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

AhR inhibits adipose differentiation independently of dioxin

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 )

View larger version (88K): [in a new window]   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 -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.

View larger version (61K): [in a new window]   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.

View larger version (25K): [in a new window]   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, PPAR2 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, PPAR2 and aP2 mRNA were determined by northern blotting. AS, cells expressing antisense mRNA; S, cells expressing AhR sense mRNA.

View larger version (62K): [in a new window]   Fig. 4. Restoration of the differentiation potential of cells expressing AhR sense mRNA by treatment with PPAR2 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.

View larger version (97K): [in a new window]   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.

View larger version (13K): [in a new window]   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.

View larger version (34K): [in a new window]   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.

View larger version (28K): [in a new window]   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).