miRNA Expression Profiles of Mouse Round Spermatids in GRTH/DDX25-Mediated Spermiogenesis: mRNA-miRNA Network Analysis.

GRTH/DDX25 is a testis-specific DEAD-box family of RNA helicase, which plays an essential role in spermatogenesis and male fertility. There are two forms of GRTH, a 56 kDa non-phosphorylated form and a 61 kDa phosphorylated form (pGRTH). GRTH-KO and GRTH Knock-In (KI) mice with R242H mutation (lack pGRTH) are sterile with a spermatogenic arrest at step 8 of spermiogenesis due to failure of round spermatids (RS) to elongate. We performed mRNA-seq and miRNA-seq analysis on RS of WT, KI, and KO to identify crucial microRNAs (miRNAs) and mRNAs during RS development by establishing a miRNA-mRNA network. We identified increased levels of miRNAs such as miR146, miR122a, miR26a, miR27a, miR150, miR196a, and miR328 that are relevant to spermatogenesis. mRNA-miRNA target analysis on these DE-miRNAs and DE-mRNAs revealed miRNA target genes involved in ubiquitination process (Ube2k, Rnf138, Spata3), RS differentiation, and chromatin remodeling/compaction (Tnp1/2, Prm1/2/3, Tssk3/6), reversible protein phosphorylation (Pim1, Hipk1, Csnk1g2, Prkcq, Ppp2r5a), and acrosome stability (Pdzd8). Post-transcriptional and translational regulation of some of these germ-cell-specific mRNAs by miRNA-regulated translation arrest and/or decay may lead to spermatogenic arrest in KO and KI mice. Our studies demonstrate the importance of pGRTH in the chromatin compaction and remodeling process, which mediates the differentiation of RS into elongated spermatids through miRNA-mRNA interactions.

Single-Cell Transcriptomic Profiling of the Mouse Testicular Germ Cells Reveals Important Role of Phosphorylated GRTH/DDX25 in Round Spermatid Differentiation and Acrosome Biogenesis during Spermiogenesis.

Gonadotropin-regulated testicular RNA helicase (GRTH)/DDX25 is a member of DEAD-box family of RNA helicase essential for the completion of spermatogenesis and male fertility, as evident from GRTH-knockout (KO) mice. In germ cells of male mice, there are two species of GRTH, a 56 kDa non-phosphorylated form and 61 kDa phosphorylated form (pGRTH). GRTH Knock-In (KI) mice with R242H mutation abolished pGRTH and its absence leads to infertility. To understand the role of the GRTH in germ cell development at different stages during spermatogenesis, we performed single-cell RNA-seq analysis of testicular cells from adult WT, KO and KI mice and studied the dynamic changes in gene expression. Pseudotime analysis revealed a continuous developmental trajectory of germ cells from spermatogonia to elongated spermatids in WT mice, while in both KO and KI mice the trajectory was halted at round spermatid stage indicating incomplete spermatogenesis process. The transcriptional profiles of KO and KI mice were significantly altered during round spermatid development. Genes involved in spermatid differentiation, translation process and acrosome vesicle formation were significantly downregulated in the round spermatids of KO and KI mice. Ultrastructure of round spermatids of KO and KI mice revealed several abnormalities in acrosome formation that includes failure of pro-acrosome vesicles to fuse to form a single acrosome vesicle, and fragmentation of acrosome structure. Our findings highlight the crucial role of pGRTH in differentiation of round spermatids into elongated spermatids, acrosome biogenesis and its structural integrity.

Prolactin receptor gene transcriptional control, regulatory modalities relevant to breast cancer resistance and invasiveness.

