Arylhydrocarbon receptor (AhR) is involved in negative regulation of adipose differentiation in 3T3-L1 cells

AhR inhibits adipose differentiation independently of dioxin

Shigeki Shimba*, Taira Wada and Masakatsu Tezuka

Department of Hygienic Chemistry, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
* Author for correspondence (e-mail: shimba{at}pha.nihon-u.ac.jp )

Accepted April 26, 2001


    SUMMARY
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The arylhydrocarbon receptor (AhR) is the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although a physiological ligand for the AhR has yet to be identified, and the precise physiological roles of the AhR are unknown, it may play important roles not only in the regulation of xenobiotic metabolism but also in the maintenance of homeostatic functions. We have previously reported that the level of AhR protein decreased with ongoing adipose differentiation in 3T3-L1 cells. Studies using a TCDD-resistant clone of 3T3-L1 cells suggested that the AhR may be involved in the negative regulation of adipose differentiation. To confirm this hypothesis, 3T3-L1 fibroblast cells were stably transfected with a vector expressing high levels of full-length sense AhR mRNA, antisense AhR mRNA or a control vector. Comparison of the differentiation potency of these clones with that of control cells showed that overexpression of the AhR suppressed morphological differentiation, as well as induction of adipocyte-related genes, whereas decreased expression of the AhR induced much greater morphological differentiation and expression of adipocyte-related genes. Activation of PPAR{gamma}2 with ligands such as troglitazone, ciglitazone and indomethacin restored the ability of the AhR-overexpressing cells to differentiate. The cells overexpressing the AhR exhibited the higher p42/p44 MAP kinase activity compared with the control cells. Treatment with PD98059 or U0126 also abrogated the inhibitory action of the AhR on adipogenesis. We also present data showing that activation of the AhR slowed clonal expansion. During clonal expansion, the AhR inhibited the pRB phosphorylation and the downregulation of p107 expression. Taken together, these results strongly suggest that AhR is a negative regulator of adipose differentiation in 3T3 L1 cells.

Key words: Arylhydrocarbon receptor, Adipose differentiation, 3T3-L1 cells


    INTRODUCTION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Unliganded AhR exists in the cytoplasm in the form of a complex with 90 kDa heat-shock protein and immunophilin-related protein (Denis et al., 1988Go; Predew, 1988Go; Ma and Whitlock, 1997Go; Carver and Bradfield, 1997Go). Upon binding to the ligand, AhR dissociates from the complex and translocates to the nucleus, followed by the formation of a heterodimer with the AhR nuclear translocator (Arnt; Reyes et al., 1992Go). This heterodimer binds to a specific enhancer sequence (termed xenobiotic response element; XRE) located upstream of target genes such as CYP1A1 (Hankinson, 1995Go; Schmidt and Bradfield, 1996Go; Sogawa and Fujii-Kuriyama, 1997Go). AhR and Arnt are members of a family of transcription factors that contain basic helix-loop-helix structure (bHLH)/PAS domain. The PAS domain is found in a variety of proteins that play roles in development and adaptation to the environment, such as neurogenesis (Nambu et al., 1991Go), regulation of circadian rhythms (Sassone-Corsi, 1997Go) and response to hypoxia (Smenza, 1998Go).

The AhR is postulated to play important roles not only in the regulation of xenobiotic metabolism but also in the maintenance of homeostatic functions. Although a physiological ligand for the AhR has yet to be identified, several reports have shown constitutive activation of the AhR in the absence of exogenous ligand (Singh et al., 1996Go; Chang and Puga, 1998Go; Crawford et al., 1997Go). AhR knockout mice exhibit decreased liver size, hepatic portal fibrosis, decreased constitutive expression of certain xenobiotic-metabolizing enzymes such as CYP1A2, and decreased body size over the first 4 weeks of life, relative to their littermate controls (Fernandes-Salguero et al., 1995Go; Schmidt et al., 1996Go; Mimura et al., 1997Go). Stable transfection of AhR cDNA into AhR-defective mouse hepatoma cells has shown that the AhR plays important roles in control of cell cycle progression and differentiation, and that no exogenous ligands are required for the function (Ma and Whitlock, 1996Go). Absence of the AhR accelerates entry into senescence in fibroblast cells (Alexander et al., 1998Go). The treatment of cultured embryos with AhR antisense oligonucleotides resulted in a significantly lower incidence of both blastocyst formation and mean embryo cell number (Peters and Wiley, 1995Go). In cell differentiation, the AhR is increased during differentiation toward keratinocytes and monocytes (Wanner et al., 1995Go; Hayashi et al., 1995Go). By contrast, AhR protein is found to decrease with ongoing adipose differentiation, resulting in the loss of functional response to xenobiotics (Shimba et al., 1998Go). Consequently, it is likely that the AhR is involved in some aspects of the developmental and differentiation processes.

