Figure 7.

TR4 Mediates the Expression of LHR

A, Regulation of LHR promoter activity in cultured cells. TR4 activates the LHR promoter luciferase reporter genes pLHR(−173)-Luc and pLHR (−2040)-Luc, which contain the DR1 motif. CV-1 cells were cotransfected with LHR promoter luciferase reporter and PCMX or TR4 (PCMX-TR4). *, P < 0.05 vs. control. B, ChIP of mouse TM3 cells using TR4-specific antisera. PCR amplification was performed of mLHR promoter (−5 to −301) that includes the region containing the DR1 motif. Lane 1, marker; lane 2, input control; lane 3, control IP without normal mouse IgG; lane 4, control IP with normal mouse IgG; lane 5, PCR product obtained from immunoprecipitates using TR4 antisera no. 15, which is specific for TR4. C, cAMP assay. TM3 cells were transfected with pRetro-shiTR4 and control vector. After 24 h, TM3-shiTR4 and TM3 control cells were incubated in serum-free medium for 1 h and then further cultured in the presence of 100 ng/ml LH with or without 10 μm forskolin for 2 h. The cells were harvested, and TR4 expression level was determined by Western blotting. The intracellular cAMP levels were measured as described in Materials and Methods. The experiment was repeated twice. A Student's t test was used to compare the mean of cAMP levels between the vector control group and TR4siRNA groups treated with LH. *, P < 0.05.