Maria L. Dufau, MD, PhD, Head, Molecular Endocrinology Section
Chon-Hwa Tsai Morris, PhD, Staff Scientist
Jianping Meng, MD, Research Fellow
Ying Zhang, PhD, Research Fellow
Yuging Dong, PhD, Visiting Fellow
Yi Lee, MD, PhD, Visiting Fellow
Aamer Qazi, PhD, Visiting Fellow
Yi Sheng, MD, PhD, Visiting Fellow
Naheed Fatima, PhD, Guest Researcher

We investigate the molecular basis of peptide hormone control of gonadal function, with particular emphasis on the structure and regulation of luteinizing hormone and prolactin receptor genes and on the regulatory mechanisms involved in the hormonal control of steroidogenesis. Our studies focus on the regulation of the promoter domain of the LH receptor gene and its control by both nuclear orphan receptors (histone acetylation, deacetylation, and methylation) and second messengers as well as on the complex genomic structure of the prolactin receptors (PRLR) and the multiple control of their transcription. We are particularly interested in the function of two novel short forms of alternative spliced transcripts of the prolactin receptors and their relevance to physiological regulation and breast cancer. We also investigate hormone-regulated membrane coupling and intracellular events involved in the modulation of steroid biosynthesis in the testis as well as novel gonadotropin-regulated genes of relevance to testicular and ovarian function and other reproductive processes.

Transcriptional regulation of the LH receptor

 Zhang, Fatima, Dufau

The luteinizing hormone receptor (LHR), a G protein- coupled receptor essential for reproductive function and predominantly located in the plasma membrane of gonadal cells, mediates gonadotropin signaling and triggers intracellular responses that participate in gonadal maturation and function and the regulation of steroidogenesis and gametogenesis. We previously demonstrated regulation of the Sp1/Sp3-driven TATAless 180 bp promoter of the human LH receptor (hLRH) by the nuclear orphan receptors EAR2 and EAR3/COUP-TF1 (inhibitory) and TR4 (stimulatory), which bind competitively and with high affinity to an imperfect direct-repeat motif composed of an estrogen response element half-site and a second degenerate half-site (DR) located within the promoter 5' of two functional SP1/ SP3 sites and exhibit differential binding to the rat and human LHR promoters. EAR2 and EAR3/COUP-TFI bind with two- to three-fold higher affinity to the human promoter than to the rat promoter because of the presence of a G 3' to the second DR site in the human sequence, which causes weaker inhibition of the rat promoter activity by these orphan receptors. In addition, TR4 is not stimulatory in the rat owing to a single base-pair mismatch in the second DR site that abolishes its binding. Changes in endogenous levels of EAR2 and EAR3 during gonadotropin stimulation of rat granulosa cells in culture correlate with derepression of promoter activity.

We recently reported that local chromatin structure modulated by histone acetylation and deacetylation plays a role in human LHR gene regulation, namely, the most proximal Sp1(I) site of the two active Sp1/Sp3 sites of the hLHR gene promoter serves as a docking site to recruit a histone deacetylase- mSin3A complex to the promoter, which causes silencing of the LHR gene via histone hypoacetylation- induced chromatin condensation, a mechanism that is independent of the pathway involving unliganded hormone receptors/ orphan receptors and co-repressor NcoR/SMRT. However, this and/or epigenetic chromatin modifications may be involved in the second messenger activation of hLHR expression, which we are currently investigating.
 

We aim to establish the functional connection between orphan receptors and the Sp1/Sp3 complex and their impact on the basal transcription machinery. Given that the orphans operate in repressive/inductive states of the hLRH of the granulosa cells of early follicles (repressive) but not in those of mid- to late-ovulatory follicles and luteal follicles (relieved of repression by reduction of the expressed orphan inhibitors), the studies are relevant to understanding hLHR orphan control during the menstrual cycle at different stages of granulosa/luteal cell development.

We identified the proximal Sp1(I) site as critical for the EAR2, EAR3/COUP-TFI repression in both human and rat, as its mutation reduced inhibition by EAR2 and abolished inhibition by EAR3/COUP-TFI (EAR3). With EAR3 exerting a complete Sp1/Sp3-dependent inhibitory effect, cotransfection analyses in SL2 cells showed that both Sp1 and Sp3 are required for repression. Mutual recruitment of EAR3 and Sp1/Sp3 bound to their cognate sites further supported functional cooperation between Sp1 and DR domains. Deletion of the EAR3 N-terminal and DNA binding domains that reduced EAR3's interaction with Sp1 also reduced its inhibition on hLHR gene transcription. Furthermore, we demonstrated interaction of TFIIB with Sp1/Sp3 at the Sp1(I) site, in addition to its association with EAR3/COUP-TFI and the TATA-less core promoter region. The interaction relied on Sp1 site- bound Sp1/Sp3 complex and adaptor protein(s) present in JAR cell nuclear extracts. We further demonstrated that EAR3 specifically reduced association of TFIIB with the Sp1(I) site without interfering in EAR3's interaction with the hLHR core promoter. The C-terminal region of EAR3, which did not participate in its interaction with Sp1, was required for its inhibitory function and may affect the association of TFIIB with Sp1. Moreover, perturbation of the association of TFIIB with Sp1 by EAR3 was reflected in reduced recruitment of RNA PoL II to the promoter. Overexpression of TFIIB counteracted the inhibitory effect of EAR3 and activated hLHR gene transcription in an Sp1-site dependent manner. These findings indicate that TFIIB is a critical component in regulatory control of EAR3 and Sp1/Sp3 on the initiation complex. Such cross-talk

among EAR3, TFIIB, and Sp1/Sp3 reveals that repression of hLHR gene transcription by nuclear orphan receptors is achieved via perturbation of communication between Sp1/Sp3 at the Sp1-1 site and the basal transcription initiator complex.

Zhang Y, Dufau ML. Dual mechanism of regulation of transcription of luteinizing hormone receptor

gene by nuclear orphan receptors and histone deacetylase complexes. J Steroid Biochem Mol Biol 2003;85:401-441.

Zhang Y, Dufau ML. EAR2 and EAR3/COUP-TFI regulate transcription of the rat LH receptor. Mol

Endocrinol 2001;15:1891-1905.

Zhang Y, Dufau ML. Repression of the luteinizing hormone receptor gene promoter by cross talk

among EAR3/COUP-TFI, Sp1/Sp3, and TFIIB. Mol Cell Biol 2003;23:6958- 6972.

Zhang Y, Dufau ML. Silencing of transcription of the human luteinizing hormone receptor gene by

histone deacetylase-mSin3A Complex. J Biol Chem 2002;277:33431-33438.

Cell-specific and hormone-regulated expression of gonadotropin-regulated testicular RNA helicase resulting from alternative utilization of translation initiation codons in the rat testis

Sheng,Tsai-Morris, Lee, Dufau

The gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) cloned from rat Leydig cell, mouse testis, and human testis cDNA libraries is a novel member of the DEAD-box protein family of RNA helicases that possess ATPase and RNA helicase activity and is the rst member found to be regulated by a hormone. The mRNA of the GRTH gene is transcriptionally up-regulated by gonadotropin (hCG) via cyclic AMP- induced androgen formation in testicular Leydig cells. To evaluate protein levels of the GRTH gene regulated by gonadotropin, we raised a specific rabbit polyclonal antibody to a GRTH peptide. Immunocytochemical and Western blot analysis identified the GRTH protein as a developmentally regulated protein in Leydig cells and in germ cells (pachytene spermatocytes and round spermatids) of the rat testis. Recent studies have identified three ATGs with potential for the generation of multiple species. Germ cells primarily used the rst ATG codon (+1) and contained major proteins of 61/56 kDa species, whereas Leydig cells preferentially used the second ATG codon (+343) with expression of 48/43 kDa species. A third ATG was weakly used and yielded a 33 kDa species only in germ cells. The androgen receptor antagonist utamide prevented an increase in GRTH 43-kDa protein in Leydig cells caused by hCG treatment. In round spermatids, hCG caused a significant decrease of 61 kDa species and an induction of 48/43 kDa species, whereas we observed no changes in pachytene spermatocytes. Reversal of this hormone-induced switch of expression by utamide indicated a role of androgen in utilization of the second ATG. Our studies have demonstrated a cell-specic and hormone-dependent alternative usage of ATG codons in the testis. They have also revealed that both the androgen-dependent alternative use of ATG codons in the rat testis and the androgen-dependent transcription of GRTH expression are accompanied by a marked increase in the 43-kDa species. The Leydig cell is tonically regulated by gonadotropin, which promotes testosterone formation in these cells. Autocrine actions of testosterone in Leydig cells and paracrine actions at tubule sites (round spermatids) cause increases in GRTH and promote the utilization of the second ATG codons in both cells types. These findings indicate that the expression of GRTH proteins is regulated by gonadotropin/androgen at the translational level.

Sheng Y, Tsai-Morris C-H, Dufau ML. Cell-specic and hormone-regulated expression of gonadotro-

pin-regulated testicular RNA helicase gene (GRTH/Ddx25) resulting from alternative utilization of translation initiation codons in the rat testis. J BiolChem 2003;278:27796-27803.

Prolactin receptors

Qazi, Meng, Dong, Tsai-Morris, Dufau

Prolactin is a polypeptide hormone with highly diversified biological actions in reproduction, lactation, maternal behavior, steroidogenesis, growth, metabolism, water-salt balance, and immune regulation. Its actions are mediated by a single-transmembrane domain receptor of the cytokine/growth hormone family. The expression of the human prolactin receptor is controlled by multiple promoters that use distinct mechanisms to regulate gene expression. The human gene uses five promoters for transcription of noncoding exons-1 as well as a generic promoter/exon-1 termed hPIII/E13, which is present in rodent and human and appears to be the most commonly used promoter. Prolactin stimulates the proliferation of human breast cancer cells and exhibits additive effects to that of estrogen in T-47D and MCF7 cells. Estradiol activates the hPIII promoter in a dose-dependent manner, an action that is prevented by the specific estrogen receptor antagonist ICI 182,780. Further, estradiol stimulates the expression in T-47D cells of two prolactin receptor isoforms, the generic form and a human-specific form with alternative exons-1. These findings support the role of estradiol in regulating human prolactin receptor expression in human breast cancer cells. The receptor is widely distributed and exists in several forms, including long and intermediate forms as well as two short forms identified in our laboratory as S1a and S1b, which are derived from alternative splicing and possess a truncated intracellular domain. The truncated forms are dominant-negative repressors of the function of the long form and are present in several normal tissues and human breast cancer and cell lines. We have demonstrated differential expression of these forms in cancer profiling arrays and by real-time PCR quantitation using human breast tissues. Both approaches revealed significant decreases in the ratio of short to long forms in the breast tumor tissue. Given that the short forms act as dominant negative regulators of the stimulatory actions of the long form in vitro, their relatively reduced expression in cancer could cause unopposed prolactin-mediated stimulatory actions of the long form.

Acting through its cognate receptor, prolactin activates the tyrosine kinase Jak2, which is essential for prolactin-induced differentiation and the activation of Stat5 in mammary epithelial cells. Receptor dimerization can be either constitutive or ligand-induced. Recognizing that the action of the short forms could result from their heterodimerization with the long form of the receptor, we studied the complex formation between the short and long isoforms. To investigate the formation of homodimers and heterodimeric forms of the long and short forms of the receptor, we used 3' tagged fusion constructs of various prolactin receptor forms with V5, FLAG, or GFP. As a 3' tagged construct transfected into HEK293 cells, the long form exhibited prolactin-induced beta-casein activity comparable to the wild type. In addition, when each of the 3' tagged short forms of receptor was cotransfected with the long forms, the wild-type or the 3' tagged constructs inhibited prolactin-induced activation of the beta-casein promoter activity by the long form of the receptor. The long form formed a homodimer under nonreducing conditions while the monomer was detected under reducing conditions, with prolactin not affecting the homodimerization. Similarly, under nonreducing conditions, S1a or S1b isoforms formed a homodimer unaffected by the presence of prolactin. Coimmunoprecipitation of the cotransfected long form and each of the 3' tagged short forms (S1a and S1 b) in HEK-293 cells showed heterodimerization of the individual short forms with the long form. We thus conclude that human prolactin receptors can form homodimers (long form, S1a and S1b) independent of ligand treatment. We also demonstrated that heterodimerization between the long and short forms of the receptor is ligand-independent and propose that prolactin receptor homo- and/or heterodimers are constitutively present. Furthermore, we conclude that the association between two different short isoforms of the prolactin receptor could exert a modulatory role on prolactin-induced signaling through the long form of the receptor. The hormonal stimulus could act on the preformed homodimer of the long form to induce the signal by causing changes in conformation of the cytoplasmic domain. Heterodimerization with either short form could abrogate the structural changes induced by the hormone that are required for activation of the dimeric long form of the receptor.

Hu ZZ, Zhuang L, Meng J, Tsai-Morris C-H, Dufau ML. Complex 5' genomic structure of the human

prolactin receptor: multiple alternative exons 1 and promoter utilization. Endocrinology 2002;143:2139-2142.

Leondires MP, Hu ZZ, Dong J, Tsai-Morris C-H, Dufau ML. Estradiol stimulates expression of two

human prolactin receptor isoforms with alternative exons-1 in T47D breast cancer cells. J Steroid Biochem Mol Biol 2002;82:263-268.

For further information, contact dufau@helix.nih.gov