Several lines of evidence suggest that one of the possible roles of the AhR is negative regulation of adipose differentiation. We and other groups have demonstrated that TCDD treatment suppresses the conversion of 3T3-L1 fibroblast cells into adipose cells (Philips et al., 1995Go; Brodie et al., 1996a; Brodie et al., 1996b; Shimba et al., 1998Go). A cell clone that lacks the ability to transport the AhR into the nucleus is resistant to TCDD (Shimba et al., 1998Go). Moreover, depletion of the AhR during adipogenesis results in the loss of the inhibitory effects of TCDD (Shimba et al., 1998Go). These results suggest that the inhibitory effects of TCDD on adipogenesis depend on the AhR. In vivo, AhR-null mice exhibited fatty metamorphosis in the liver over the first 2 weeks of life (Schmidt et al., 1996Go). Furthermore, the AhR is not expressed in liver fat storage cells (Riebniger and Schrenk, 1998Go). Collectively, AhR activation can have profound effects on adipose differentiation.

In this study, we have investigated the role of the AhR in adipose differentiation in 3T3-L1 cells. We show that expression of high levels of AhR sense RNA is able to inhibit significantly the accumulation of lipid, as well as the induction of adiposity-specific genes. In contrast, lowering AhR levels in 3T3-L1 cells, using antisense AhR mRNA, induces much greater differentiation. These results strongly suggest that the AhR plays a role in the negative regulation of adipose differentiation in 3T3-L1 cells.


    MATERIALS AND METHODS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
3T3-L1 cells, obtained from the Human Science Research Resources Bank (Osaka), were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf serum. For induction of adipose differentiation, cells were grown to confluence. The cells were then fed with differentiation medium (a 3:1 mixture of DMEM and Ham's F12 containing 10% FBS, 1.6 mM insulin, 0.0005% transferrin, 180 µM adenine, 20 pM triiodothyronine, 0.25 µM dexamethasone (DEX) and 500 µM isobutylmethylxanthine (IBMX)). After 3 days, the cells were re-fed with fresh differentiation medium without DEX and IBMX, and maintained over the following days.

Stable transfection
To construct the AhR mRNA expression vector, full-length murine AhR cDNA was subcloned into the mammalian expression vector pRc/CMV2 (Invitrogen), which contains a selective marker, the neomycin resistance gene. Similarly, antisense AhR mRNA expression vector was constructed by inserting full-length AhR cDNA in the antisense orientation. The cells were transfected by using Lipofectin (Life Technologies) and were allowed to grow in nonselective medium for 48 hours. The cells were then cultured in medium containing G-418 (450 µg/ml). After 2 to 3 weeks, clones were isolated and expanded individually. The expression of AhR protein in each clone was analyzed by western blotting.

Immunoblot analysis
The cells grown in 60 mm dishes were rinsed with ice-cold phosphate-buffered saline (PBS). The rinsed cells were scraped off the dish, placed in a microcentrifuge tube, and centrifuged at 5000 g for 1 minute. The resulting pellets were suspended in the lysis buffer (50 mM Hepes KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 1 mM DTT, 0.5 mM PMSF, 0.0002% leupeptin and 20% glycerol), vortex-mixed, and rocked at 4°C for 60 minutes. The suspensions were centrifuged for 15 minutes at 10000 g and the resulting supernatants were then frozen until further analysis. The protein concentration of the extracts was determined according to the method of Bradford, using bovine serum albumin as standard (Bradford, 1976Go). Protein samples were denatured by heating to 90°C in SDS-reducing buffer and were resolved by electrophoresis on 10% SDS-polyacrylamide gels. After transfer to a nitrocellulose membrane, the filters were probed with the antibodies. Color visualization was performed with secondary antibodies conjugated with alkaline phosphatase and nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate substrate solution (Promega). Protein expression was quantified with the use of National Institutes of Health Image 1.61 software as described previously (Pollenz, 1996Go).

