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 the regulatory mechanism(s) 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, deactylation, and methylation) and second messengers as well as on the complex genomic structure of the prolactin receptors (PRLR) and the multiple promoter 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 and ovary as well as novel gonadotropin-regulated genes of relevance to testicular and ovarian function and other reproductive processes. Two such genes are an RNA helicase and a Long Chain Acyl CoA synthetase that are developmentally regulated and present in the Leydig cell and specific tubule cells. These genes are of interest for their potential relevance to steroidogenesis and stage-specific translation in spermatogenesis.

Transcriptional Regulation of the LH Receptor
Zhang, Dufau
The luteinizing hormone receptor (LHR), a G protein–coupled receptor, is essential for reproductive function and is predominantly located in the plasma membrane of gonadal cells. It mediates gonadotropin signalling and triggers intracellular responses that participate in gonadal maturation and function as well as in the regulation of steroidogenesis and gametogenesis. The LHR gene is also expressed in several non-gonadal tissues, including the uterus and placenta, where its functions have not been determined. We previously demonstrated that the nuclear orphan receptors EAR2 and EAR3/COUP-TF1 (inhibitory) and TR4 (stimulatory) regulate the Sp1-/Sp3-driven TATA-less 180 bp promoter of the human LH receptor (hLRH). The orphan receptors bind competitively and with high affinity to an imperfect direct-repeat motif containing an estrogen response element half-site and a second degenerate half-site (DR) located within the promoter 5' of two functional Sp1/Sp3 sites. The orphan receptors 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 as a consequence of the presence of a G 3' to the second DR site in the human sequence and, accordingly, exert weaker inhibition on rat promoter activity. In addition, TR4 is inactive in the rat promoter due 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.

Modification of chromatin structure by histone deacetylases (HDACs) is an important mechanism in the modulation of eukaryotic gene transcription. Investigation of the regulation of the human LHR by histone deacetylases in human choriocarcinoma cells (JAR cells) showed that LHR transcription was markedly induced when histone deacetylase activity was inhibited by trichostatin A (TSA) and that histone deacetylation caused significant repression of the hLHR gene transcription in these cells. Acetylated histones H3 and H4 accumulated in TSA-treated cells and were shown to be predominantly associated with the LHR promoter (-181/+27 bp from ATG at +1) and only minimally with the adjacent 5' region (-489/-235 bp). In addition, TSA significantly enhanced the recruitment of RNA polymerase II to the promoter. This promoter-specific localization of histone acetylation could provide a more accessible environment for the recruitment of components(s) of the general transcriptional machinery and of RNA Pol II. We identified only one of the two Sp1 sites essential for basal promoter activity as critical for the TSA effect (Sp1-1 site, -79/-71 bp from ATG +1), but the binding of Sp1/Sp3 to the site remained unchanged in the absence or presence of TSA. The orphan receptor DR site did not participate in TSA induction. We used DNA precipitation assays (DAPA) to determine whether Sp1/Sp3 were candidates to target non–DNA binding proteins. Our findings demonstrated the formation of a multi-protein complex including DNA binding and non-binding protein(s). Using co-immunoprecipitation assays to determine the association order of the individual components, we demonstrated that the multi-protein complex was recruited to the LHR promoter via interaction with Sp1 and Sp3. HDAC1 and HDAC2 docked directly to Sp1-bound DNA and indirectly to Sp3-bound DNA through RbAp48 while mSin3A interacted with hDACs. Investigation of the mode of repression of hLHR gene transcription by the HDACs/mSin3A complex in co-transfection studies revealed that HDACs 1 and 2 strongly inhibited hLRH promoter activity induced by TSA. Transfection of mSin3A caused minor inhibition of hLHR promoter activity, presumably because of its cooperation with endogenous HDACs. HDAC1 and HDAC2 potently repressed hLHR gene transcription, and mSin3A potentiated the inhibition caused by HDAC1. These findings demonstrated that the HDAC-mSin3A complex has an important role in the regulation of hLHR gene transcription by interaction with Sp1/Sp3 and by region-specific changes in histone acetylation and Pol II recruitment within the hLHR promoter. We have identified the hLHR gene promoter as a target for regulatory repression by the HDACs-mSin3A complex during the control of hLHR gene transcription. Expression of the LHR gene is subject to tonic repression by deacetylation of its gene promoter. The regulated derepression of such inhibitory control of the LHR gene, through as yet unidentified signal inputs, may provide functional regulation during the induction and cyclic variations in the differentiation, growth, and development of gonadal cells.

Figure 9

Recruitment of HDACs-mSin3A Complex to the Human LHR Promoter
Sp1-1 and Sp1-2 are functional sites for promoter activity. Both sites bind to Sp1 and Sp3 and contribute to basal promoter activity. The HDAC-mSin3A complex is recruited directly to Sp1 the site and indirectly through RbAp48 to Sp3 bound to this site. DR: imperfect estrogen receptor half-site direct repeat for binding of orphan receptors (OR) EAR2, EAR3/COUP-TF1 binding/function (inhibitory), and TR4 (stimulatory). TSS: transcriptional start sites (vertical lines).

