Maria L. Dufau, MD, PhD, Head, Section on Molecular Endocrinology
Chon-Hwa Tsai-Morris, PhD, Staff Scientist
Ying Zhang, PhD, Research Fellow
Juying Dong, PhD, Postdoctoral Fellow
Ravi K. Gutti, PhD, Postdoctoral Fellow
Mingjuan Liao, PhD, Postdoctoral Fellow
Yuji Maeda, MD, PhD, Postdoctoral Fellow
Aamer Qazi, PhD, Postdoctoral Fellow

We investigate the molecular basis of peptide hormone control of gonadal function, with particular emphasis on the structure and regulation of the luteinizing hormone receptor (LHR) and prolactin receptor (PRLR) genes. We also study the regulatory mechanism(s) involved in the progress of spermatogenesis and the control of Leydig cell function. Our studies focus on the regulation of human LHR gene transcription (nuclear orphan receptors, epigenetic mechanisms, DNA methylation, and second messengers) as well as on the multiple-promoter control of hPRLR gene transcription. We are elucidating the function of two inhibitory short forms of the prolactin receptors and their relevance to physiological regulation and breast cancer. We also investigate novel gonadotropin-regulated genes of relevance to the progression of testicular gametogenesis, Leydig cell function, and other endocrine processes.

Epigenetic control of luteinizing hormone receptor transcription

Zhang, Liao, Dufau

We previously demonstrated that transcription of the LHR gene is subject to repression by histone deacetylation at the gene's promoter region, where a histone deacetylase (HDAC )/mSin3A complex is anchored at a proximal Sp1 site (Zhang and Dufau, J Biol Chem 2002;277:33431). This Sp1 site is an essential mediator of trichostatin A (TSA)-induced LHR gene activation. We also showed that epigenetic silencing and activation of LHR is achieved through coordinated regulation at both histone and DNA levels in cancer cells. The LHR gene promoter is markedly silenced in JAR and MCF-7 cells while it is in an active state in simian virus 40-transformed normal placenta PLC cells. The HDAC inhibitor TSA evoked robust but significantly lower activation of the LHR gene in JAR than in MCF7 cells. This effect was localized to the 176bp promoter region, which is highly methylated in JAR and lightly methylated in MCF7 cells. Consequently, TSA and the DNA-demethylating reagent 5-azacytidine (5-AzaC) caused marked synergistic activation of the LHR gene in JAR cells but not in MCF7 cells. Multiple, site-specific lysine acetylation of H3/H4 is associated with such LHR gene activation. We observed a maximally activated state of the LHR gene in JAR cells (induced by TSA+AzaC) and MCF7 cells (induced by AzaC), whereas, in LHR-expressing PLC cells—in which the promoter is unmethylated, associated with hyperacetylated histones, and consequently unresponsive to drug treatment—LH expression was at a basal level. While DNA methylation did not affect the histone code of the LHR promoter, demethylation of the LHR CpG sites was necessary for maximal stimulation of the LHR gene. Release of the repressive HDAC/mSin3A complex required both TSA-induced changes of histone modifications and, concurrently, a demethylated promoter. Overall, these findings identified a regulatory mechanism whereby concurrent dissociation of repressors and association of activators and basal transcriptional components resulting from coordinated histone hyperacetylation and DNA methylation lead to derepression of LHR gene expression. The fact that TSA-induced histone acetylation and gene activation in JAR cells prevailed in the absence of changes in Sp1/Sp3 expression or binding activity, disassociation of the HDAC/mSin3A complex from the Sp1 site, or demethylation of the promoter indicated a different mechanism involved in TSA-induced derepression.

FIGURE 4.1
Model of transcriptional derepression and activation of hLHR gene by TSA.
The LH gene promoter is hypermethylated in JAR cells (left, top) but demethylated in MCF-7 cells (left, bottom). In the absence of TSA, the LHR gene promoter is repressed in both cell types, and the HDAC/mSin3A complex and p107 are both anchored by Sp1(left). No Sp1 phosphorylation was observed under these repressive conditions. TSA treatment of JAR cells induced LH gene promoter-localized histone hyperacetylation without affecting the promoter's DNA methylation status. TSA caused Sp1 phosphorylation that was dependent on the activity of PI3K/ PKCzeta and release of p107, but not on the activity of the HDAC/mSin3A inhibitory complex from the LHR promoter. This chain of events causes partial derepression with a 40-fold induction of promoter activity (top, right). Upon promoter demethylation and hHDAC/mSin3A disassociation by TSA (HDAC inhibitory) and 5-azacytidine (DNA methylation inhibitor) treatment, we observed maximal derepression of LHR expression. In MCF-7 cells, TSA induced maximal derepression of LHR gene expression (160-fold), which was caused by histone hyperacetylation and DNA demethylation at the gene's promoter, release of the HDAC/mSin3A complex, and dissociation of p107 resulting from Sp1 phosphorylation by PKCzeta and PI3K (bottom right). Me, 13 methylated CpG sites within the promoter of the LHR; Ac, acetylated histones; P, phosphorylated; De-Me, promoter DNA demethylation.

Our most recent studies revealed that phosphatidylinositol 3-kinase/protein kinase C zeta (PI3K/PKCzeta)-mediated Sp1 phosphorylation accounts for Sp1 site-dependent LHR activation. TSA caused marked phosphorylation of Sp1 at serine 641 in JAR and MCF7 cells. Blockade of PI3K or PKCzeta activity by specific inhibitors, kinase mutants, or small interfering RNA abolished the effect of TSA on the LHR gene and Sp1 phosphorylation. PKCzeta was shown to associate with Sp1, an association enhanced by TSA. Sp1 phosphorylation at serine 641 was required for the release of the pRB homologue p107 from the LHR 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. Our findings demonstrate that phosphorylation of Sp1 by PI3K/PKCzeta is critical for TSA-activated LHR gene expression. Our results also reveal 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.

Gonadotropin-regulated testicular genes

Tsai-Morris, Sheng, ¹ Li, ² Gutti, Maeda, Dufau

Gonadotropin-regulated acyl CoA synthetase (GR-LACS) is a 79 kD protein that we cloned in our laboratory from a rat cDNA library (Tang et al., Proc Natl Acad Sci USA 2001;98:6581). The protein, which is transcriptionally downregulated by gonadotropin and capable of activating long-chain fatty acids, is a new member of the long-chain fatty acyl-CoA synthetase family. GR-LACS has sequence identity with two conserved regions of the LACS family (ATP/AMP-binding domain and fatty acid acyl-CoA synthetase FACS signature motif) but shares low overall amino acid sequence similarity with all other known members of the family (23-28 percent). GR-LACS is abundantly expressed in Leydig cells and minimally in germinal cells of the adult testis. Given that GR-LACS is constitutively expressed in the steroid-producing rat testicular Leydig cells and downregulated during desensitization by gonadotropin, we hypothesize that GR-LACS may contribute to the provision of energy and biosynthesis of steroid precursors and participate through acyl-CoA's multiple functions in the regulation of the male gonad. GR-LACS protein is expressed in the rodent brain and gonads, but in the adrenal cortex only in the mouse. In both species, it is found in most regions of the brain and is highly expressed in the hippocampus. In the ovary of both species, it is associated with follicles undergoing atresia (that is, those undergoing transition between preantral to antral stages and subordinate antral follicles not selected for ovulation). Thus, in further explorations of the mechanisms associated with follicle development, GR-LACS could serve as a marker for atresia.

We observed in the rat brain and mouse ovary a distinct GR-LACS protein species of 64 kDa that was more abundant than the 79 kDa long-form species and was a minor form of the73 kDa species. In a rat brain cDNA library, we identified two novel species resulting from alternative splicing of the GR-LACS gene: a short form 1 (S1) lacking exon 8 and short form 2 (S2) lacking exons 6-8. Expression studies revealed that the sizes of the S1/S2 proteins are comparable to those of the endogenous variant species. Neither S form has FACs activity, suggesting that exon 8 is essential for enzymatic function, although AMP binding sites (exon 7) and a FACS signature motif (exon 11), which are the conserved functional motifs common to FACS family members, were still present in S1 while only the FACS signature motif was preserved in S2. Even though no particular functional motif is known to be associated with exon 8 sequences, exon 8 clearly exhibits functional importance for FACs activity, given that the newly identified S1 variant that exclusively lacks exon 8 exhibited no FACS activity. In addition, S1 exhibited a significant dominant negative effect on the FACS activity of the long-form GR-LACS while S2 displayed a lesser but significant inhibitory effect. However, given its endogenous abundance in the brain and its higher expression level than that of the long form, S2 could be a more effective regulator of long-form activity than S1. GR-LACS variants may regulate the long form's activity in the brain.

We previously identified a novel gonadotropin-regulated testicular helicase (GRTH/Ddx25). The enzyme is present in the nucleus and cytoplasm of pachytene spermatocytes and round spermatids and binds to mRNA species as an integral component of messenger RNP particles, with storage in chromatoid bodies located in the cytoplasm of spermatids (Tsai-Morris et al., 2004). GRTH-targeted null male mice are sterile as a consequence of spermatid arrest at stage 8 of spermatogenesis, demonstrating marked diminution of chromatoid bodies and failure to elongate. The transcription of messages in spermatids of these mice during stages 1 through 8 did not undergo alteration, but their translation was selectively abrogated. Our current studies are defining the function of GRTH as an RNA binding protein as well as the helicase's storage and translational function during sperm progression. We recently determined the subcellular distribution of the GRTH protein in cytoplasm and nucleus of mouse Leydig and germinal cells. N-terminal and C-terminal antibodies recognize, respectively, a cytoplasmic species of 61 kDa and a nuclear species of 56 kDa, with both species generated from use of the first ATG codon. The observed differences in molecular-weight species are attributable to phosphorylation of the 61 kDa cytoplasmic form. Evidence for a phospho-form of the 61 kDa protein came from its enrichment by phosphoprotein affinity column fractionation and its conversion to the lower molecular-weight species (56 kDa) after treatment with calf intestinal alkaline phosphatase. The 61 kDa species became more abundant in GRTH-expressing cells after treatment with cAMP and was significantly augmented by overexpression of the catalytic subunit of protein kinase C alpha, whereas stimulation was prevented by co-expression of the PKA inhibitor cDNA. The results indicate that cAMP-dependent PKA participates in post-translational modification. The endogenous phosphorylation status of the GRTH protein in the rat and mouse testis was revealed in Western blots of 61 kDa GRTH species precipitated by the phospho-Thr antibody. The association of these protein species with specific cellular compartments could be important for GRTH-dependent regulatory functions. In addition to its storage function, the cytoplasmic species of GRTH associates with polyribosomes to regulate the translational activity of specific subsets of expressed genes.

