The Luteinizing Hormone Receptor (LHR): The LHR is a G-coupled seven-transmembrane receptor which is expressed primarily in the gonads where it mediates luteinizing hormone signals that regulate cyclic ovarian changes and testicular function. Our findings demonstrated that LHR gene expression at the transcriptional level is regulated by complex and diverse networks, in which coordination and interactions between these regulatory effectors are essential for silencing/activation of LHR expression. The proximal Sp1 site of the LHR promoter recruits histone deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcriptional expression. Site specific acetylation/methylation induce cell-specific phosphatase release. This serves as an on mechanism for Sp1 phosphorylation by PI3K/PKCzeta at Ser641 causing p107 repressor decruitment from Sp1 and LHR transcriptional activation. Maximal derepression of the LHR gene is dependent on complete DNA demethylation of the promoter, histone hyperacetylation and release of repressors (p107 and HDAC/Sin3A). This causes recruitment of TFIIB and Pol II and transcriptional activation. Our more recent studies demonstrated that Positive Cofactor 4 (PC4) has an important role in the formation/assembly of general transcriptional machinery in TSA-mediated LHR transcription. It is recruited by Sp1 following TSA treatment and acts as a coactivator of Sp1. The coactivator domain of PC4 and DNA binding domain of Sp1 are essential for their interaction. However, PC4 does not participate in TSA induced chromatin structural alteration since silencing of PC4 does not prevent release of HDAC/Sin3A and phosphatase from Sp1. These two events were demonstrated to be dependent on DNA methylation and chromatin decompactation resulting from histone acetylation at the promoter. Although TFIIB recruitment is largely dependent on PC4 we have ruled out TFIIB as target to link Sp1 to the transcriptional machinery. Since interaction with other members of this initiation and or/mediator complex is anticipated we are currently investigating PC4s linking function using various strategies. We have excluded acetylation of PC4 either basally or TSA- induced in the transcriptional activation process. However, immunoprecipitation studies demonstrated that TSA specifically induced acetylation of two PC4 interacting proteins (Mr 10 and 15 kDa). We are proceeding with their identification to determine their role in LHR gene transcription. Gonadotropin regulated genes: Gonadotropin Regulated Testicular RNA helicase (GRTH/DDX25) GRTH /DDX25 discovered in our laboratory is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and germ cells. It is a multi-functional protein that is essential for the completion of spermatogenesis. Males lacking GRTH are sterile due to the absence of sperm resulting from failure of round spermatids to elongate. Normal basal levels of testosterone in serum and LCs excluded abnormal steroidogenesis as responsible for the arrest of spermiogenesis. However, testosterone production was highly magnified in LCs of GRTH null mice (KO) compared to WT upon stimulation by gonadotropin both in vivo and in vitro. Our recent studies have revealed that GRTH has an important regulatory role in gonadotropin-induced androgen production by LCs. These cells in KO mice had reduced lipid droplets and swollen mitochondria (site of conversion of cholesterol,supplied by lipid droplets, to pregnenolone) with increased cholesterol content at the inner mitochondrial membrane. This resulted from the increase in StAR, a protein that transports cholesterol to the inner mitochondrial membrane and HMGCR a key enzyme in cholesterol biosynthesis. The half-life of StAR mRNA was significantly increased in the KO mice vs Wild type (WT) and association of StAR mRNA with GRTH protein was observed in WT mice. Major testosterone increases in KO over WT was observed upon hCG stimulation which resulted from the availability in KO of basal accumulated cholesterol as substrate of P450scc for pregnenolone production and other distal enzymes of the androgen pathway. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis provides insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male. GRTH participates in the export of specific mRNAs relevant to spermatogenesis from nuclear to cytoplasmic sites of germ cells including the Chromatoid body of spermatids (storage/processing site) and polyribosomes where it participates in translation of relevant messages. In recent studies we demonstrated the involvement of GRTH in the export of its own message from the nucleus to cytoplasmic sites in addition to previously reported mRNAs of spermatogenic genes. Association of GRTH mRNA with GRTH protein was observed in testis extracts of WT mice. Blockade of nuclear export with inhibitor leptomicin b caused significant nuclear accumulation of GRTH mRNA, with mayor decrease at the cytoplasmic level and its exclusion from the CB. This study has demonstrated the essential participation of the GRTH export/transport function to the CB and other cytoplasmic sites. Prolactin receptor (PRLR): The prolactin receptor is a member of the lactogen/cytokine receptor family which mediates the diverse cellular actions of prolactin (PRL) in several target tissues. PRL is a major factor in the proliferation and differentiation of breast epithelium and is essential in the stimulation and maintenance of lactation. It has been also implicated in the development of breast cancer. In humans hPRLR expression hPRLR is controlled at the transcriptional level by multiple promoters (one generic, also present in rat and mouse PIII, and five human specific hE1N1-hE1N5) that were defined and characterized in our laboratory. The transcription of PRLR in breast cancer cells by the preferentially utilized promoter PIII which lacks an estrogen responsive element is directed by E2/ERa through complex formation with SP1 and C/EBPb that associate with cognate elements and TFIIB and Pol II recruitment. BRET revealed ERa constitutive homodimers. E2 enhances through stabilization of the dimer, E2/ERa-dimer interaction with SP1 and C/EBPb. Chromatin immunoprecipitation and small interfering RNA knockdown of members of the complex in breast cancer cells (ERa+) demonstrated the endogenous recruitment of components of the complex onto the PIII promoter of the hPRLR gene. SP1 is the preferred transfactor for the recruitment of ERa to the complex that facilitates the C/EBPb association. The E2/ERa-induced hPRLR transcription was demonstrated in ERa-negative cells. Recruitment of ERa or C/EBPb but not Sp1 was E2 inducible. Cells with depletion of C/EBPb showed the recruitment of Sp1 and ERa. Only SP1 was recruited to PIII when ERa was knocked down. The ERa-SP1-C/EBPb complex was formed by the association of the DNA binding domain of ERa with ZF motifs of SP1 followed by recruitment of C/EBPb through its LeuZipper (LZ). Interactions between ZF of SP1 and LZ of C/EBPb stabilizes the ERa-SP1-C/EBPb complex. Our studies have demonstrated that the enhanced complex formation of ERa dimer with SP1 and C/EBPb dimers at the PIII promoter by E2 has an essential role in the transcriptional activation of the hPRLR gene in breast cancer cells
Showing the most recent 10 out of 24 publications