The prolactin receptor (PRLR) is a member of the lactogen/cytokine receptor family, which mediates multiple actions of prolactin (PRL). PRL is a major hormone in the proliferation/differentiation of breast epithelium that is essential for lactation. It is also involved in breast cancer development, tumor growth and chemoresistance. Human PRLR expression is controlled at the transcriptional level by multiple promoters. Each promoter directs transcription/expression of a specific non-coding exon 1, a common non-coding exon 2 and coding exons E3-11. The identification of exon 11 of PRLR led to finding of alternative spliced products and two novel short forms (SF) that can inhibit the long form (LF) of PRLR activity with relevance in physiological regulation and breast cancer. Homo and heterodimers of LF and SF are formed in the absence of PRL that acts as a conformational modifier. Heterodimerization of SF with LF is a major mechanism through which SF inhibits some signaling pathways originating at the LF. Biochemical/molecular modeling approaches demonstrated that the human PRLR conformation stabilized by extracellular intramolecular S-S bonds and several amino acids in the extracellular D1 domain of PRLR SF are required for its inhibitory actions on PRLR LF-mediated functions. Studies in breast cancer cells demonstrated that the transcription of PRLR was directed by the preferentially utilized PIII promoter, which lacks an estrogen responsive element. Complex formation of non-DNA bound ERα dimer with Sp1 and C/EBPß dimers bound to their sites at the PRLR promoter is required for basal activity. Estradiol induces transcriptional activation/expression of the PRLR gene, and subsequent studies revealed the essential role of autocrine PRL released by breast cancer cells and CDK7 in estradiol-induced PRLR promoter activation and upregulation. Other studies revealed stimulation of the PRLR promoter activity and PRLR LF protein by PRL in the absence of estrogen via the STAT5/phospho-ERα activation loop. Additionally, EGF/ERBB1 can induce the transcription of PRLR independent of estrogen and prolactin. The various regulatory modalities contributing to the upregulation of PRLR provide options for the development of therapeutic approaches to mitigate its participation in breast cancer progression and resistance.

Chromatoid Bodies in the Regulation of Spermatogenesis: Novel Role of GRTH.

Post-transcriptional and translational control of specialized genes play a critical role in the progression of spermatogenesis. During the early stages, mRNAs are actively transcribed and stored, temporarily bound to RNA binding proteins in chromatoid bodies (CBs). CBs are membrane-less dynamic organelles which serve as storehouses and processing centers of mRNAs awaiting translation during later stages of spermatogenesis. These CBs can also regulate the stability of mRNAs to secure the correct timing of protein expression at different stages of sperm formation. Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is an essential regulator of spermatogenesis. GRTH transports mRNAs from the nucleus to the cytoplasm and phospho-GRTH transports mRNAs from the cytoplasm to the CBs. During spermiogenesis, there is precise control of mRNAs transported by GRTH from and to the CBs, directing the timing of translation of critical proteins which are involved in spermatid elongation and acrosomal development, resulting in functional sperm formation. This chapter presents our current knowledge on the role of GRTH, phospho-GRTH and CBs in the control of spermiogenesis. In addition, it covers the components of CBs compared to those of stress granules and P-bodies.

Blockade of GRTH/DDX25 Phosphorylation by Cyclic Peptides Provides an Avenue for Developing a Nonhormonal Male Contraceptive.

Gonadotropin-regulated testicular RNA helicase (GRTH)/DDX25 is a DEAD-box RNA helicase essential for the completion of spermatogenesis. Our previous studies indicated that blocking the GRTH phospho-site or perturbing the GRTH/protein kinase A (PKA) interface could provide an avenue for developing a nonhormonal male contraceptive. In this study, cyclic peptides were rationally designed and synthesized as promising therapeutic agents. The peptides showed effective delivery into COS-1 and germ cells and a dose-dependent inhibitory effect on GRTH phosphorylation. The peptides inhibit GRTH phosphorylation in the presence of PKA, and binding to the helicase resulted in thermal stabilization of non-phospho GRTH. Increased efficiency in fluorescence resonance energy transfer (FRET) assay revealed their interaction with GRTH. Cyclic peptide exposure of cultures from mice seminiferous tubules resulted in significant inhibition of phospho GRTH. These peptides did not exhibit toxicity. Effective delivery and targeted decrease of in vitro expression of phospho GRTH by cyclic peptides provide a promising angle to develop effective compounds as a nonhormonal male contraceptive.