Prolactin receptor

Dong, Qazi, Tsai-Morris, Dufau

Prolactin exerts diverse functions in target tissues through its membrane receptors and is a potent mitogen in normal and neoplastic breast cells. Prolactin acts through the long form of the receptor (LF) to cause differentiation of mammary epithelium and to initiate and maintain lactation through activation of the Jak2/Stat5 pathway and subsequent transcriptional events. Prolactin is a tumor promoter in rodents and has been implicated in the development of breast cancer. Our laboratory has identified two novel short forms (SFs) of the human prolactin receptor (hPRL) with abbreviated cytoplasmic domain (S1a and S1b) that are products of alternative splicing and inhibit the activation induced by PRL through the LF. The ratio of SFs to LFs is significantly lower in tumor tissue and breast cancer cell lines than in normal breast and control mammary cells. Therefore, the relatively low expression of short forms in cancer could cause gradations of unopposed prolactin-mediated long form-stimulatory function and could contribute to breast tumor development/progression. Furthermore, we have shown that the inhibitory effect of SFs on LF-stimulatory activity induced by PRL can be attributed to the formation of heterodimers. In addition, our work revealed heterodimer formation that is independent of hormone, which does not induce additional dimeric forms. Even though SF homodimers and their heterodimers with LF are competent to bind to hormone and mediate JAK2 activation through box 1 in the cytoplasmic domain, the SF heterodimer partner lacks cytoplasmic sequences essential for activation of the JAK2/STAT5 pathway, thereby preventing the heterodimeric LF from mediating activation of PRL-induced genes. Furthermore, our results indicated that homo- and heterodimers of the hPRLR are constitutively present and that the bivalent hormone acts on the preformed LF homodimer to induce the active signal-transduction configuration. Our findings modify the concept of the initial step of PRL's action from dimer-inducer to a conformational modifier.

FIGURE 4.2
Novel non-estrogen responsive element-dependent transcriptional mechanism governing estradiol-induced hPRLR expression in breast cancer cells. Model of complex formation of ERalpha/E2 binding to C/EBPbeta and Sp1 bound to their cognate elements and co-activator assembly on the hPRLR promoter. TSS, transcriptional start sites; PIC, pre-initiation complex; open circle, putative adaptor proteins; Ac, acetylated histones.

Our previous studies on the hPRLR gene revealed its complex genomic structure, which is distinguished by six alternative non-coding exons 1 and promoter use, including the preferentially used, generic promoter1/exon 1 (PIII/hE13), which is also present in rat and mouse, and five human-specific exon-1/promoters (hE1N1-5). In subsequent studies, we showed that E2 induces increases in PRLR non-coding exon-1 hE13 transcripts directed by the preferentially used promoter III (hPIII) in breast cancer cells. Also, in transfection studies, E2 activated hPIII, which lacks an estrogen-responsive element. This promoter contains functional Sp1 and C/EBP sites that bind, respectively, to Sp1/Sp3 and CEBPbeta. Abolition of the E2 effect by mutation of Sp1 or C/EBP elements within PIII indicated the cooperation of these transfactors in E2-induced transcription of the hPRLR. E2-activated estrogen receptor alpha (ERalpha), through interaction with Sp1/Sp3 and C/EBPbeta DNA protein complexes, caused transcriptional activation of the hPIII promoter and consequently of hPRLR expression in cancer cells. The ligand-binding domain of ERalpha was essential for its physical interaction with C/EBPbeta; E2 promoted such interaction while ERalpha's DNA-binding domain was required for transactivation of PIII. Other studies revealed tethering of C/EBPbeta to Sp1 by the E2--activated ERalpha, favoring interaction with its cognate element, and recruitment of co-activators p300, SRC-1, and pCAF to the complex, with consequent region-specific changes in histone acetylation. These hormone/receptor-induced associations and chromatin changes favored TFIIB and RNA polymerase recruitment and the activation of hPIII-directed hPRLR transcription. Stromal and adipose tissue, which are major sources of estrogen in postmenopausal women, could exert paracrine control of prolactin and prolactin receptor expression in adjacent mammary epithelial cells and stimulate breast tumor growth.

¹ Yi Sheng, MD, PhD, former Postdoctoral Fellow
² Jie Li, former Postdoctoral Fellow

For further information, contact dufaum@mail.nih.gov.