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.

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.

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.

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.

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.

Complex formation and interactions between transcription factors essential for human prolactin receptor gene transcription.

The protein association of estrogen receptor α ERα with DNA-bound SP1 and C/EBPß is essential for the 17ß-estradiol (E2)-induced activation of human prolactin receptor (hPRLR) gene transcription. Protein-protein interaction and complex formation at the hPIII promoter of hPRLR was investigated. The basic region and leucine zipper (bZIP) of C/EBPß, zinc finger (ZF) motifs of SP1, and the DNA binding domain of ERα were identified as regions responsible for the interactions between transfactors. The E2-induced interaction was confirmed by bioluminescence resonance energy transfer (BRET) assays of live cells. The combination of BRET/bimolecular luminescence complementation assay revealed that ERα exists as a constitutive homodimer, and E2 induced a change(s) in ERα homodimer conformation favorable for its association with C/EBPß and SP1. Chromatin immunoprecipitation and small interfering RNA knockdown of members of the complex in breast cancer cells demonstrated the endogenous recruitment of components of the complex onto the hPIII promoter of the hPRLR gene. SP1 is the preferred transfactor for the recruitment of ERα to the complex that facilitates the C/EBPß association. The E2/ERα-induced hPRLR transcription was demonstrated in ERα-negative breast cancer cells. This study indicates that the enhanced complex formation of ERα dimer with SP1 and C/EBPß by E2 has an essential role in the transcriptional activation of the hPRLR gene.

Coactivator function of positive cofactor 4 (PC4) in Sp1-directed luteinizing hormone receptor (LHR) gene transcription.

The LHR has an essential role in sexual development and reproductive function, and its transcription is subjected to several modes of regulation. In this study, we investigated PC4 coactivator function in the control of LHR transcription. Knockdown of PC4 by siRNA inhibited the LHR basal promoter activity and trichostatin A (TSA)-induced gene transcriptional activation and expression in MCF-7 cells. While overexpression of PC4 alone had no effect on the LHR gene, it significantly enhanced Sp1- but not Sp3-mediated LHR transcriptional activity. PC4 directly interacts with Sp1 at the LHR promoter, and this interaction is negatively regulated by PC4 phosphorylation. The coactivator domain (22-91 aa) of PC4 and DNA binding domain of Sp1 are essential for PC4/Sp1 interaction. ChIP assay revealed significant occupancy of PC4 at the LHR promoter that increased upon TSA treatment. Disruption of PC4 expression significantly reduced TSA-induced recruitment of TFIIB and RNAP II, at the promoter. PC4 functions are beyond TSA-induced phosphatase release, PI3K-mediated Sp1 phosphorylation, and HDAC1/2/mSin3A co-repressor release indicating its role as linker coactivator of Sp1 and the transcriptional machinery. These findings demonstrated a critical aspect of LHR modulation whereby PC4 acts as a coactivator of Sp1 to contribute to the human of LHR transcription.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25): a multifunctional protein essential for spermatogenesis.

Male germ cell maturation is governed by the expression of specific protein(s) in a precise temporal sequence during development. Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a member of the Glu-Asp-Ala-Glu (DEAD)-box protein family, is a testis-specific gonadotropin/androgen-regulated RNA helicase that is present in germ cells (meiotic spermatocytes and round spermatids) and Leydig cells. GRTH is essential for completion of spermatogenesis as a posttranscriptional regulator of relevant genes during germ cell development. Male mice lacking GRTH are sterile with spermatogenic arrest due to failure of round spermatids to elongate, where striking structural changes and reduction in size of chromatoid bodies are observed. GRTH also plays a central role in preventing germ cell apoptosis. In addition to its inherent helicase unwinding/adenosine triphosphatase activities, GRTH binds to specific mRNAs as an integral component of ribonuclear protein particles. As a shuttle protein, GRTH transports target mRNAs from nucleus to the cytoplasm for storage in chromatoid bodies of spermatids, where they await translation during spermatogenesis. GRTH is also associated with polyribosomes to regulate target gene translation. The finding of a missense mutation associated with male infertility, where its expression associates with loss of GRTH phosphorylation, supports the relevance of GRTH to human germ cell development. We conclude that the mammalian GRTH/DDX25 is a multifunctional RNA helicase that is an essential regulator of spermatogenesis and is highly relevant for studies of male infertility and contraception.

Unlocking repression of the human luteinizing hormone receptor gene by trichostatin A-induced cell-specific phosphatase release.