Crosstalk between PRLR and EGFR/HER2 Signaling Pathways in Breast Cancer.

Prolactin receptor (PRLR) and epidermal growth factor receptor (EGFR/ERBB) signaling pathways activated by prolactin (PRL) and epidermal growth factor (EGF), have a major role in the mammary gland development and in the etiology of breast cancer, respectively. ER+ breast tumors comprise up to 75% of all breast cancers and 10% of these are HER2+. Elevated levels of PRLR in breast tumors, high circulating levels of PRL and increased expression of ERBB1/2 in patients that become resistant to endocrine therapy have shown to be associated with higher risk of cancer progression. In this review, we examine the role of crosstalk between PRLR and ERBB1/2 signaling pathways in the activation of unliganded ERα, cyclin-D1 and other oncogenic factors (MYC, FOS, JUN) in breast cancer. PRL/PRLR and EGF/EGFR induces phosphorylation of ERα through activation of MEK/MAPK and PI3K/AKT signaling pathways. PRL in breast cancer cells via PRLR/JAK2 can also induce phosphorylation of ERBB2/HER2, which in turn activates the downstream RAS/MEK/ERK pathway required for ERα phosphorylation. EGFR, independent of PRL/PRLR, can activate STAT5 indirectly via c-SRC and drive the expression of target genes involved in cell proliferation and survival. The crosstalk between PRLR and HER2, where PRL induces HER2 signaling can be an alternative route for ERα activation to induce transcription of PRLR and other ER target genes. We believe that overexpression of EGFR/HER2 and PRLR in breast tumors could maximize the actions of their ligands, and further induce cell proliferation promoting malignancy. This could also explain the resistance to endocrine therapy resulting in tumor growth.

Linking Phospho-Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) to Histone Ubiquitination and Acetylation Essential for Spermatid Development During Spermiogenesis.

GRTH/DDX25 is a testicular RNA helicase expressed in germ cells that plays a crucial role in completion of spermatogenesis. Previously, we demonstrated a missense mutation (R242H) of GRTH gene in Japanese infertile patients (5.8%) with non-obstructive azoospermia. This mutation upon expression in COS-1 cells revealed absence of the 61 kDa phosphorylated GRTH in cytoplasm and the presence of the 56 kDa non-phosphorylated GRTH in the nucleus. GRTH knock-in (KI) mice carrying the human GRTH (R242H) mutation, lack phosphorylated GRTH, and sperm due to failure of round spermatid elongation during spermiogenesis. To determine the impact of phosphorylated GRTH on molecular events/pathways participating in spermatid development during spermiogenesis, we analyzed transcriptome profiles obtained from RNA-Seq of germ cells from KI and WT mice. RNA-Seq analysis of 2624 differentially expressed genes revealed 1404 down-regulated and 1220 up-regulated genes in KI mice. Genes relevant to spermatogenesis, spermatid development and spermatid differentiation were significantly down-regulated. KEGG enrichment analysis showed genes related to ubiquitin-mediated proteolysis and protein processing in endoplasmic reticulum pathway genes were significantly down-regulated while the up-regulated genes were found to be involved in Focal adhesion and ECM-receptor interaction pathways. Real-Time PCR analysis confirmed considerable reduction in transcripts of ubiquitination related genes Ube2j1, Ube2k, Ube2w, Rnf8, Rnf133, Rnf138, Cul3 and increased expression of Ccnd2, Col1a, Lamb1, Cav1, Igf1, Itga9 mRNA's in KI mice compared to WT. Also, marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase), their modified Histone substrates (H2AUb, H2BUb) and H4Ac, H4K16Ac were observed in KI mice. GRTH-IP mRNA binding studies revealed that Rnf8 and Ube2J1 mRNAs from WT mice associated with GRTH protein and the binding is greatly impaired in the KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice. Absence of phosphorylated GRTH impairs UBE2J1, RNF8 and MOF-dependent histone ubiquitination and acetylation essential for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis.

Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific mRNAs in Chromatoid Bodies During Spermatogenesis.

GRTH/DDX25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H) show loss of the phospho-species from cytoplasm with preservation of the non-phospho form in the cytoplasm and nucleus. GRTH KI mice are sterile and lack elongated spermatids and spermatozoa, with spermatogenic arrest at step 8 of round spermatids which contain chromatoid body (CB) markedly reduced in size. We observed an absence of phospho-GRTH in CB of GRTH KI mice. RNA-Seq analysis of mRNA isolated from CB revealed that 1,421 genes show differential abundance, of which 947 genes showed a decrease in abundance and 474 genes showed an increase in abundance in GRTH KI mice. The transcripts related to spermatid development, differentiation, and chromatin remodeling (Tnp1/2, Prm1/2/3, Spem1/2, Tssk 2/3/6, Grth, tAce, and Upf2) were reduced, and the transcripts encoding for factors involved in RNA transport, regulation, and surveillance and transcriptional and translational regulation (Eef1a1, Ppp1cc, Pabpc1, Ybx3, Tent5b, H2al1m, Dctn2, and Dync1h1) were increased in the CB of KI mice and were further validated by qPCR. In the round spermatids of wild-type mice, mRNAs of Tnp2, Prm2, and Grth were abundantly co-localized with MVH protein in the CB, while in GRTH KI mice these were minimally present. In addition, GRTH binding to Tnp1/2, Prm1/2, Grth, and Tssk6 mRNAs was found to be markedly decreased in KI. These results demonstrate the importance of phospho-GRTH in the maintenance of the structure of CB and its role in the storage and stability of germ cell-specific mRNAs during spermiogenesis.

Corrigendum: Gonadotropin Regulated Testicular RNA Helicase, Two Decades of Studies on Its Structure Function and Regulation From Its Discovery Opens a Window for Development of a Non-hormonal Oral Male Contraceptive.

[This corrects the article DOI: 10.3389/fendo.2019.00576.].

Gonadotropin Regulation Testicular RNA Helicase, Two Decades of Studies on Its Structure Function and Regulation From Its Discovery Opens a Window for Development of a Non-hormonal Oral Male Contraceptive.

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25) is member of the DEAD-box family of RNA helicases present in Leydig and germ cells. GRTH is the only family member regulated by hormones, luteinizing hormone, through androgen action. Male mice with knock-out of the GRTH gene are sterile, lack sperm with arrest at round spermatids. GRTH participates on the nuclear export and transport of specific mRNAs, the structural integrity of Chromatoid Bodies of round spermatids, where mRNAs are processed and stored, and in their transit to polyribosomes, where it may regulate translation of relevant genes. GRTH has a central role in the control of germ cell apoptosis and acts as negative regulator of miRNAs which regulate expression of genes involved in the progress of spermatogenesis. In Leydig cells, GRTH gene transcription is regulated by LH via autocrine actions of androgen/androgen receptor and has regulatory effects in steroidogenesis. In germ cells, androgen actions are indirect via receptors in Sertoli cells. Transgenic mice carrying GRTH 5' flanking region-GFP permitted to discern regions in the gene which directs its expression upstream, in germ cells, and downstream in Leydig cells, and the androgen-regulated transcription at interstitial (autocrine), and germ cell (paracrine) compartments. Further evidence for paracrine actions of androgen/androgen receptor is their transcriptional induction of Germ Cell Nuclear Factor as requisite up-regulator of GRTH gene transcription in round spermatids, linking androgen action to two relevant germ cell genes essential for the progress of spermatogenesis. A missense mutation of R to H at amino acid 242 of GRTH found in 5.8% of a patient population with azoospermia causes loss of the cytoplasmic phospho-GRTH species with preservation of the non-phospho form in transfected cells. Mice with knock-in of the human mutation, lack sperm due to arrest at round spermatids. This model permits to discern the function of phospho-GRTH. The GRTH phospho-site resides at a Threonine structurally adjacent to the mutant site found in patients. Molecular modeling of this site elucidated the amino acids that form the GRTH/PKA interphase and provide the basis for drug design for use as male contraceptive.