Oil red O staining
To judge the states of adipose differentiation by visual inspection, cultures were fixed with 10% formalin in PBS for 2 hours, rinsed three times with distilled water and then air-dried. The fixed cells were stained with 0.5% oil red O solution for 1 hour. After staining, the cultures were rinsed several times with 70% ethanol.

Glycerophosphate dehydrogenase (GPDH) activity
Cells grown in 35 mm culture dishes were rinsed twice with ice-cold PBS, scraped into 0.2 ml of the extraction buffer (25 mM Tris-HCl (pH 7.5), 1 mM EDTA), and homogenized with a Teflon pestle drill apparatus. The homogenate was centrifuged for 10 minutes at 4°C. GPDH activity was assayed in the supernatant by monitoring the decrease in absorbance at 340 nm of NADH in the presence of dihydroxyacetone phosphate (Wise and Green, 1979Go).

Analysis of RNA
Total RNA was extracted from the clones with TRIzol reagent (Life Technologies) according to the manufacturer's instructions and was analyzed by northern blotting. Probes corresponding to C/EBPs, PPAR, aP2 and CYP1B1 were prepared by RT-PCR techniques using Bca PLUS RTase and LA taq with GC buffer (Takara Biomed, Japan).

In vitro kinase assay
Active p42/p44 MAP kinase was immunoprecipitated in extraction buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na3VO4, 1 µg leupeptin, 1 mM PMSF) from the cells. Immunoprecipitates were washed twice in extraction buffer followed by two washes in kinase buffer (25 mM Tris-HCl (pH 7.5), 1 mM glycerophosphate, 0.1 mM Na3VO4, 2 mM DTT, 10 mM MgCl2). Kinase reaction was incubated in the presence of Elk-1 protein (2 µg) and 200 µM ATP at 30°C for 30 minutes followed by the addition of SDS-reducing buffer. Samples were run on a 10% SDS-polyacrylamide gels and visualized by western blotting using anti-phospho Elk-1 antibody.

Flow cytometry
Cells were collected by mild trypsinization and gentle centrifugation and were fixed in 70% ethanol. The fixed cells were washed twice with PBS and resuspended in Propidium Iodine (PI) solution (10 mg of DNase-free RNase A and 100 mg of PI per ml of PBS). DNA content in the cells was analyzed with FACScan (Becton Dickinson).


    RESULTS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of the AhR ligand on adipose differentiation
We and others have demonstrated that TCDD inhibits adipose differentiation in 3T3-L1 cells (Philips et al., 1995Go; Brodie et al., 1996Go,Brodie et al., 1996Go; Shimba et al., 1998Go). Although most of the actions of TCDD depend upon the AhR, TCDD has AhR-independent actions (Fernandez-Salguero et al., 1996Go). Therefore, in a first set of experiments, we examined the effects of other AhR ligands, ß-naphthoflavone (BNF) and {alpha}-naphthoflavone (ANF), on adipose differentiation in 3T3-L1 cells (Fig. 1). Adipose staining with oil red O showed that treatment with TCDD allowed the cells to accumulate a minimum amount of lipid droplets, as reported previously (Philips et al., 1995Go; Brodie et al., 1996Go,Brodie et al., 1996Go; Shimba et al., 1998Go). BNF, which has less affinity for the AhR compared with TCDD, also inhibited adipose differentiation when used at a 1000-fold higher concentration than TCDD. ANF, which can bind to the AhR but is unable to transform the AhR to its DNA-binding form, had no effect on adipogenesis in 3T3-L1 cells. The effects of TCDD and BNF on adipogenesis were reversed by cotreatment with ANF. These results strongly suggest that activation of the AhR is involved in negative regulation of adipose differentiation.



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Fig. 1. Effect of AhR ligands on adipose differentiation in 3T3-L1 cells. Cells were treated with 10 nM TCDD, 10 µM ß-naphthoflavone (BNF), or 10 µM {alpha}-naphthoflavone (ANF) at the time of DEX and IBMX addition. The AhR ligands were dissolved in dimethylsulfoxide (DMSO) and control cells were given the same amounts (2 µl) of DMSO. The cultures were allowed to differentiate for 6 days. The state of adipose differentiation was measured by staining with oil red O.

