Crucial Role of Estrogen Receptor-α Interaction with Transcription Coregulators in Follicle-Stimulating Hormone and Transforming Growth Factor β1 Up-Regulation of Steroidogenesis in Rat Ovarian Granulosa Cells

This study was to explore estrogen receptor (ER) involvement in FSH and TGFβ1-stimulated steroidogenesis in rat ovarian granulosa cells. We first determined the specific involvement of ERα and ERβ in the process, and then investigated the molecular interaction of ERα and transcription coregulators in FSH and TGFβ1 up-regulation of steroidogenic gene expression. Primary culture of ovarian granulosa cells from antral follicles of gonadotropin-primed immature rats was used. Interestingly, a selective ERα antagonist methyl-piperidino-pyrazole (MPP) [like ER antagonist ICI-182,780 (ICI)] decreased FSH ± TGFβ1-stimulated progesterone production, whereas an androgen receptor antagonist hydroxyflutamide and particularly a selective ERβ antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol had no significant effect. Consistent with this, a selective ERβ agonist diarylpropionitrile (unlike 17β-estradiol) also had no effect on FSH ± TGFβ1-stimulated progesterone production. Furthermore, a selective ERα agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (like 17β-estradiol) enhanced FSH-stimulated progesterone production, and this was abolished by pretreatment with MPP. Immunoblotting and chromatin immunoprecipitation analyses indicate that MPP/ICI suppression of FSH ± TGFβ1 action is partly attributed to the reduced ERα-mediated expression of Hsd3b and Cyp11a1 genes, but not steroidogenic acute regulatory protein. Furthermore, FSH ± TGFβ1 increased ERα association with histone acetylases (CBP and SRC-1) and coactivator of peroxisome proliferator-activated receptor γ (PGC-1α), and MPP/ICI dramatically reduced these interactions. In addition, FSH ± TGFβ1 increased CBP, SRC-1, and PGC-1α binding to Hsd3b and Cyp11a1 genes. Together, we demonstrate for the first time that ERα interaction with transcription coregulators, histone acetylases (CBP/SRC-1), and PGC-1α is crucial to FSH and TGFβ1-up-regulated expression of Hsd3b and Cyp11a1, and, thus, progesterone production in rat ovarian granulosa cells.

Interaction of Histone Acetylases and Deacetylases In Vivo

ABSTRACT Having opposing enzymatic activities, histone acetylases (HATs) and deacetylases affect chromatin and regulate transcription. The activities of the two enzymes are thought to be balanced in the cell by an unknown mechanism that may involve their direct interaction. Using fluorescence resonance energy transfer analysis, we demonstrated that the acetylase PCAF and histone deacetylase 1 (HDAC1) are in close spatial proximity in living cells, compatible with their physical interaction. In agreement, coimmunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase, GCN5, in HeLa cells. We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST. This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro. The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo.