TGF-β mediates suppression of adipogenesis by estradiol through connective tissue growth factor induction

Abstract

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.

Fig. 1.

Estrogen repression of adipogenesis as assessed by accumulation of neutral lipid droplets in ST2 cells. A and B, ST2 cells were cultured in base medium with or without the indicated treatments (E, 10 nm 17β-estradiol; EtOH, 0.1% ethanol; Rosi, rosiglitazone). Cultures were fed on d 3 with the same media and fixed after 6 d. Cultures were treated throughout the culture time. Cells were stained for lipid with Oil Red O as outlined. A, Image of stained cultures (×200). B, The stain was extracted and the amount of stain quantitated by measurement at OD 500 as detailed in Materials and Methods. Data are the mean of three replicates. *, P < 0.05 compared with base; **, P < 0.05 compared with rosiglitazone+EtOH. C, hFOB/ER9 cells were cultured and analyzed as outlined for the ST2 cells in A and B. *, P < 0.05 compared with EtOH treatment. D, ST2 cells were treated with base medium with rosiglitazone (Rosi), rosiglitazone and vehicle (Rosi EtOH), rosiglitazone and 10 nm 17β-estradiol (Rosi E), rosiglitazone and 100 nm 4-hydroxytamoxifen (Rosi OHT), or rosiglitazone, 10 nm 17β-estradiol, and 100 nm 4-hydroxytamoxifen (Rosi E OHT). Cells were stained and quantitated as above. *, P < 0.05 compared with Rosi. E, Cells were harvested (D0) or cultured in base plus rosiglitazone medium for 6 d with a refeeding on d 3. RNA was harvested and analyzed by real-time PCR for the indicated estrogen receptor. Expression is relative to d 0 (D0). *, P < 0.05 compared with D0.

Fig. 2.

Estradiol repression of adipogenic gene expression. A–E, ST2 cells were harvested at the beginning of culture (D0) or treated with rosiglitazone. ST2 cells cultured with rosiglitazone were cotreated with vehicle (0.1% EtOH) or 10 nm estradiol for the indicated time. RNA was harvested and analyzed by real-time PCR for the indicated mRNA. The experiment was repeated three times, and these results are representative of these experiments. *, P < 0.05 compared with vehicle at the same time point. F, ST2 cells were treated with base medium (BASE), rosiglitazone (Rosi), rosiglitazone and 10 nm 17β-estradiol (Rosi+E), rosiglitazone and 100 nm 4-hydroxytamoxifen (Rosi+OHT), or rosiglitazone, 10 nm 17β-estradiol, and 100 nm 4-hydroxytamoxifen (Rosi+E+OHT). RNA was harvested and analyzed by real-time PCR for the indicated mRNA. *, P < 0.05 compared with base medium; **, P < 0.05 compared with rosiglitazone treatment; ***, P < 0.05 compared with rosiglitazone and 17β-estradiol treatment.

Fig. 3.

A–E, Delayed estradiol treatment blunts suppression of adipogenic gene expression. ST2 cells were harvested at the beginning of culture (D0) or treated with rosiglitazone. On d 3, the cells were fed with rosiglitazone-containing medium with the addition of either vehicle (0.1% EtOH) or 10 nm 17β-estradiol until d 9 with a refeeding on d 6. RNA was harvested and analyzed by real-time PCR for the indicated mRNA. *, P < 0.05 compared with D0; **, P < 0.05 compared with either D0 or rosiglitazone plus vehicle treatment.

Fig. 4.

Estradiol effects on adipogenesis are dependent on TGF-β expression. ST2 cells were cultured in base plus rosiglitazone medium for 6 d with a refeeding on d 3 with or without the indicated treatments: 0.1% ethanol (EtOH), 0.1% DMSO, 10 nm 17β-estradiol (E), 2 ng/ml TGF-β1, and 2 × 10⁻⁷ m SB 431542 (TGF-β inhibitor). Cultures were treated throughout the culture time. A, Cultures were fixed and stained for lipid with Oil Red O, extracted, and quantitated as outlined. B–F, RNA was harvested and analyzed by real-time PCR for the indicated mRNA. Expression is relative to EtOH plus DMSO vehicles. *, P < 0.05 compared with EtOH plus DMSO vehicles.

Fig. 5.

Estradiol-mediated stimulation of TGF-β leads to increased CTGF expression. A, ST2 cells harvested (d 0) or cultured in base plus rosiglitazone medium for up to 6 d with a refeeding on d 3 with or without the indicated treatments: vehicles with 0.1% ethanol plus 0.1% BSA, 10 nm 17β-estradiol, and 2 ng/ml TGF-β1. Cultures were treated throughout the culture time. RNA was harvested and analyzed by real-time PCR for relative CTGF mRNA expression compared with d 0. *, P < 0.05 compared with vehicles at the same time point. B, ST2 cells were cultured for 6 d in base plus rosiglitazone medium with or without the indicated treatments with a refeeding on d 3: 0.1% ethanol (EtOH), 0.1% DMSO, 10 nm 17β-estradiol (E), 2 ng/ml TGF-β1, and 2 × 10⁻⁷ m SB 431542 (TGF-β inhibitor). RNA was harvested and analyzed by real-time PCR for relative CTGF mRNA expression compared with no additional treatment. *, P < 0.05 compared with vehicle; **, P < 0.05 compared with 17β-estradiol without the TGF-β inhibitor.

Fig. 6.

A–C, Estradiol modulates TGF-β gene expression. ST2 cells were cultured in base plus rosiglitazone with 0.1% ethanol (Rosi) or rosiglitazone plus 10 nm 17β-estradiol for the indicated time with a refeeding on d 3. Cultures were treated throughout the culture time. RNA was harvested and analyzed by real-time PCR for the indicated TGF-β isoform. Values are normalized to d 0. *, P < 0.05 compared with d 0 for both treatments; **, P < 0.05 between rosiglitazone and rosiglitazone plus estradiol.