Our previous studies demonstrated that the histone deacetylase inhibitor, trichostatin A (TSA), induces derepression of the human luteinizing hormone receptor (LHR) gene by de-recruitment of the pRB homologue p107 repressor from the promoter in JAR and MCF-7 cancer cells. TSA initiates a mechanism whereby the phosphatidylinositol 3-kinase/protein kinase zeta (PKCzeta) cascade phosphorylates Sp1 at Ser-641, which is essential for the release of the repression of LHR transcription. The present studies have revealed that dissociation of serine/threonine protein phosphatases PP2A and PP1 from the LHR promoter mediates TSA-induced activation of LHR gene transcription in a cell-specific manner. Changes in chromatin structure induced by TSA cause the release of PP2A in JAR cells or of PP1 in MCF-7 cells, which is associated with Sp1 directly or through histone deacetylase 1/2, respectively, at the promoter. This favors the phosphorylation of Sp1 mediated by the phosphatidylinositol 3-kinase/PKCzeta pathway, which in turn causes the release of the p107 inhibitor from Sp1 and marked transcriptional activation of the LHR. These findings reveal the importance of phosphatases in the control of LHR transcription, where the balance between phosphatidylinositol 3-kinase/PKCzeta and phosphatases could be critical for up- and down-regulation of LHR gene expression in physiological and pathological settings.

Gonadotropin-regulated testicular helicase (DDX25), an essential regulator of spermatogenesis, prevents testicular germ cell apoptosis.

Gonadotropin-regulated testicular helicase (GRTH)/DDX25 is an essential post-transcriptional regulator of spermatogenesis. In GRTH null mice severe apoptosis was observed in spermatocytes entering the metaphase of meiosis. Pro- and anti-apoptotic factors were found to be under GRTH regulation in comparative studies of spermatocytes from wild type and GRTH(-/-) knock-out (KO) mice. KO mice displayed decreased levels of Bcl-2 and Bcl-xL (anti-apoptotic factors), an increase in Bid, Bak, and Bad (pro-apoptotic), reduced phospho-Bad, and release of cytochrome c. Also, an increase on Smac, a competitor of inhibitor apoptotic proteins that release caspases, was observed. These changes caused an increase in cleavage of caspases 9 and 3, activation of caspase 3 and increases in cleavage products of PARP. The half-life of caspase 3 transcripts was markedly increased in KO, indicating that GRTH had a negative role on its mRNA stability. IkappaBalpha, which sequesters NF-kappaB from its transcriptional activation of pro-apoptotic genes, was highly elevated in KO, and its phospho-form, which promotes its dissociation, was reduced. The increase of HDAC1 and abolition of p300 expression in KO indicated a nuclear action of GRTH on the NF-kappaB-mediated transcription of anti-apoptotic genes. It also regulates the associated death domain pathway and caspase 8-mediated events. GRTH-mediated apoptotic regulation was further indicated by its selective binding to pro- and anti-apoptotic mRNAs. These studies have demonstrated that GRTH, as a component of mRNP particles, acts as a negative regulator of the tumor necrosis factor receptor 1 and caspase pathways and promotes NF-kappaB function to control apoptosis in spermatocytes of adult mice.

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.

LH receptor (LHR) gene transcription is subject to repression/derepression through various modes and multiple effectors. Epigenetic silencing and activation of the LHR is achieved through coordinated regulation at both histone and DNA levels. The LHR gene is subject to repression by deacetylation and methylation at its promoter region, where a HDAC/mSin3A repressor complex is anchored at Sp1 sites. The present studies revealed that protein kinase C (PKC) alpha/ERK signaling is important for the activation of LHR promoter activity, and the increase of endogenous transcripts induced by phorbol-12-myristate-13-acetate (PMA) in HeLa cells. Whereas these effects were attributable to PKCalpha activity, the ERK pathway was the downstream effector in LHR activation. PMA caused a significant enhancement of Sp1 phosphorylation at serine residue (s), which was blocked by PKCalpha or ERK inhibition. The interaction of activated phosphorylated ERK with Sp1 and ERK's association with the LHR promoter points to Sp1 as a direct target of ERK. After Sp1 phosphorylation, the HDAC1/mSin3A repressor complex dissociated from Sp1 sites, histone 3 was acetylated, and transcription factor II B and RNA polymerase II were recruited. In addition, overexpression of a constitutively active PKCalpha (PKCalpha CA) strongly activated LHR transcription in MCF-7 cells (devoid of PKCalpha), induced Sp1 phosphorylation at serine residue (s) and caused derecruitment of HDAC1/mSin3A complex from the promoter. These effects were negated by cotransfection of a dominant-negative PKCalpha. In conclusion, these studies have revealed a novel regulatory signaling mechanism of transcriptional control in which the LHR is derepressed through PKCalpha/ERK-mediated Sp1 phosphorylation, causing the release of HDAC1/mSin3A complex from the promoter.