Characterization of the Phosphorylation Site of GRTH/DDX25 and Protein Kinase A Binding Interface Provides Structural Basis for the Design of a Non-Hormonal Male Contraceptive.

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8% of Japanese patient population with azoospermia. Transfection of the mutant GRTH construct in COS-1 cells leads to loss of the 61 kDa cytoplasmic phospho-species. Mice with knock-in of the human GRTH mutation are sterile and lack sperm with normal androgen and mating behavior. These findings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specific phospho-antibody, we have identified T239, structurally adjacent to the patient's mutant site as the GRTH phospho-site. Molecular modelling provided structural basis for the role of R242 and other critical solvent-exposed residues at the GRTH/PKA interface (E165/K240/D237), on the control of GRTH phosphorylation at T239. Single or double mutations of these residues caused marked reduction or abolition of the phospho-form. These effects can be ascribed to critical disruptions of intramolecular H-bonds at the GRTH/PKA interface, which leads to modest but consequential structural changes that can affect PKA catalytic efficiency. Inhibition of phosphorylation may be achieved by small, drug-like molecules that bind to GRTH and reconfigure the GRTH/PKA interface.

Targeted knock-in mice with a human mutation in GRTH/DDX25 reveals the essential role of phosphorylated GRTH in spermatid development during spermatogenesis.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis specific member of the DEAD-box family of RNA helicases expressed in meiotic and haploid germ cells which plays an essential role in spermatogenesis. There are two species of GRTH the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in azoospermic men that when expressed in COS1-cells lack the phospho-form of GRTH. To investigate the role of the phospho-GRTH species in spermatogenesis, we generated a GRTH knock-in (KI) transgenic mice with the R242H mutation. GRTH-KI mice are sterile with reduced testis size, lack sperm with spermatogenic arrest at round spermatid stage and loss of the cytoplasmic phospho-GRTH species. Electron microscopy studies revealed reduction in the size of chromatoid bodies (CB) of round spermatids (RS) and germ cell apoptosis. We observed absence of phospho-GRTH in the CB of RS. Complete loss of chromatin remodeling and related proteins such as TP2, PRM2, TSSK6 and marked reduction of their respective mRNAs and half-lives were observed in GRTH-KI mice. We showed that phospho-GRTH has a role in TP2 translation and revealed its occurrence in a 3' UTR dependent manner. These findings demonstrate the relevance of phospho-GRTH in the structure of the chromatoid body, spermatid development and completion of spermatogenesis and provide an avenue for the development of a male contraceptive.

A versatile mouse model of epitope-tagged histone H3.3 to study epigenome dynamics.

The variant histone H3.3 is incorporated into the genome in a transcription-dependent manner. This histone is thus thought to play a role in epigenetic regulation. However, our understanding of how H3.3 controls gene expression and epigenome landscape has remained incomplete. This is partly because precise localization of H3.3 in the genome has been difficult to decipher particularly for cells in vivo To circumvent this difficulty, we generated knockin mice, by homologous recombination, to replace both of the two H3.3 loci (H3f3a and H3f3b) with the hemagglutinin-tagged H3.3 cDNA cassette, which also contained a GFP gene. We show here that the hemagglutinin-tagged H3.3 and GFP are expressed in the majority of cells in all adult tissues tested. ChIP-seq data, combined with RNA-seq, revealed a striking correlation between the level of transcripts and that of H3.3 accumulation in expressed genes. Finally, we demonstrate that H3.3 deposition is markedly enhanced upon stimulation by interferon on interferon-stimulated genes, highlighting transcription-coupled H3.3 dynamics. Together, these H3.3 knockin mice serve as a useful experimental model to study epigenome regulation in development and in various adult cells in vivo.