 

Isolation of cells overexpressing the AhR or expressing antisense AhR
To directly assess the participation of the AhR in adipogenesis, 3T3-L1 cells were stably transfected with a vector expressing high levels of full-length sense AhR mRNA, antisense AhR mRNA or a control vector. After selection and expansion of stable clones, the level of the AhR protein was examined by western blotting. On the basis of AhR protein level, two clones expressing control vector mRNA, three clones expressing AhR sense mRNA and three clones expressing antisense AhR mRNA were chosen for subsequent studies. As shown in Fig. 2A, the cells transfected with the vector expressing sense AhR mRNA produced more AhR protein compared with the control vector-transfected cells and untreated cells, as analyzed by western blotting. In contrast, expression of the full-length AhR antisense mRNA resulted in a substantial decrease in the levels of the AhR protein. To confirm the activity of AhR in these clones, they were treated with 3-MC (1 µM) and the induction of CYP1B1 mRNA was analyzed. Note that CYP1A1, which is extensively used as the marker for AhR-mediated cellular response, is not induced in fibroblastic cells, including 3T3-L1 cells (Gradin, 1999Go), and therefore CYP1B1 expression was examined in this study. In the sense cells, high levels of CYP1B1 mRNA were expressed in the presence and absence of 3-MC (Fig. 2B). By contrast the antisense cells failed to induce CYP1B1 mRNA, although they expressed a basal level of CYP1B1 mRNA. These results indicate that the increased levels of AhR in the sense cells, and the decreased levels of AhR in the antisense cells, are of sufficient magnitude to affect AhR-mediated signaling activity.



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Fig. 2. Isolation of cells expressing AhR sense mRNA or antisense mRNA. (A) Western blot analysis of AhR was performed on whole cell extracts (5 µg of protein for cells expressing AhR sense mRNA (S) and 15 µg of protein for cells expressing AhR antisense mRNA (AS)) that were resolved by electrophoresis on 10% SDS/polyacrylamide gel. (B) The cell clones were treated with 1 µM 3-methylcholanthrene (3-MC) for 12 hours. Total RNA was purified and the level of CYP1B1 mRNA was determined by northern blotting. Results are representative of three separate experiments. The analysis was reproduced twice with similar results. AS, cells expressing antisense mRNA; S, cells expressing AhR sense mRNA; TfR, Transferrin receptor; V, cells expressing vector mRNA.

 

The AhR is involved in negative regulation of adipose differentiation
To evaluate the differentiation potency of the clones, the cells were cultured to confluence and then treated with a standard differentiation-induction medium containing DEX, IBMX and insulin. The extent of differentiation was estimated by adipose staining with oil red O and measurement of activity of glycerophosphate dehydrogenase (GPDH), a marker enzyme of adipogenesis. The sense and antisense cell clones, as well as control cells, showed no signs of lipid accumulation if cultured in the absence of differentiation-inducing agents (data not shown). When cell clones were treated with differentiation medium, wild type 3T3-L1 cells and the cells expressing vector mRNA exhibited a similar degree of lipid accumulation and induction of GPDH activity (Fig. 3A, middle lane; Fig. 3B). However, the cells overexpressing AhR accumulated a minimum amount of lipid droplets and very little GPDH activity was induced. Conversely, the antisense cells showed somewhat more lipid accumulation and marked induction of GPDH activity compared with the control cells (Figs 3A,B). These results were confirmed by measuring the expression of adipocyte-related genes, such as aP2, CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator activator receptor (PPAR) {gamma}2. As expected, expression of these genes was greatly induced in the control cells after 6 days of induction (Fig. 3C). Consistent with the morphological observations and GPDH activity, a minimum amount of aP2 and C/EBP {alpha} mRNA was induced in the AhR sense cells. Interestingly, the induction of PPAR{gamma}2 was not substantially affected by overexpression of the AhR. However, enhanced induction of these genes was observed in the antisense cells (Fig. 3C). These effects of AhR on the expression of adipocyte-specific genes are similar to those of TCDD (Liu et al., 1996Go).



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Fig. 3. Effects of the AhR on the differentiation potential of the clones. The cell clones were passaged to confluence and treated with the differentiation cocktail (DEX, IBMX and insulin). After 3 days, the cells were re-fed with fresh differentiation medium without DEX and IBMX, and maintained for several days. (A) On day 6, cells were fixed and stained with oil red O. The dishes in the middle lane show the cells expressing vector mRNA (V) (left and right) and wild-type 3T3-L1 cells (WT) (center). AS, cells expressing antisense mRNA; S, cells expressing AhR sense mRNA. (B) Induction of GPDH activity in the clones. Enzyme extracts were prepared at days 0 and 6 from the three independent clones. GPDH activity was measured as described in Materials and Methods. The results presented are the means from three separate determinations on three independent clones (n=9). (C) Expression of C/EBP{alpha}, PPAR{gamma}2 and aP2 mRNA in the clones. The cell clones were treated as described in the legend to Fig. 4. On days 0 and 6, total RNA was extracted and the expressions of C/EBP{alpha}, PPAR{gamma}2 and aP2 mRNA were determined by northern blotting. AS, cells expressing antisense mRNA; S, cells expressing AhR sense mRNA.