Phosphatidylinositol 3-kinase/protein kinase Czeta-induced phosphorylation of Sp1 and p107 repressor release have a critical role in histone deacetylase inhibitor-mediated derepression [corrected] of transcription of the luteinizing hormone receptor gene.

We have demonstrated that silencing of luteinizing hormone receptor (LHR) gene transcription is mediated via a proximal Sp1 site at its promoter. Trichostatin A (TSA) induced histone acetylation and gene activation in JAR cells that prevailed in the absence of changes in Sp1/Sp3 expression, their binding activity, disassociation of the histone deacetylase/mSin3A complex from the Sp1 site, or demethylation of the promoter. This indicated a different mechanism involved in TSA-induced derepression. The present studies have revealed that phosphatidylinositol 3-kinase/protein kinase Czeta (PI3K/PKCzeta)-mediated Sp1 phosphorylation accounts for Sp1 site-dependent LHR gene activation. TSA caused marked phosphorylation of Sp1 at serine 641 in JAR and MCF-7 cells. Blockade of PI3K or PKCzeta activity by specific inhibitors, kinase-deficient mutants, or small interfering RNA abolished the effect of TSA on the LHR gene and Sp1 phosphorylation. PKCzeta was shown to associate with Sp1, and this association was enhanced by TSA. Sp1 phosphorylation at serine 641 was required for the release of the pRb homologue p107 from the LHR gene promoter, while p107 acted as a repressor of the LHR gene. Inhibition of PKCzeta activity blocked the dissociation of p107 from the LHR gene promoter and markedly reduced Sp1 phosphorylation and transcription. These results have demonstrated that phosphorylation of Sp1 by PI3K/PKCzeta is critical for TSA-activated LHR gene expression. These studies have revealed a novel mechanism of TSA action through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKCzeta-induced Sp1 phosphorylation-dependent manner.

A novel estradiol/estrogen receptor alpha-dependent transcriptional mechanism controls expression of the human prolactin receptor.

Prolactin exerts diverse functions in target tissues through its membrane receptors, and is a potent mitogen in normal and neoplastic breast cells. Estradiol (E(2)) induces human prolactin receptor (hPRLR) gene expression through stimulation of its generic promoter (PIII). This study identifies a novel E(2)-regulated non-estrogen responsive element-dependent transcriptional mechanism that mediates E(2)-induced hPRLR expression. E(2) stimulated transcriptional activity in MCF7A(2) cells transfected with PIII lacking an estrogen responsive element, and increased hPRLR mRNA and protein. The abolition of the E(2) effect by mutation of Sp1 or C/EBP elements that bind Sp1/Sp3 and C/EBPbeta within PIII indicated the cooperation of these transfactors in E(2)-induced transcription of the hPRLR. DNA affinity protein assay showed that E(2) induced estrogen receptor alpha (ERalpha) binding to Sp1/Sp3 and C/EBPbeta DNA-protein complexes. The ligand-binding domain of ERalpha was essential for its physical interaction with C/EBPbeta, and E(2) promoted this association, and its DNA binding domain was required for transactivation of PIII. Co-immunoprecipitation studies revealed tethering of C/EBPbeta to Sp1 by E(2)-activated ERalpha. Chromatin immunoprecipitation analysis showed that E(2) induced recruitment of C/EBPbeta, ERalpha, SRC1, p300, pCAF, TFIIB, and Pol II, with no change in Sp1/Sp3. E(2) also induced promoter-associated acetylation of H3 and H4. These findings demonstrate that an E(2)/ERalpha, Sp1, and C/EBPbeta complex with recruitment of coactivators and TFIIB and Pol II are required for E(2)-activated transcriptional expression of the hPRLR through PIII. Estradiol produced in breast stroma and adipose tissue, which are major sources of estrogen in post-menopausal women, could up-regulate hPRLR gene expression and stimulate breast tumor growth.

Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization.

Prolactin (PRL) acts through the long form (LF) of the human PRL receptor (hPRLR) to cause differentiation of mammary epithelial cells through activation of the Janus kinase-2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway and subsequent transcriptional events. To determine whether the inhibitory action of hPRLR short forms (SFs; S1a and S1b) on PRL-induced signal transduction through the LF results from heterodimerization, we studied complex formation among variant forms of the hPRLR. 3'-Tagged fusion constructs, with activities comparable to the wild-type species, were used to investigate homodimer and heterodimer formation. The LF and both SFs of the hPRLR formed homodimers under nonreducing conditions, independently of PRL, but formed only monomers under reducing conditions. Coimmunoprecipitation of the cotransfected LF with the SFs (S1a or S1b) in transfected cells showed ligand-independent heterodimerization of individual SFs with the LF. Bioluminescence resonance energy transfer analysis demonstrated homo- and heterodimeric associations of hPRLR variants in human embryonic kidney 293 cells. Biotin-avidin immunoprecipitation analysis revealed that hPRLR forms are cell surface receptors and that SFs do not influence the steady state or half-life of the LF. Significant homo- and heterodimerization of biotinylated membrane hPRLR forms was observed. These findings indicate that homo- and heterodimers of hPRLR are constitutively present, and that the bivalent hormone acts on the preformed LF homodimer to induce the active signal transduction configuration. Although SF homodimers and their heterodimers with LF mediate JAK2 activation, the SF heterodimer partner lacks cytoplasmic sequences essential for activation of the JAK2/signal transducer and activator of transcription 5 pathway. This prevents the heterodimeric LF from mediating activation of PRL-induced genes.

Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription.

We have previously demonstrated that transcription of the luteinizing hormone receptor (LHR) gene is subject to repression by histone deacetylation at its promoter region, where a histone deacetylase (HDAC)/mSin3A complex is anchored at a proximal Sp1 site. The present studies have shown that epigenetic silencing and activation of the LHR gene is achieved through coordinated regulation at both the histone and DNA levels. The HDAC inhibitor trichostatin A (TSA) evoked robust but significantly lower activation of the LHR gene in JAR than in MCF-7 cells. This effect was localized to the 176-bp promoter region, which is highly methylated in JAR and lightly methylated in MCF-7 cells. Consequently, TSA and the DNA demethylating reagent 5-azacytidine (5-AzaC) caused marked synergistic activation of the LHR gene in JAR but not in MCF-7 cells. Multiple site-specific lysine acetylation of H3/H4 is associated with such LHR gene activation. Methylation or acetylation of H3 at K9 is present at the silenced and derepressed LHR promoter, respectively. While DNA methylation levels did not affect the histone code of the LHR gene promoter, demethylation of the promoter CpG sites was necessary for maximal stimulation of this gene. Mechanistically, the combined actions of TSA and 5-AzaC, but not either 5-AzaC or TSA alone, resulted in complete demethylation of the LHR gene promoter in JAR cells. Release of the repressive HDAC/mSin3A complex from the LHR gene promoter in both cell types required both TSA-induced changes of histone modifications and, concurrently, a demethylated promoter. Also, Dnmt1 was largely dissociated from the LHR gene promoter in the presence of TSA or TSA plus 5-AzaC, and binding of MBD2 in JAR cells was diminished upon conversion of the promoter to a demethylated state. Such changes induced a more permissive chromatin where recruitment of polymerase II and TFIIB to the promoter was significantly increased. The activated state of the LHR gene induced by TSA and 5-AzaC in JAR and MCF-7 cells was observed basally in LHR-expressing PLC cells, in which the promoter is unmethylated and associated with hyperacetylated histones. Consequently, PLC cells are unresponsive to drug treatment. These findings have elucidated a regulatory mechanism whereby concurrent dissociation of repressors and association of activators and basal transcriptional components, resulting from coordinated histone hyperacetylation and DNA demethylation, lead to derepression of the LHR gene expression.

The gonadotropin-regulated long-chain acyl CoA synthetase gene: a novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity.

The 79 kD gonadotropin-regulated testicular long chain acyl-CoA synthetase gene (GR-LACS) is a hormone-regulated member of the acyl-CoA synthetase family that is expressed abundantly in Leydig cells and to a lesser extent in germinal cells of the adult testis. GR-LACS possesses an ATP/AMP binding domain and the fatty acyl-CoA synthetase (FACS) signature motif. To gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determined the genomic organization of the gene, including the upstream flanking sequences. The mouse GR-LACS gene spans over at least 45 kb and the coding region is encoded by exons 1-14. All exon-intron junction sites correspond to the consensus splice sequence GT-AG. Exon 7 and 11 comprise the conserved ATP/AMP binding domain and the FACS signature motif, respectively. Primer extension and S1 nuclease analyses demonstrated four transcriptional start sites located at -266/-216 bp 5' to the ATG codon. The minimal promoter domain resides within -254/-217 bp 5' to ATG codon, and upstream sequences to -404 bp (-1035/-405 bp) contribute to the inhibition of transcription in the expressing mouse Leydig tumor cells. Removal of -217/-1 bp, containing a 23 nt GC rich sequence (-112/-90) with an Sp1/Sp3 binding element, within the 1st exon of this TATA-less promoter, significantly reduced GR-LACS gene transcription. Transcriptional activity was abolished by a 2 nt mutation of this element. Thus, functional analyses of this promoter domain indicate that transcription of GR-LACS gene requires an Sp1/Sp3 binding element downstream of the transcriptional start sites which is essential for basal promoter activity.