The Luteinizing Hormone Receptor (LHR) is expressed primarily in the gonads where it mediates signals that regulate ovarian and testicularfunction. LHR is transcriptional regulated by 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 by TSA causes phosphatase release that serves as a switch for Sp1 phosphorylation, recruitment of TFIIB and Pol II and transcriptional activation Positive coactivator 4(PC4)which is recruited by Sp1 acts as its coactivator has an essential role in the formation/assembly of PIC and TFIIB and Pol II recruitment in TSA mediated LHR transcription. Our recent studies demonstrated association between PC4 and acetylated histone3 in TSA induced LHR derepression in MCF7 cells. MS/MS analysis revealed that PC4 associates with histone variant H3.3 acetylated at several Lys residues. The presence of these modifications was further confirmed by immunoprecipitation of FlagPC4-H3 complex with detection by site specific antibodies in Western blots. ChiP/reChiP analysis showed an increased recruitment of complexes of PC4/acetylated H3 at these sites to the LHR promoter upon TSA stimulation. Further, IP studies with Flag demonstrated interaction of PC4 with H3.3 induced by TSA using H3.3 specific antibody and the presence of the complex PC4-H3.3 at the LHR promoter was demonstrated by reChiP. Depletion of endogenous PC4 or H3.3 by siRNA caused marked reduction of TSA induced formation of the complex,its recruitment to the LHR promoter and transcriptional activation of the LHR gene. Taken together, these findings indicate a critical role of PC4 association with acetylated H3.3 in TSA-induced Sp1 activated LHR transcription. To elucidate the physiological impact of PC4 on Sp1 directed LHR transcription in gonads,using-Cre-lox technology we have generated the PC4(flox/flox)homozygous mice to be bred with female Cyp17iCre for conditional deletion of the PC4 gene in Leydig cells and expect to observe phenotypic changes related to LHR function. Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25, is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and meiotic germ essential for the completion of spermatogenesis. Males lacking GRTH are sterile due azoospermia resulting from failure of round spermatids to elongate. Our studies have demonstrated its participation on the nuclear export/transport of specific mRNAs,in the structural integrity of the Chromatoid Body(CB) storage/processing of relevant mRNAs and their transit/association to the actively translating polyribosomes where it may regulate translational initiation of genes. GRTH is regulated by LH through androgen (A)/A receptor (AR)at the transcriptional level in LCs (direct)with impact in steroidogenesis, and germ cells (indirect through AR in Sertoli cells) where its expression is both cell-and stage specific. More recently, Tg-mice model carrying GRTH 5' flanking regions-GFP generated in our laboratory provided in vivo systems that permitted differential elucidation of regions in the GRTH gene that directs its expression (upstream) in germ cells and downstream in LCs and its regulation by A/AR in LC (directly) and indirectly in germ cells. Functional binding sites for germ cell specific transcription factor (GCNF)are present in round spermatids (RS) and spermatocytes(SP) and its regulation by A/AR identified in the distal region-of the GRTH gene which is operative selectively in RS. The studies provided evidence for actions of A on GCNF cell specific regulation of GRTH expression in germ cells. Also, GRTH associates exerts negative autocrine regulation of GCNF. Our in vivo/in vitro models link A actions to germ cells through GCNF as an A regulated transfactor that controls transcription/expression of GRTH, provided a connection of A action to two relevant germ cell genes essential for the progress of spermatogenesis and established their regulatory interrelationship. Our early studies revealed that missense mutation of R to H at aa 242 of GRTH found in 5.8% of patients with complete loss of sperm causes loss of the 61 kDa cytoplasmic phospho-species with preservation of the non-phospho form. This finding provided an avenue to elucidate the function of phospho-GRTH in spermatogenesis. We generated a humanized mutant GRTH knock-in (KI) mice. Very recent studies revealed that mutant hGRTH mice are sterile with reduction on testicular size, lack sperm with arrest at step 8 of round spermatids and complete loss of the phospho-GRTH species with preservation of the non phospho-form. This mice model will permit to discern the biological and biochemical impact of the phospho species in GRTH function. Prolactin receptor (PRLR) mediates the diverse cellular actions of Prolactin (PRL)including proliferation and differentiation of