the effects of TNF{alpha} are not mediated by suppression of the normal autonomous decrease in Pref-1 expression, which is a prerequisite for differentiation.

In contrast to its lack of effect on Pref-1 expression, TNF{alpha} prevents PPAR{gamma} mRNA expression from increasing in adipogenic cells. The regulatory roles of PPAR{gamma} in adipogenesis in general (reviewed in Ref. 15) and in the expression of some adipocyte-specific genes (17, 27) have been defined. PPAR{gamma} and C/EBP{alpha}-binding sites are present in the promoter regions of the aP2 (17) and PEPCK genes (27). In addition, TNF{alpha} causes a rapid decrease in the expression of PPAR{gamma} mRNA (Fig. 3AGo) and corresponding protein (Fig. 5AGo), as well as a parallel decrease in DNA-binding activity in adipocytes (Fig. 5BGo). It is noteworthy that the time-dependent decrease in both mRNA expression and DNA-binding activity of PPAR{gamma} and C/EBP{alpha} (33, 34) occurs well before the disappearance of adipocyte-specific genes, such as FSP27 and aP2 (33, 34, 35), suggesting that the decrease in DNA-binding activities of PPAR{gamma} and C/EBP{alpha} in response to TNF{alpha} may lead to the decreased expression of adipocyte-specific genes, thus accounting for the reversal of the adipocyte phenotype.

TNF{alpha} is a potent activator of phospholipase A2 in preadipocytes and adipocytes, leading to formation of a variety of arachidonate metabolites, notably 5-lipoxygenase products (47). Recently a PG J2 metabolite has been identified as both a PPAR{gamma} ligand and an inducer of adipogenesis (48, 49), suggesting a novel mechanism of action for PGs of the J2 series through a nuclear receptor signaling pathway. It will be of interest to examine whether any of these TNF{alpha}-induced arachidonate metabolites act as antagonists of PPAR{gamma} activation.

Several reports have indicated that polypeptide hormones, such as TGFß and bFGF, as well as protein kinase C-activating agents and calcium ionophores, are capable of reversing adipocyte differentiation (32, 37). We have shown that TGFß, bFGF, and TPA plus ionomycin effectively suppress PPAR{gamma} mRNA expression in adipocytes with concomitant decrease in adipocyte-specific gene expression. Whether these agents share similar cellular signaling pathways is unknown at this time. Consistent with the effects of TNF{alpha}, none of these agents are able to cause significant reexpression of Pref-1 mRNA in adipocytes. Thus, Pref-1 seems to function as a nonreversible molecular checkpoint at the start of the adipocyte differentiation process whose expression is independent of all tested agents. These results demonstrate that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological characteristics and patterns of gene expression.


   Acknowledgments
 
We thank Marc Narve for critical reading of the manuscript and providing helpful comments; and Caroline Davies, Robyn Davis, and Maria Schaefer for oligonucleotide synthesis and DNA sequencing.


   Footnotes
 
1 This research was supported by NIH Grant GM-25821 (to G.M.R.) and by Affymax Research Institute. Back

Received December 4, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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