The Luteinizing Hormone Receptor (LHR): The LHR is expressed primarily in the gonads where it mediates LH signals that regulate ovarian and testicular function. The LHR gene transcription is regulated by complex and diverse networks, in which coordination and interactions between regulatory effectors are essential for silencing/activation of LHR expression. The proximal Sp1 site of the promoter recruits histone (H) deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcription. Site specific acetylation/methylation-induced phosphatase release serves as an on switch for Sp1 phosphorylation at Ser641 that causes p107 repressor release from Sp1, recruitment of TFIIB and Pol II and transcriptional activation. Maximal derepression of the gene is dependent on DNA demethylation of the promoter, H3/H4 acetylation and HDAC/Sin3A release. Positive Cofactor 4 (PC4) has an important role in the formation/assembly of PIC in TSA-mediated LHR transcription. It is recruited by Sp1 following TSA treatment and acts as its coactivator. However, PC4 does not participate in TSA release of phosphatases, Sp1 phosphorylation or release repressor/complexes. Although TFIIB recruitment is dependent on PC4 we have ruled out TFIIB as its direct target and acetylation of PC4 in the activation process. However, TSA induced acetylation of a PC4 interacting proteins, identified as Acetylated H3 by MS, and its presence in the complex in association to chromatin at the promoter was demonstrated by ChiP/reChiP. The role of these interactions on chromatin structure and their participation in the assembly of the PIC and transcriptional activation are under investigation. To further investigate the role of PC4 in vivo we have initiated generation of a Leydig and granulosa/luteal cell specific PC4 null mice. Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25): GRTH is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and meiotic germ cells. It is a multi-functional protein 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. In addition, to its intrinsic RNA helicase activity, GRTH is a shuttling protein that exports specific mRNAs from the nucleus to cytoplasmic sites. Our studies have demonstrated the essential participation of the GRTH export/transport of mRNAs in the structural integrity of the Chromatoid Body (storage/processing of mRNAs) and their transit/association to actively translating polyribosomes where it may regulate translational initiation of genes. We have identified mRNAs which are associated with GRTH and regulated at polysomal sites of cell populations of the mouse testis. The reduction in mRNAs associated at polysomal sites in the differential studies (KO vs WT) not detected at total cellular level but in the cytoplasm with abolition of protein expression are reflective of the importance of the transport function of GRTH to relevant sites and underscore its impact in protein synthesis. GRTH is regulated by LH through androgen (A) at the transcriptional level in LCs (direct) and germ cells (presumably indirect) of the testis where its expression is both cell- and stage specific. This helicase displays a novel negative autocrine control of androgen production in LCs by preventing overstimulation of the LH-induced androgen pathway through enhanced degradation of StAR protein. Our studies have described the mechanism by which Androgen (A)/Androgen Receptor (AR) regulates the expression of the GRTH gene in the Leydig cell via a short range chromosomal loop. A/AR signaling in LC s through its activation of GRTH transcription, participates in an autocrine regulation mechanism with a major impact on Leydig cell steroidogenic function. Our development of transgenic mice model carrying GRTH 5'flanking regions-GFP reporter have provided a unique in vivo system that permitted differential elucidation of regulatory regions in the GRTH gene that directs its expression (upstream) in germ cells (pachytene spermatocytes and round spermatids) and downstream in LCs. Binding sites for germ cells specific transcription factors (GCNF, Dmrt1/7) were identified in the distal region. In contrast, the proximal region directs basal GRTH expression and androgen-induced intracrine expression in Leydig cells through a functional ARE. In the transgenic animal model the AR antagonist, flutamide, blocked GRTH/GFP expression in LCs and germ cells demonstrating direct intracrine regulation by Andr/AR in LCs and indirect through effects in germ cells through paracrine regulation by the actions Andr/AR present in Sertoli cells. This