 



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Fig. 4. Restoration of the differentiation potential of cells expressing AhR sense mRNA by treatment with PPAR{gamma}2 ligand. Cells expressing AhR sense mRNA (S) and cells expressing vector mRNA (V) were treated with differentiation cocktail (IBMX, DEX and insulin; MDI), 10 µM ciglitazone or a combination of both. Ciglitazone remained in the medium after DEX and IBMX were removed. On day 6, cells were fixed and stained with oil red O. Similar results were obtained with independent clones.

 
The combination of PPAR{gamma} activator and conventional differentiation cocktail restores the ability of AhR-overexpressing cells to differentiate
Activation of PPAR{gamma} with ligands such as thiazolidinediones can promote adipose differentiation. Therefore, we next examined whether activation of PPAR{gamma} can restore the differentiation potential of the sense cells. As the PPAR{gamma} activator, we used indomethacin and two thiazolidinediones: ciglitazone and troglitazone. In the sense cells, conventional differentiation cocktail (IBMX, DEX and insulin) or ciglitazone alone had no effect on adipogenesis (Fig. 4). However, combination of the differentiation cocktail and ciglitazone allowed the sense cells to accumulate lipid droplets to the same extent as the control cells (Fig. 4). Similarly, cotreatment with the conventional differentiation cocktail and troglitazone or indomethacin restores the ability of the sense cells to differentiate (data not shown). These results suggest that AhR influences the early stage of the differentiation cascade, before the activation of PPAR.

The inhibitory effect of the AhR on adipogenesis is mediated by p42/p44 MAP kinase
In adipose differentiation, p42/p44 kinase activation is required for clonal expansion, whereas hyperactivation of p42/p44 MAP kinase cascade results in inhibition of differentiation (Sale et al., 1995Go; Font de Mora et al., 1997Go). As shown in Fig. 5A, p42/p44 MAP kinase activity was gradually decreased during adipogenesis in the control cells. By contrast, p42/p44 MAP kinase activity in the sense cells was maintained throughout day 5, and, as a result, the activity in the sense cells was higher than that in the control cells on day 3-5 (Fig. 5A). Western blot analysis showed that the p42/p44 protein was present in equal amounts through all the time points (Fig. 5B). In addition, there was no substantial differences in the p42/p44 protein level in between the control cells and the sense cells, indicating that the apparent differences in kinase activity is not simply due to variation in the protein level (Fig. 5B). To examine whether the higher activity of p42/p44 MAP kinase observed accounts for the lower potency of differentiation in the sense cells, cell clones were treated with PD98059 or U0126, the specific inhibitors of upstream of p42/p44 MAP kinase. Co-treatment with the conventional differentiation cocktail and PD98059 or U0126 restores the ability of the sense cells to differentiate (Fig. 5C). Treatment of the cells with SB203580, a specific inhibitor of p38 MAP kinase, inhibited adipogenesis (Fig. 5C). These results indicate that the hyperactivation of p42/p44 MAP kinase, at least partly, mediates the inhibitory effect of the AhR on adipose differentiation.



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Fig. 5. Involvement of MAP kinase activation in the inhibitory effects of the AhR on adipogenesis. (A) Cells expressing AhR sense mRNA (S) and cells expressing vector mRNA (V) were treated with differentiation cocktail (MDI). Cell lysates were prepared at the indicated day. The lysates obtained were immunoprecipitated with anti-phospho p42/p44 MAP kinase antibody and the kinase activity was assayed as described in Materials and Methods. (B) The lysates obtained as described above were resolved by electrophoresis on 10% SDS/polyacrylamide gel. Western blot analysis of p42/p44 MAP kinase was performed with the specific antibodies against p42/p44 MAP kinase, which detect total p42/p44 MAP kinase (phosphorylation-state independent) protein. (C) On day 0, cells expressing AhR sense mRNA (S) and cells expressing vector mRNA (V) were treated with differentiation cocktail (MDI) and 10 µM PD98059, 1 µM U0126 or 10 µM SB203580. On day 6, cells were fixed and stained with oil red O. Similar results were obtained with independent clones.