Interaction of positive coactivator 4 with histone 3.3 protein is essential for transcriptional activation of the luteinizing hormone receptor gene.

The luteinizing hormone receptor (LHR) is essential for sexual development and reproduction in mammals. We have established that Sp1 has a central role in derepression of LHR gene transcription induced by Trichostatin A (TSA) in MCF7 cells. Moreover, the co-activator PC4 which associates directly with Sp1 at the LHR promoter is essential for TSA-mediated LHR transcription. This study explores interactions of PC4 with histone proteins, which presumably triggers chromatin modifications during LHR transcriptional activation. TSA treatment of MCF7 cells expressing PC4-Flag protein induces acetylation of histone 3 (H3) and immunoprecipitation (IP) studies revealed its interaction with PC4-Flag protein. MS/MS analysis of the protein complex obtained after IP from TSA treated samples detected H3.3 acetylated at K9, K14, K18, K23 and K27 as a PC4 interacting protein. The association of PC4 with H3.3 was corroborated by IP and re-ChIP using H3.3 antibody. Similarly, IP and re-ChIP showed association of PC4 with H3 acetylated protein. Knockdown of PC4 in MCF7 cells reduced H3.3 enrichment, H3 acetylation at the Lys sites and LHR promoter activity in TSA treated cells despite an increase in H3 and H3.3 protein induced by TSA, linking PC4 to H3 acetylation and LHR transcription. Depletion of H3.3 A/B in MCF7 cells impair chromatin accessibility and enrichment of Pol II and TFIIB at the LHR promoter and its activation, resulting in marked reduction of LHR gene expression. Together, these findings point to the critical role of PC4 and its association with acetylated H3.3 in TSA-induced LHR gene transcription.

Essential role of endogenous prolactin and CDK7 in estrogen-induced upregulation of the prolactin receptor in breast cancer cells.

Our early studies have shown that Estradiol (E2)/Estrogen Receptor α (ER) in a non-DNA dependent manner through complex formation with C/EBPß/SP1 induced transcriptional activation of the generic hPIII promoter and expression of the Prolactin Receptor (PRLR) receptor in MCF-7 cells. Subsequent studies demonstrated effects of unliganded ERα with requisite participation of endogenous PRL on the activation of PRLR transcription. Also, EGF/ERBB1 in the absence of PRL and E2 effectively induced upregulation of the PRLR. In this study we have delineated the transcriptional mechanism of upregulation of PRLR receptor induced by E2 incorporating knowledge of the various transcriptional upregulation modalities from our previous studies. Here, we demonstrate an essential requirement of STAT5a induced by PRL via PRLR receptor which associates at the promoter and its interaction with phoshoERα S118. Knock-down of PRL by siRNA significantly reduced E2-induced PRLR promoter activity, mRNA and protein expression, recruitment of ERα to the complex at promoter, C/EBPß association to its DNA site and productive complex formation at hPIII promoter. The specific CDK7 inhibitor (THZ1) that attenuates E2-induced ERα phosphorylation at S118 abrogated E2-induced PRLR promoter activation. Further studies demonstrated that E2 induced cell migration was inhibited by PRL siRNA and THZ1 indicating its dependence on PRL/PRLR and CDK7, respectively. Our studies have demonstrated the essential role of endogenous PRL and CDK7 in the upregulation of PRLR by E2 and provide insights for therapeutic approaches that will mitigate the transcription/expression of PRLR and its participation in breast cancer progression fueled by E2 and PRL via their cognate receptors.