model permits to elucidate mechanism for androgen action in germ cells that would permit the identification of androgen regulated factors that control expression of a critical gene(s) require for GRTH expression in germ cells involved in the progress of spermatogenesis. This could lead to development of contraceptive strategies, that block sperm formation without impacting other aspects of androgen action. Prolactin receptor (PRLR): The PRLR is a member of the lactogen/cytokine receptor family which mediates the diverse cellular actions of PRL. PRL is a major factor in the proliferation and differentiation of breast epithelium and is essential for lactation. It has been also implicated in the development of breast cancer, tumoral growth and chemoresistance. hPRLR expression is controlled at the transcriptional level by multiple promoters (one generic, PIII, and five human specific hPN1-hPN5) that were defined and characterized in our laboratory. Each promoter directs transcription/expression of a specific non-coding Exon 1 (E1-3, hEN1-hEN5), a common non-coding exon 2 and coding exons (E3-E11). The transcription of PRLR in breast cancer cells is directed by the preferentially utilized PIII that lacks an estrogen responsive element. BRET studies revealed ERa constitutive homodimers. Complex formation of ERa dimer (non-DNA bound)with Sp1 and C/EBPb dimers bound to their cognate sites at the PIII promoter is required for basal (constitutive ERa homodimers)and E2-induced transcriptional activation/expression of the human PRLR gene. Prolactin in tumoral breast causes cell proliferation via activation of its cognate receptor. Exacerbation of prolactin's actions resulting from increased receptor number can explain resistance to estrogen inhibitors in breast cancer. Our studies reveal stimulation of prolactin receptor promoter activity, mRNA and protein in MCF7 cells by prolactin in absence of estradiol abolished by mutation of a GAS site, Stat5 siRNA, or estrogen receptor- antagonist. This indicates the participation of the estrogen receptor in prolactin receptor transcription via prolactin/prolactin receptor/Stat5. Increased recruitment of estrogen receptor- to Sp1 and C/EBP bound at promoter sites is essential for prolactin-induced receptor transcription. Direct evidence is provided for local actions of prolactin independent of estradiol in the up-regulation of prolactin receptor transcription/expression via a STAT5/estrogen receptor activation-loop. These studies are of relevance in refractory states to aromatase inhibitors where cancer progression could be fueled by endogenous prolactin.

Project Start
Project End
Budget Start
Budget End
Support Year
38
Fiscal Year
2013
Total Cost
$1,192,561
Indirect Cost
City
State
Country
Zip Code
Kang, J-H; Hassan, S A; Zhao, P et al. (2014) Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin. Biochim Biophys Acta 1840:2272-80
Kavarthapu, Raghuveer; Tsai-Morris, Chon-Hwa; Fukushima, Masato et al. (2013) A 5'-flanking region of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) gene directs its cell-specific androgen-regulated gene expression in testicular germ cells. Endocrinology 154:2200-7
Xie, Y-L; Hassan, S A; Qazi, A M et al. (2009) Intramolecular disulfide bonds of the prolactin receptor short form are required for its inhibitory action on the function of the long form of the receptor. Mol Cell Biol 29:2546-55
Sheng, Yi; Tsai-Morris, Chon-Hwa; Li, Jie et al. (2009) Lessons from the gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) null mouse model: a role in steroidogenesis, but not result in X-ALD phenotype. J Steroid Biochem Mol Biol 114:44-56
Liao, Mingjuan; Zhang, Ying; Dufau, Maria L (2008) Protein kinase Calpha-induced derepression of the human luteinizing hormone receptor gene transcription through ERK-mediated release of HDAC1/Sin3A repressor complex from Sp1 sites. Mol Endocrinol 22:1449-63
Tsai-Morris, Chon-Hwa; Koh, Eitetsu; Dufau, Maria L (2008) Differences in gonadotropin-regulated testicular helicase (GRTH/DDX25) single nucleotide polymorphism between Japanese and Chinese populations. Hum Reprod 23:2611-3
Zhang, Ying; Liao, Mingjuan; Dufau, Maria L (2008) Unlocking repression of the human luteinizing hormone receptor gene by trichostatin A-induced cell-specific phosphatase release. J Biol Chem 283:24039-46
Gutti, Ravi K; Tsai-Morris, Chon-Hwa; Dufau, Maria L (2008) Gonadotropin-regulated testicular helicase (DDX25), an essential regulator of spermatogenesis, prevents testicular germ cell apoptosis. J Biol Chem 283:17055-64