Unlocking Repression of the Human Luteinizing Hormone Receptor Gene by Trichostatin A-induced Cell-specific Phosphatase Release

Ying Zhang,Mingjuan Liao,Maria L Dufau

doi:10.1074/jbc.m801878200

Abstract

Our previous studies demonstrated that the histone deacetylase inhibitor, trichostatin A (TSA), induces derepression of the human luteinizing hormone receptor (LHR) gene by de-recruitment of the pRB homologue p107 repressor from the promoter in JAR and MCF-7 cancer cells. TSA initiates a mechanism whereby the phosphatidylinositol 3-kinase/protein kinase zeta (PKCzeta) cascade phosphorylates Sp1 at Ser-641, which is essential for the release of the repression of LHR transcription. The present studies have revealed that dissociation of serine/threonine protein phosphatases PP2A and PP1 from the LHR promoter mediates TSA-induced activation of LHR gene transcription in a cell-specific manner. Changes in chromatin structure induced by TSA cause the release of PP2A in JAR cells or of PP1 in MCF-7 cells, which is associated with Sp1 directly or through histone deacetylase 1/2, respectively, at the promoter. This favors the phosphorylation of Sp1 mediated by the phosphatidylinositol 3-kinase/PKCzeta pathway, which in turn causes the release of the p107 inhibitor from Sp1 and marked transcriptional activation of the LHR. These findings reveal the importance of phosphatases in the control of LHR transcription, where the balance between phosphatidylinositol 3-kinase/PKCzeta and phosphatases could be critical for up- and down-regulation of LHR gene expression in physiological and pathological settings.

Highlights

The luteinizing hormone receptor (LHR),2 whose expression is controlled by hormones, growth factors, and other activators during development [1], has an essential role in mammalian reproduction
Our recent findings have demonstrated that PI3K/PKC␨ have an essential role in the regulation of LHR gene transcription in human JAR choriocarcinoma and MCF-7 breast cancer cells [10]
The efficacy of the inhibitor compounds used was confirmed by their negative effects on trichostatin A (TSA)-induced LHR gene promoter activation (Fig. 2C). These results have demonstrated that the release of PP1 or PP2A caused by TSA is independent of Sp1 phosphorylation induced by PI3K/PKC␨

Summary

EXPERIMENTAL PROCEDURES

Expression Vectors, and Antibodies—TSA was obtained from Calbiochem. The human LHR promoter/luciferase reporter gene wild type and Sp1-I site (Ϫ79 bp to ATG) mutant constructs have been described in our previous studies [4]. Other studies (not shown) revealed that both Sp1 N-terminal and C-terminal segments were able to interact with PP2AC Taken together these results have revealed that the observed cell typeselective binding of PP2A and PP1 to the LHR gene promoter in JAR and MCF-7 cells occurred in Sp1 or its proximity, respectively, where the association of Sp1 protein with PP2A in JAR or with PP1 in MCF-7 cells enables these phosphatases to exert their regulatory effects on Sp1 phosphorylation levels in the control of silencing or activation of the LHR gene expression. The reduction of p107 release was consistent with the mayor reduction of Sp1 phosphorylation observed by overexpression of the PP1C or PP2AC in the individual cell types (Fig. 5A) These results further confirm that the negative regulation of PP1 or PP2A on the LHR gene expression is mediated through their dephosphorylation effect on Sp1, which cause the release of repressor p107

DISCUSSION

Findings

ADDITIONS AND CORRECTIONS