GONADAL RECEPTORS AND MECHANISM OF ACTION OF PEPTIDE HORMONES IN STEROIDOGENIC CELLS
     
Maria L. Dufau, M.D., Ph.D., Principal Investigator
Chon-Hwa Tsai-Morris, Ph.D., Staff Scientist
Ying Zhang, Ph.D., Research Fellow
*Zhang-Zhi Hu, M.D., Research Fellow
Baojin Fu, M.D., Postdoctoral Fellow
Yi Sheng, M.D., Ph.D., Postdoctoral Fellow
†Pei-Zhong Tang, M.D., Ph.D., Postdoctoral Fellow
Naheed Fatima, Ph.D., Guest Researcher
Azra Khanum, Ph.D., Guest Researcher
Jianping Meng, M.D., Guest Researcher
Maria Dufau
 
The Section on Molecular Endocrinology investigates the molecular basis of peptide hormone action, with particular emphasis on the control of gonadal function. The major objective of the laboratory is to characterize the structure and regulation of luteinizing hormone (LH) and prolactin (PRLR) receptor genes and the regulatory mechanism(s) involved in the hormonal control of steroid biosynthesis. Current studies include the regulation of the promoter domain of the TATA-less LH receptor and the mechanism of its control by orphan nuclear receptors and cAMP, unraveling the complex genomic structure of the prolactin receptors, elucidating the multiple promoter control of PRLR transcription, and analyzing the functions of two novel receptors encoded by alternatively spliced transcripts of the prolactin receptors and investigating their roles in physiological regulation and breast cancer. We also investigate the hormone-regulated membrane coupling and intracellular events involved in the modulation of steroid biosynthesis in the testis and ovary. Other goals include the identification of new gonadotropin-regulated genes of relevance to testicular and ovarian function and other reproductive processes, including, for instance, a recently identified RNA helicase and a long-chain fatty acyl CoA synthetase that are developmentally regulated and expressed in the Leydig cell and specific tubule cells. These genes are undergoing investigation for their relevance to stage-specific translation in spermatogenesis and their roles in testicular steroidogenesis. Other studies include the molecular characterization of direct and indirect actions of androgens and estrogens produced during gonadotropin stimulation in the control of steroidogenic enzyme transcription/and expression. Current research also includes studies on cell-to-cell communication in the testis, developmental aspects of Leydig cell maturation, and the induction of regulatory mechanisms in the Leydig cell.

Transcriptional Regulation of the LH Receptor
Zhang, Dufau
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that plays an essential role in gonadal development and differentiation. Our previous studies demonstrated regulation of the Sp1/Sp3-driven TATA-less promoter of the human LH receptor by the orphan receptors EAR2 and EAR3/COUP-TF1 (inhibitory) and TR4 (stimulatory). We showed that orphan receptors 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), which is located within the 180 bp promoter 5' two functional SP1 sites. Current studies are investigating the differential binding of orphan receptors to rat and human LHR promoters and their modulation of LHR transcription in rat granulosa cells differentiated in culture by hormone treatment. The process resembles the induction of the LHR gene in granulosa cells of the human ovary and permits analysis of the role of orphan receptors during gonadal cell differentiation from early stages to luteinization. So far, the studies have demonstrated repression of rat LHR gene transcription by EAR2 and EAR3/COUP-TFI through their binding (albeit with half the affinity of that of their human counterparts) to the rat DR motif with one nucleotide difference from the core human DR. The one base pair mismatch (A/C) in the second half-site of the rat DR caused loss of TR4 binding and function but had no effect on EAR2 and EAR3/COUP-TFI. The lower binding affinity and inhibitory capacity for EAR2 and EAR3 in the rat was associated with a smaller inhibitory effect than that observed in the human. This resulted from the lack of a guanine in the rat DR, which is present 3' next to the human DR core motif. Thus, our studies have identified sequence-specific requirements for the binding of EAR2, EAR3/COUP TFI, and TR4 to the DRs that explain their differential regulation of the rat and human LHR genes. In addition, we have shown that the rat promoter is only partly acceptable as a model for the study of regulation of the LH promoter transcription. In contrast to the human, the negative regulation induced by EAR2 and EAR3 is unopposed by TR4 in the rat. To address the functional contribution of the orphan receptors to LHR gene transcription, we performed studies in cultured rat ovarian granulosa cells in which LHR expression is induced by the actions of FSH and E2 and is subsequently up-regulated by LH. The process closely resembles the induction of the LHR gene in human granulosa cells that are not, however, readily available for functional studies. hCG treatment of granulosa cell cultures markedly reduced the inhibition of the rat LHR in granulosa cells and reduced EAR2 and EAR3 protein levels. Abolition of the orphan receptor-mediated inhibition of the rLHR upon hCG treatment via derepression may contribute to the elevated LHR expression required for progression of granulosa cell maturation.