breast epithelium and lactation. It has an important role in the etiology and progression of breast cancer. hPRLR expression is control at the transcriptional level by the preferentially utilized PIII promoter which lacks an ERE. Complex formation of ERa homodimer (non-DNA bound) with Sp1 and C/EBPb dimers bound to their sites at the PIII promoter is required for basal and estradiol (E2)- induced transcription of the PRLR gene. Exacerbation of PRLs actions resulting from increased receptor number can explain aspects of resistance to E2 inhibitor therapy and invasion in breast cancer. Our recent studies have revealed three distinct regulatory modalities (A,B,C)in breast cancer cells relevant to breast cancer resistance and invasiveness induced A) by endogenous PRL in the absence of E2 B) by EGF in the absence of E2 and PRL and C) by E2 with PRL requirement. All these modalities of PRLR of transcription/expression which participate in the upregulation of the PRLR (A,B,C)found to share commonalities (1,2). These are the central role of ERa phosphorylation via JAK2/PI3K/MAPK/ERK or JAK2/HER pathways induced PRL/PRLR (A), or by CDK7 kinase induced by E2 (C) or by EGF/ERBB1 signalling (B). Another aspect in common is the requisite phosphorylation/ activation of Stat5 (pStat5) by PRL/PRLR/JAK2 (A&C)- (1),and by EGF receptor tyrosine kinase induced by EGF (B)-(2). Also, it is of much relevance the requisite interaction between pERa liganded or unliganded (associated to Sp1 and C/EBPb) with pStat5 bound to a GAS element at the PIII PRLR promoter (A,B,C). This interaction is require for productive stabilization of the ERa-Sp1-C/EBPb complex essential for activation of PRLR promoter and transcription/ expression of the PRLR receptor. In regard to modality (C) we demonstrated that the specific CDK7 kinase inhibitor (THZ1) significantly attenuates E2-induced phosphorylation at S118 and abrogated E2-induced PRLR transcription/expression. Moreover, the E2-induced cell migration was inhibited PRL siRNA and THZ1 indicating its dependence on PRL/PRLR and CDK7 respectively (1). Our findings have provided novel mechanistic avenues for the increase of PRLR in cancer to maximize the actions of endogenous/ exogenous PRL that upon resistance to hormonal therapy could promote progression and metastasis in breast cancer. THZ1 treatment singly or in combination with other inhibitory approaches targeting receptor function and/or the various signaling pathways with impact on ERa and Stat5 phosphorylation could effectively ablate transcription/expression and prevent PRLR fueled by tumor PRL deleterious effect in breast cancer.

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U.S. National Inst/Child Hlth/Human Dev
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Kavarthapu, Raghuveer; Dufau, Maria L (2016) Role of EGF/ERBB1 in the transcriptional regulation of the prolactin receptor independent of estrogen and prolactin in breast cancer cells. Oncotarget :
Chason, Rebecca J; Kang, Jung-Hoon; Gerkowicz, Sabrina A et al. (2015) GnRH agonist reduces estrogen receptor dimerization in GT1-7 cells: evidence for cross-talk between membrane-initiated estrogen and GnRH signaling. Mol Cell Endocrinol 404:67-74
Kavarthapu, Raghuveer; Dufau, Maria L (2015) Germ Cell Nuclear Factor (GCNF/RTR) Regulates Transcription of Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in Testicular Germ Cells--The Androgen Connection. Mol Endocrinol 29:1792-804
Yang, Ruifeng; Tsai-Morris, Chon-Hwa; Kang, Jung Hoon et al. (2015) Elucidation of RNA binding regions of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) to transcripts of a chromatin remodeling protein essential for spermatogenesis. Horm Mol Biol Clin Investig 22:119-30
Kavarthapu, Raghuveer; Tsai Morris, Chon-Hwa; Dufau, Maria L (2014) Prolactin induces up-regulation of its cognate receptor in breast cancer cells via transcriptional activation of its generic promoter by cross-talk between ER? and STAT5. Oncotarget 5:9079-91
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
Tsai-Morris, Chon-Hwa; Sato, Hisashi; Gutti, Ravi et al. (2012) Role of gonadotropin regulated testicular RNA helicase (GRTH/Ddx25) on polysomal associated mRNAs in mouse testis. PLoS One 7:e32470
Villar, Joaquin; Tsai-Morris, Chon-Hwa; Dai, Lisheng et al. (2012) Androgen-induced activation of gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) transcription: essential role of a nonclassical androgen response element half-site. Mol Cell Biol 32:1566-80
Dufau, Maria L; Sato, Hisashi; Gutti, Ravi et al. (2011) Gonadotropin-regulated testicular helicase (GRTH/DDX25): a master post-transcriptional regulator of spermatogenesis. Adv Exp Med Biol 707:23-9

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