 

AhR activation inhibits clonal expansion
As shown by the previous result, AhR influences a very early stage in the differentiation pathway. Thus, we examined the effect of the AhR on clonal expansion, which is the earliest event in adipogenesis. In a first set of experiments, wild-type 3T3-L1 cells were treated with 10 nM TCDD and cell proliferation was determined. Treatment of the cells with TCDD suppressed the growth of 3T3-L1 cells (Fig. 6A). Similarly, overexpression of the AhR slowed cell proliferation, whereas lowering of AhR protein levels stimulated cell growth (Fig. 6B). Cell cycle analysis revealed that, after 18 hours of stimulation with differentiation medium, most of the sense cells remained in G0/G1 phase, whereas half of the control cells had entered S phase (Fig. 7). In antisense cells, somewhat enhanced entry into S phase was observed relative to that seen in the control cells (Fig. 7).



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Fig. 6. Activation of the AhR delays clonal expansion in 3T3-L1 cells. (A) 3T3-L1 cells were grown to confluence. The cells were treated with 10 nM TCDD in the differentiation medium. Control cells were given the same amount (2 µl) of DMSO. Cell numbers were determined by taking hemocytometer cell counts. Each time point represents a triplicate determination, and each experiment was performed twice with similar results. (B) Cell growth curve during the clonal expansion period in the various clones. Data represent means of determinations made on three independent clones. The analysis was reproduced twice with similar results. Symbols are defined in the figure.

 


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Fig. 7. Cell cycle analysis of the cell clones. Confluent cell clones were treated with differentiation cocktail for 18 hours. The cells were fixed and stained with PI. The percentage of cells in each phase was determined by flow cytometry. Data represent means of determinations made on three independent clones. The analysis was reproduced twice with similar results. AS, cells expressing antisense mRNA; S, cells expressing AhR sense mRNA; V, cells expressing vector mRNA.

 

Overexpression of the AhR inhibits the Rb protein phosphorylation and the down-regulation of p107
The retinoblastoma protein (pRB) plays key role in regulating clonal expansion associated with adipogenesis (Chen et al., 1996Go). Also, a direct interaction of pRB with the AhR has been reported (Ge and Elferink, 1998Go). Therefore, to explore the mechanism by which the AhR inhibits clonal expansion, we analyzed phosphorylation state in pRB. Immunoblot of pRB in Fig. 8A showed that the AhR had no effect on pRB phosphorylation on day 0 (quiescent cells). Upon induction of differentiation, pRB in control cells and antisense cells was almost entirely hyperphosphorylated on day 1 (Fig. 8A). This pRB phosphorylation pattern observed is similar to that in previous report (Shao and Lazar, 1997Go). The degree of pRB phosphorylation in the antisense cells was slightly higher than that in the control cells. By contrast, the sense cells exhibited minimal degree of pRB phosphorylation (Fig. 8A). These data suggest that the delay of cell cycle shown in Fig. 7 is, at least partly, due to the lower phosphorylation state of pRB caused by the AhR.



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Fig. 8. AhR inhibits pRB phosphorylation and down-regulation of p107. (A) Nuclear extracts were prepared from the cell clones at the indicated day. 50 µg of protein were resolved by electrophoresis on 10% SDS/polyacrylamide gel, transferred and analyzed by western blot. AS, cells expressing antisense mRNA; S, cells expressing AhR sense mRNA; V, cells expressing vector mRNA. (B) Western blots of p107 in cells expressing AhR sense mRNA (S) and cells expressing vector mRNA (V). (C) Western blots of p130 in cells expressing AhR sense mRNA (S) and cells expressing vector mRNA (V).

 

In addition to pRB, the related member p107 and p130 have been shown to play a critical role in differentiation and proliferation (Richon et al., 1997Go). Upon stimulation of differentiation, the expression of p107 was increased on day 1 and was entirely diminished on day 4 (Fig. 8B). The activated AhR had little effect on the transient increase of the p107 expression on day 1 but stabilized the expression of p107 throughout day 4 (Fig. 8B). By contrast, the expression of p130 was not affected by the AhR activation (Fig. 8C).