Gonadotropin Regulation of Receptors, Steroidogenic Enzymes, and Novel Genes
Tang, Tsai-Morris, Khanum, Sheng, Dufau
Treatment with high doses of gonadotropins causes LHR-independent negative regulation of steroidogenic enzymes (steroidogenic desensitization) and up-regulation of a novel gonadotropin-regulated RNA-helicase (GRTH). A previously unidentified protein that is constitutively present in Leydig cells and down-regulated by gonadotropin was recently cloned and characterized as a gonadotropin-regulated long-chain acyl CoA synthetase (GR-LACS). The 79-kDa cytoplasmic protein is expressed in the pubertal and adult Leydig cells of the rat testis and shares sequence identity with two conserved regions of the LACS and luciferase families but displays low overall amino acid similarities with other members of the LACS family (23 to 28 percent). GR-LACS mRNA is also expressed in spermatogonia and Sertoli cells, but less strongly. It is also observed in the ovary and brain, mainly in the hippocampus. However, an peptide antibody raised against the N-terminal portion of the protein was unable to detect protein expression outside the testis sites, perhaps reflecting very low levels of the protein or lack of translation of the mRNA form. On the other hand, the protein may be an alternatively spliced variant form or arise from the use of an alternate initiation-translation site. These possibilities are now under investigation. In vivo treatment with a desensitizing dose of hCG known to cause down-regulation of steroidogenic enzymes produces a major reduction of GR-LACS mRNA (by 80 percent) for a 24- to 48-hour period. However, this reduction was not paralleled by changes in protein expression. The results stand in contrast to previous findings related to steroidogenic enzymes, for which mRNA changes were mostly comparable to those of protein expression. The indication is that the GR-LACS protein is efficiently translated. Whether the discrepancy relates to the increased levels of the GRTH remains to be determined. The expressed GR-LACS protein present in the cytoplasm of transfected cells displayed acyl CoA synthetase activity for long-chain fatty acid substrates. In addition to its potential contributions to energy production and testicular steroidogenesis, GR-LACS could provide long-chain acyl-CoA esters with regulatory effects on enzyme activity, membrane function, and gene expression.

Prolactin Receptors
Hu, Meng, Zhuang, Dufau
Previous studies in our laboratory have mapped and resolved the genomic structure of the human prolactin gene (over 200 Kb) and have demonstrated that the gene has a complex structure and is amenable to alternative splicing. The studies also detected the presence of 10 exons (multiple noncoding exons 1 and the common noncoding exon 2 and exons 3 to 10 coding for the long form of the receptor). In addition, a novel exon-11 of the human prolactin receptor was recently found to be distinct from its rodent counterpart, and two novel forms of the human prolactin receptor (S1a and S1b, which are derived from alternative splicing of exons 10 and 11) were identified. The new forms of the human prolactin receptor resemble the conventional receptor in terms of similar extracellular and transmembrane domains but differ in terms of unique truncated intracellular domains. The short forms, which were found in several normal tissues and in breast cancer cell lines, are expressed as cell surface receptors and possess binding affinities comparable to those of the long form. However, unlike the long form, neither of the short forms mediates the prolactin-induced activation of the beta-casein gene promoter that is exhibited by the long form of the receptor. In contrast, the forms act as dominant negative repressors of the function of the long and intermediate receptor isoforms that mediate cell growth responses in human breast cancer cells. The short forms with unique C-termini may exhibit distinct signaling pathways in addition to modulating signaling from the long form of the receptor.

Designed to visualize subcellular localization of the short forms as well as of the long form, confocal microscopy studies of hPRLR-GFP fusion proteins transiently expressed in COS1 cells found that the long GFP and S1b forms were expressed at relatively high levels and were localized on the cell membrane as well as at intracellular sites. The outcome is consistent with our previous studies in ovarian tissue, which demonstrated that a major pool of receptors is immobilized even in membrane preparations and can bind to prolactin only upon detergent solubilization. The S1a form was located mostly at the cellular membrane but was expressed at a much lower level than the long and S1b forms, consistent with results obtained by other approaches (immunological, binding studies). In other studies that use hormone binding competition assays, we examined whether the inhibitory effect of the short forms resulted from ligand competition or intrinsic inhibitory action between the long and short forms. The results showed that the inhibition of the long form–mediated activation of the b-casein gene by the short forms was independent of the ligand concentration and that therefore the short formspresumably acted as intrinsic dominant negative inhibitors of the long form. Previous studies by other groups have demonstrated that a stimulatory effect of prolactin in human breast cancer cells and a mutated prolactin molecule acting as a receptor antagonist have inhibitory roles and induce apoptosis in human cancer cells. The new receptors may have important roles in the diversified actions of prolactin in human tissues and are of potential therapeutic relevance to the control of mammary cancer cell growth and immunoregulation. Therefore, our work can provide links to development of therapeutic strategies in cancer research at two levels: at the transcription level by reducing or causing a tissue-specific control of expression of the receptor and at the signal transduction level by inhibiting the transduction function of the long form of the receptor by peptide or nonpeptide antagonists that mimic the function of the new forms of the prolactin receptor.

Effects of Disruption of the Growth Hormone Receptor Gene on Testicular Function
Tsai-Morris, Dufau, and extramural collaborators
Recent studies in GH receptor knock-out mice have provided an in vivo demonstration that LH action on testosterone secretion is significantly impaired because of a decrease in the number of testicular LH receptors. The reduced LH action is accompanied by diminished responsiveness of testicular steroidogenesis and decreased ability to convert androstenedione to testosterone. These findings are probably attributable to the absence of circulating IGF-I in the GH-deficient mice, suggesting that IGF-I has a major role in the regulation of testicular endocrine function.

 
 

PUBLICATIONS

  1. Chandrashekar B, Bartke Awoniyi CA, Tsai-Morris CH, Dufau ML, Russell LD, Kopchick JJ. Testicular endocrine function in GH receptor gene disrupted mice. Endocrinology 2001;142:3443-3450.
  2. Dufau ML, Tsai-Morris CH, Tang PZ, Khanum A. Regulation of steroidogenic enzymes and a novel testicular RNA helicase. J Steroid Biochem Mol Biol 2001;76:187-197.
  3. Hu ZZ, Meng J, Dufau ML. Isolation and characterization of two novel forms of the human prolactin receptor generation by alternative splicing of a newly identified exon 11. J Biol Chem 2001;276:41086-41093.
  4. Tang PZ, Tsai-Morris CH, Dufau ML. Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase. Proc Natl Acad Sci USA 2001;98:6581-6586.
  5. Zhang Y, Dufau ML. EAR2 and EAR3/COUP-TFI regulate transcription of the rat luteinizing hormone receptor. Mol Endocrinol 2001;15:1891-1905. Zhang Y, Dufau ML. Nuclear orphan receptors regulate transcription of the gene for the human luteinizing hormone receptor. J Biol Chem 2000;75:2763-2770.

    *Current address: National Biomedical Foundation, Georgetown University Medical Center, Washington, D.C.
    †Current address: Department of Gene Discovery, Huntsville, AL.