Expression of a Novel Gonadotropin-Regulated Testicular RNA Helicase
Sheng, Tsai-Morris, Dufau
The gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) that we cloned from the rat Leydig cell, mouse, and human testis cDNA libraries is a novel member of the DEAD-box protein family of RNA helicases that possesses ATPase and RNA helicase activity; it is the first 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. Immuno-cytochemical analysis of GRTH protein levels revealed that GRTH protein is predominantly present in Leydig cells. It is also expressed to a lesser extent in germ cells of the adult rat; we identified the highest immunosignals in pachytene spermatocytes and round spermatids at stages VIII and IX. Western blot analysis displayed a 43 kDa GRTH protein band in Leydig cells, where its levels significantly increased at 24 hours and returned to control levels four days after in vivo treatment with hCG. The androgen receptor antagonist Flutamide markedly suppressed basal and hCG-induced expression of GRTH protein. We observed high expression of GRTH protein in pachytene spermatocytes while low levels were present in round spermatids. The compartmen-talized localization of GRTH in the tubules indicates that the protein may be involved in the meiotic process during spermatogenesis. Our studies have demonstrated marked up-regulation of GRTH protein expression by hCG via androgen formation in Leydig cells at agonist doses that cause down-regulation of LH receptors and steroidogenic enzymes. We have also shown that the sites of protein expression and pattern of regulation by hCG and androgen are highly correlated with the mRNA changes observed in our previous studies.

Prolactin Receptors
Meng, Leondires, Dong, Hu, Tsai-Morris, Dufau
The polypeptide hormone prolactin has 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 receptor is widely distributed and exists in several forms, including long and intermediate forms, and two short forms with abbreviated cytoplasmic domains that inhibit the function of the long form. Prolactin receptors (PRLR) are expressed in normal and neoplastic human tissues and mediate prolactin-induced proliferative actions in human breast cancer cells that are additive to those of estrogen. Circulating prolactin of pituitary origin and that synthesized locally in the breast could contribute to tumor biogenesis. Adipose cells are a major source of estrogen in post-menopausal women and could exert paracrine control of prolactin and PRLR synthesis in the epithelial cells of the breast. Current evidence and the ability of prolactin to stimulate the growth of rodent mammary tumors indicate that prolactin could contribute to the development of such tumors and possibly to the development of human breast cancer.

To determine the control mechanism(s) underlying human PRLR gene expression, we defined the 5' exonic gene structure and initiated studies on promoter identification and regulation. The expression of the PRLR is controlled by a highly complex regulatory system at the transcriptional level. Of the three promoters used for transcription of non-coding exons-1 in the rat (PI, PII, and PIII) and the two in the mouse (PI and PIII), only PIII is shared with the human. Resolution of the complete genomic structure of the hPRLR gene revealed that transcription of the hPRLR is controlled by multiple promoters that use distinct mechanisms to regulate gene expression. The gene has five human-specific non-coding exons-1 (hE1N1-5) as well as a generic exon-1 that is present in rodents and human-hE13. We identified all alternative first exons in a genomic fragment of a PAC clone that included the 5' UTR and the coding region of the hPRLR. Their order with their corresponding 5' flanking and promoter sequences (hPIII generic; hPN1-5, human specific) was established in chromosome 5p14-13. We found that promoters used in the transcription of exon 1 generic species, hE13 (hPIII) and the human-specific hE1N1 (hPIN1), employ distinct mechanisms to control hPRLR transcription. In the human, Sp1/Sp3 are the major transcription factors that participate in hPIII promoter activation while C/EBPb can minimally support activity when Sp1 is mutated. The hPN1 promoter uses a distinct mechanism for controlling hPRLR transcription. Two putative regulatory complexes that specifically bind to Ets and NR elements may contribute to transcriptional activity; the transfactors involved remain to be determined. Our studies have excluded transfactors that are known members of the PEA3 or Fli1 /ERG or Ets subfamily for interaction with the ETS element. Furthermore, the complex formed at the NR half-site is unlikely to include any of the known monomeric orphan receptors; it does not contain the required 5' extended core motif required for high-affinity interaction with the individual monomer.

Estradiol (E2) significantly increased the expression of both hPRLR mRNA transcripts hE13 and hE1N1. In all human tissues examined (ovary, testis, liver, T47D cells), the expression levels of these alternative first exons are markedly lower than that of the hE13. In both normal breast and cancer cells lines, hPIII appears to be the more commonly used promoter of hPRLR and is therefore the main focus of our ongoing investigation of the hormonal induction of hPRLR gene expression. In transfected T47D cells, E2 activated the hPIII promoter in a dose-dependent manner, and such activation was sensitive to the specific estrogen receptor antagonist ICI 182,780. The lack of a formal ERE in these promoters suggests that the effect of estradiol is mediated through association of the activated ER with relevant DNA binding transfactor(s). These findings support the role of E2 in the regulation of hPRLR expression in human breast cancer cell lines.

Figure 10

Schematic Representation of the Prolactin Receptor Gene The location of multiple first exons is indicated. All first non-coding exons are spliced onto the common non-coding exon 2 followed by the third exon in which translation starts at ATG. Exons 4 through 11 are coding exons. The long form of the receptor is encoded by exons 3 through 10. Exon 8 encodes the transmembrane domain and exon 10 most of the intracellular domain. The intermediate form is encoded by exons 3 through 10 and contains a partial deletion of exon 10 (black box). The two short forms (S1a and S1b) with truncated cytoplasmic domains are derived from alternative splicing of exons 10 and 11. S1a encodes 376 amino acids (aa) and contains partial exon 10 and a unique 39 aa C-terminal region derived from exon 11. S1b encodes a 288 aa protein that lacks exon 10 and contains three amino acids at the C-terminus derived from exon 11 (splice sites are indicated).