    DISCUSSION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increasing evidence has accumulated in recent years to suggest that the AhR plays a critical role in embryonic development and homeostasis. In this study, we aimed to study the role of the AhR in adipose differentiation. First we established cell clones from 3T3-L1 cells by stable transfection with vectors expressing high levels of AhR sense mRNA or antisense mRNA. Comparison of the differentiation potency of these clones with that of control cells showed that overexpression of the AhR suppressed morphological differentiation, as well as induction of adipocyte-related genes, whereas lowering of levels of the AhR induced much greater morphological differentiation and expression of adipocyte-related genes. Several lines of evidence support the results in this study. First, TCDD, a most potent ligand for the AhR, inhibits adipose differentiation in 3T3-L1 cells (Philips et al., 1995Go; Brodie et al., 1996Go,Brodie et al., 1996Go; Shimba et al., 1998Go). The inhibitory effect is AhR-dependent (Fig. 1). Second, the AhR is depleted during differentiation, as is the other negative regulatory factor, RAR (Shimba et al., 1998Go). Third, and more importantly, fatty metamorphosis is observed in AhR-null mice over the first 2 weeks of life (Schmidt et al., 1996Go). In addition, the AhR is not expressed in liver fat storage cells (Riebniger and Schrenk, 1998Go). Consequently, we are led to conclude that the AhR appears to play a negative regulatory role in adipose differentiation in 3T3-L1 cells in the absence of exogenous ligand.

It is well recognized that PPAR{gamma}2 plays pivotal roles in adipogenesis (Tontonz et al., 1994Go). It is expressed in small amounts in preadipocytes, and its synthesis is increased during the process of adipogenesis. In this study, the AhR-overexpressing cells exhibited similar level of PPAR{gamma}2 expression compared with control cells, despite their reduced ability for morphological differentiation. Two possible mechanisms can explain this discrepancy between the normal level of expression of PPAR{gamma}2 mRNA and the lower potency for morphological differentiation in the AhR-overexpressing cells. First, the AhR may inhibit the production of endogenous ligand for PPAR{gamma}2. This view is compatible with the results in Fig. 4 showing that treatment with exogenous PPAR{gamma}2 ligands, such as troglitazone, ciglitazone and indomethacin, restored the ability of the AhR-overexpressing cells to differentiate. The second possibility is that the AhR signaling pathway may inhibit PPAR{gamma}2 activity. TCDD activates COUP-TF, thereby antagonizing the activity of PPAR{gamma}2 (Brodie et al., 1996Go,Brodie et al., 1996Go). We have shown in this study that the activated-AhR stimulates p42/p44 MAP kinase activity (Fig. 5A). Treatment of the cells with PD98059 or U0126 overcame the inhibitory effects of the AhR on adipogenesis (Fig. 5C). These results suggest that p42/p44 MAP kinase activity mediates negative regulatory action of the AhR on adipogenesis. A report by Hu et al. has revealed that the phosphorylation of Ser112 in PPAR{gamma}2 by MAP kinase results in the loss of transcriptional activity (Hu et al., 1996Go). Collectively, a possible mechanism is that the AhR inactivates PPAR{gamma}2 via stimulation of MAP kinase. We have also shown that the activation of the AhR affects on the expression of p107, pRB-related protein (Fig. 8B). The expression of p107 is transiently upregulated in clonal expansion stage and is downregulated after clonal expansion (Fig. 8B; Richon et al., 1997Go). Studies using p107-deficient fibroblast cells revealed that p107 suppresses PPAR{gamma}2 activity in adipose differentiation (Classon et al., 2000Go). Thus, the downregulation of p107 is essential for activation of PPAR{gamma} (Classon et al., 2000Go). However, as we presented in Fig. 8B, the activation of the AhR stabilized the expression of p107, but not that of p130 (Fig. 8B). Therefore, the stabilization of p107 expression by the AhR may result in lower PPAR{gamma}2 activity. Taken together, these results strongly suggest that PPAR{gamma}2 activation pathway is the target for the AhR. Further studies on the crosstalk between the PPAR{gamma}2 activation pathway and the AhR signaling pathway may reveal new aspects of adipogenesis-related signal transduction.

