The Luteinizing Hormone Receptor (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 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 serve as switch for Sp1 phosphorylation, 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. TFIIB recruitment is dependent on PC4 and have ruled out TFIIB as its direct target. However, TSA induced acetylation of a PC4 interacting proteins, was identified as Acetyl 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 and their participation in the assembly of the PIC and transcriptional activation are under investigation. Immunoprecipitated flag-tagged PC4/H3-complexes in transfected MCF-7 cells revealed Acetyl H3 at various K pulled-down by Flag Ab. To elucidate the physiological impact of PC4 on Sp1 directed transcription in gonads, we generated a PC4-floxed mice by breeding with transgenic mice expressing tissue specific Cyp17Cre. We are now analyzing PC4 null-mice with specific deletion in testicular Leydig cells. 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 to the absence of sperm resulting from failure of round spermatids to elongate. Besides, to its intrinsic helicase activity, it 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. 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-AR in Sertoli cells) where its expression is both cell-and stage specific. It displays negative autocrine control of A production in LCs by preventing overstimulation of the LH-induced A pathway through enhanced degradation of StAR protein. Tg-mice models carrying GRTH 5' flanking regions-GFP provided in vivo systems that permit differential elucidation of regions in the GRTH gene that directs its expression (upstream;-6.4kb/-3.6kb) 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-5270-5252 nt of the GRTH gene was operative selectively in RS. GCNF knock-down in RS preparation from testis of TG mice decreased GFP/GRTH expression upon in vivo treatment of mice with flutamide (Flu)- an AR antagonist. Moreover, Flu treatment of WT mice caused selective reduction of GRTH in RS. The studies provided evidence for actions of A on GCNF cell specific regulation of GRTH expression in germ cells. Also, GRTH associates with GCNF mRNA and its absence causes increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. 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. The study provides valuable links and facilitate what could be a difficult search for gene products in Sertoli cells affecting germ cell function and spermatogenesis (1). Transition protein 2 (Tp2), a chromatin remodeling protein whose mRNA associates with GRTH-required for spermatid elongation, fail to express in GRTH KO with impaired nuclear transport. We have determined Tp2 mRNA and GRTH protein binding regions. Nt 1-47&78-127 downstream of TGA of Tp2 bind conserved GRTH motifs(Ia/V)(3). Prolactin receptor (PRLR)-mediates the diverse cellular actions of Prolactin (PRL). PRL is a major factor in the proliferation and differentiation of breast epithelium and essential for lactation. It has an important role in the etiology and progression of breast cancer, tumoral growth and chemoresistance. hPRLR expression is controlled at the transcriptional level by the preferentially utilized PIII promoter which lacks and 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 E2-induced transcriptional activation of the PRLR gene. PRL in tumoral breast causes cell proliferation via activation of its cognate receptor. Exacerbation of PRLs actions resulting from increased receptor number can explain resistance to E2 inhibitors in breast cancer. Our recent studies demonstrated in MCF7 breast cancer ERa+ HER2+ cells, upregulation of PRLR transcription/expression induced by endogenous/exogenous PRL in absence or estrogen (E2) via the the PRLR with essential participation ERa and JAK2/STAT5, mitogen activated protein kinase (MAPK) and PI3K pathways. This occurs by interaction of phosphorylated ERa (pERa)-generated by PRL/PRLR/JAK2 actions to SP1 and CEBPb and of STA5a,b to a GAS site in PIII. We also found that ERBB2/HER2, which is overexpressed in 10 of breast cancers, phosphorylated and activated by JAK2 via PRL/PRLR induce ERa phosphorylation. Such cross-talk activation of ERBB2/HER2 signalling was identified as an alternate route in the increase of PRLR induced by PRL in breast cancer cells. Paracrine inputs have an active role in breast tumor development, progression and metastasis . Stromal fibroblasts secrete Epidermal growth factor (EGF) activates through its receptor EGFR/ERRB1 present in breast tumor cells (BTC) signaling pathways which in turn trigger requisite transcription factors and co-activators that can affect the proliferation of BTC. We have shown in MCF7 cells marked activation of PRLR gene transcription/expression by exogenous EGF independent of PRL/PRLR/JAK2 with the essential involvement of the MPK; ERK1/2 and PI3K-AKT pathways. These are mediated by EGFR Y1068,1086 and the c-SRC dependent EGFR Y845 for ERa and STAT5b phosphorylation, respectively -for their recruitment to the PRLR promoter. Aside from its independence in E2 and the activators requirements (PRL vs EGF ) there are important commonalities (requisite ERa,STAT5)in the mechanism of PRLR transcription /expression. Also,the studies revealed STAT5 interaction with ERa as essential for PRLR as essential for PRLR up-regulation. Our findings provide mechanistic avenues for the increase of PRLR that upon resistance to hormonal therapy could promote progression and metastasis in breast cancer.
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