Role of EGF/ERBB1 in the transcriptional regulation of the prolactin receptor independent of estrogen and prolactin in breast cancer cells.

Prolactin receptor (PRLR) and epidermal growth factor receptor (EGFR/ERBB1) have important roles in the physiology of the human breast and in the etiology and progression of breast cancer. Our present studies in MCF-7 cells revealed that EGF induces up-regulation of PRLR via activation of EGFR signalling pathways leading to activation of estrogen receptor α (ERα). EGF treatment of MCF-7 cells cultured in absence of estradiol induced expression of PRLR that was consistent with the activation of PRLR generic promoter (hPIII). These were abolished by ERα antagonist and siRNA, indicating involvement of ERα in EGF-induced hPIII promoter activity. MEK/MAPK and PI3K/AKT pathways participate in the phosphorylation of ERα induced by EGF/EGFR. PI3K and MEK inhibitors abolished EGF-induced PRLR promoter activity. Increased recruitment of non-DNA bound unliganded ERα to Sp1 and C/EBPß bound to their sites at hPIII induced by EGF was abrogated by ERα siRNA demonstrating the requisite role of phospho-ERα in PRLR upregulation. EGF/EGFR, independent of endogenous prolactin induced phosphorylation of STAT5b with participation of c-SRC and recruitment of STAT5b:STAT5b to a GAS site at hPIII. STAT5b interaction with ERα was essential for stable phospho-ERα recruitment to the SP1/CEBPß complex. These studies indicate a role for paracrine EGF via EGFR independent of estrogen and prolactin in the transcriptional activation of PRLR gene expression and its contribution to high levels of PRLRs in breast cancer. These by maximizing the actions of endogenous prolactin could have a role in cancer progression and resistance to endocrine therapy.

Germ Cell Nuclear Factor (GCNF/RTR) Regulates Transcription of Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in Testicular Germ Cells--The Androgen Connection.

Gonadotropin-regulated testicular RNA helicase (GRTH) (GRTH/DDX25), is a testis-specific protein essential for completion of spermatogenesis. Transgenic mice carrying 5'-flanking regions of the GRTH gene/green fluorescence protein (GFP) reporter revealed a region (-6.4/-3.6 kb) which directs its expression in germ cells (GCs) via androgen action. This study identifies a functional cis-binding element on the GRTH gene for GC nuclear factor (GCNF) (GCNF/RTR) required to regulate GRTH gene expression in postmeiotic testis GCs and explore the action of androgen on GCNF and GRTH transcription/expression. GCNF expression decreased in mice testis upon flutamide (androgen receptor antagonist) treatment, indicating the presence of an androgen/GCNF network to direct GRTH expression in GC. Binding studies and chromatin immunoprecipitation demonstrated specific association of GCNF to a consensus half-site (-5270/-5252) of the GRTH gene in both round spermatids and spermatocytes, which was abolished by flutamide treatment in round spermatids. Moreover, flutamide treatment of wild-type mice caused selective reduction of GCNF and GRTH in round spermatids. GCNF knock-down in seminiferous tubules from GRTH-transgenic mice (dark zone, round spermatid rich) caused decreased GFP expression. Exposure of tubules to flutamide caused decrease in GCNF and GFP expression, whereas androgen exposure induced significant increase. Our studies provide evidence for actions of androgen on GCNF cell-specific regulation of GRTH expression in GC. GRTH associates with GCNF mRNA, its absence caused increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. These in vivo/in vitro models link androgen actions to GC through GCNF, as regulated transfactor that controls transcription/expression of GRTH.

Elucidation of RNA binding regions of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) to transcripts of a chromatin remodeling protein essential for spermatogenesis.