Recent reports showed a direct interaction of the AhR with pRB (Ge and Elferink, 1998Go; Puga et al., 2000Go). In addition, the AhR associates preferentially with the hypophosphorylated, active form of pRB (Ge and Elferink, 1998Go). The roles of pRB in adipogenesis have been amply demonstrated. It has been shown that the ability of SV 40 large T antigen to block adipogenesis is dependent on its ability to sequester pRB (Higgins et al., 1996Go). In addition, it has been demonstrated that fibroblast cells from RB-deficient mouse embryos are unable to undergo adipose conversion, and ectopic expression of RB enables RB-/- fibroblasts to differentiate (Chen et al., 1996Go). Furthermore, pRB has been shown to physically interact with C/EBPs, to promote the binding of C/EBPß to DNA response element and to increase its transactivation capacity (Chen et al., 1996Go). The interaction between the AhR and pRB may affects on these roles of pRB in adipogenesis. Given the known activity of pRB as a cell cycle regulator, one of the roles of pRB in adipogenesis is regulation of clonal expansion (Classon et al., 2000Go). Clonal expansion has generally been regarded as a prerequisite event for adipose differentiation. Inhibition of clonal expansion by treatment with drugs such as rapamycin or TNF{alpha} results in failure of the subsequent differentiation process in 3T3-L1 cells (Yeh et al., 1995Go; Lyle et al., 1998Go). In this study we present data showing that activation of the AhR inhibits clonal expansion in adipose differentiation (Fig. 6). Cell cycle analysis revealed that the AhR-overexpressing cells lag in G0/G1 phase, with subsequent decreased entry into the S phase (Fig. 7). We also found that overexpression of the AhR inhibits the phosphorylation of pRB, whereas underexpression of the AhR stimulates pRB phosphorylation (Fig. 8A). The importance of hyperphosphorylation of pRB for the commitment of cells to undergo adipose conversion has been described previously (Shao and Lazar, 1997Go). Collectively, these results suggest that the represses of pRB phosphorylation by the AhR results in delay of clonal expansion and subsequent progress of differentiation program. In addition to the roles in clonal expansion, as described above, pRB increases the transactivation capacity of C/EBPß via a direct interaction. This tempts us to speculate that the association of the AhR with pRB limits the binding capacity of pRB to C/EBPß. In such a scenario, the activity of C/EBPß could be suppressed. C/EBPß is known to positively regulate the expression of C/EBP{alpha}. By contrast, the AhR suppresses the induction of C/EBP{alpha} (Fig. 3C; Phillip et al., 1996). Therefore, suppression of the C/EBP{alpha} expression and subsequent lower morphological differentiation in the AhR-activated cells could be due to less interaction of pRB with C/EBPß. However, this model has to be proven in the future study.

From a toxicological perspective, it is not clear whether the AhR-dependent effects of TCDD reflect the enhanced physiological function of the AhR, or the specific actions of the TCDD-AhR complex, or a combination of both. In this study, the features of AhR-overexpressing cells were observed in the absence of exogenous ligand. However, the cells exhibit several similarities with TCDD-treated cells: (1) inhibition of differentiation; (2) induction of CYP1B1; (3) alteration of expression level of adipocyte-related genes; and (4) growth delay. These results indicate that overexpression of the AhR can mimic the effects of exogenous ligand. Thus, TCDD may induce its toxicological effects, at least partly, by overactivating the physiological AhR signaling pathway. If so, the AhR may play several unidentified physiological roles in homeostasis, because TCDD causes a variety of toxic reactions in living material (Poland and Knutson, 1982Go; Safe, 1986Go; Landers and Bunce, 1991Go; Pohjanvitra and Tuomisto, 1994Go). Understanding the detailed mechanisms of the toxicological action of TCDD may reveal further physiological roles of the AhR.

In summary, this study is the first to report that the AhR participates in the negative regulation of adipose differentiation in the absence of exogenous ligands. The proposed mechanism by which the AhR inhibits adipose differentiation is that the AhR inhibits PPAR{gamma}2 activation pathway by stimulating p107 expression and/or p42/p44 MAP kinase, and the inhibition of PPAR{gamma}2 signaling pathway results in lower morphological differentiation in the AhR-activated cells. Furthermore, the AhR inhibits pRB phosphorylation, resulting in delay of clonal expansion and the subsequent progress of differentiation program. Dynamic regulation of the AhR is observed in mouse embryos (Abbott et al., 1994Go), suggesting that the AhR plays a role during development and differentiation. Therefore, the data presented in this paper will provide opportunities to carry out studies in order to better understand the physiological role of the AhR in development and differentiation.


    ACKNOWLEDGMENTS
 
This work is supported in part by a grant from the Ministry of Education, Sciences, Sports, and Culture, Japan (S. S. and M. T.), by a research grant for general joint research from Nihon University (S. S. and M. T.), and by a research grant for young researchers from Nihon University (S. S.). We thank Dr Imagawa for critically reviewing this manuscript.


    REFERENCES
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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