BACKGROUND: Gonadotropin-regulated testicular RNA helicase (GRTH) is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig and germ cells. It is a transport protein of mRNAs from nucleus to cytoplasmic sites and is essential for posttranscriptional regulation and completion of spermatogenesis. Transition protein 2 (Tp2), which associates with GRTH and is required for spermatid elongation, failed to express in GRTH null mice with impaired mRNA nuclear export. The present study determines GRTH binding motifs/regions that associate with Tp2 mRNA transcripts. MATERIALS AND METHODS: RNA-protein interaction was analyzed using biotin-labeled electrophoretic mobility gel shift assays (EMSA). 3'-biotin-labeled RNA (Tp2) was incubated with mGRTH protein (full length/sequential deletion of specific and conserved RNA helicase motifs of GRTH) expressed from in vitro TNT coupled reticulocyte lysate system. Binding specificity was further elucidated by mutagenesis and antibody supershift analysis. RESULTS: RNA-EMSA revealed that the 3' UTR of Tp2 RNA (127 nt from TGA) was retarded in presence of full length GRTH. Nucleotide sequences downstream of TGA of the Tp2 transcript (1-47 and 78-127 nt) are important for binding to GRTH. Sequential deletions/point mutations in GRTH revealed region(s) of conserved binding motifs of RNA helicases (Ia and V) essential for GRTH binding to Tp2 mRNA. CONCLUSIONS: Our studies provide insights into the association of Tp2 expression via binding to the conserved RNA binding motifs of GRTH protein and the basis for understanding GRTH in the regulation of the genes essential for germ cell elongation and completion of spermatogenesis.

GnRH agonist reduces estrogen receptor dimerization in GT1-7 cells: evidence for cross-talk between membrane-initiated estrogen and GnRH signaling.

17ß-estradiol (E2), a key participant on the initiation of the LH surge, exerts both positive and negative feedback on GnRH neurons. We sought to investigate potential interactions between estrogen receptors alpha (ERα) and beta (ERß) and gonadotropin releasing hormone receptor (GnRH-R) in GT1-7 cells. Radioligand binding studies demonstrated a significant decrease in saturation E2 binding in cells treated with GnRH agonist. Conversely, there was a significant reduction in GnRH binding in GT1-7 cells treated with E2. In BRET(1) experiments, ERα-ERα dimerization was suppressed in GT1-7 cells treated with GnRH agonist (p < 0.05). There was no evidence of direct interaction between ERs and GnRH-R. This study provides the first evidence of reduced ERα homodimerization by GnRH agonist. Collectively, these findings demonstrate significant cross-talk between membrane-initiated GnRH and E2 signaling in GT1-7 cells.

Prolactin induces up-regulation of its cognate receptor in breast cancer cells via transcriptional activation of its generic promoter by cross-talk between ERα and STAT5.

Prolactin (PRL) serves a critical role in breast cancer progression via activation of its cognate receptor. These studies reveal up-regulation of PRLR gene expression by PRL in absence of estradiol in MCF-7 and T47D breast cancer cells. PRL/PRLR via activation of STAT5 that binds a GAS-element in the PRLR gene and the participation of ERα stimulates PRLR transcription/expression. PRL/PRLR induces phosphorylation of ERα through the JAK2/PI3K/MAPK/ERK and JAK2/HER2 activated pathways. The increased recruitment of phospho-ERα, induced by PRL to Sp1 and C/EBPß at PRLR promoter sites is essential for PRL-induced PRLR transcription. This recruitment is prevented by blockade of PRL expression using RNA interference or ERα phosphorylation using specific inhibitors of PI3K and ERK. Direct evidence is provided for local actions of PRL, independent of estradiol, in the up-regulation of PRLR transcription/expression by an activation-loop between STAT5 and the phospho-ERα/Sp1/C/EBPß complex with requisite participation of signaling mechanisms. PRL's central role in the up-regulation of PRLR maximizes the action of the endogenous hormone. This study offers mechanistically rational basis for invasiveness fueled by prolactin in refractory states to adjuvant therapies in breast cancer.