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A novel human luteinizing hormone receptor (LHR) gene was isolated from a human placental genomic library. This gene (Gene II) differs from that previously isolated from a lymphocyte library (Gene I) by several base changes in the 5' flanking region and the deletion of 6 nucleotides in the coding region (+55 to +60). The sequence of the exon 1 coding region of gene II conforms to the sequence of the human ovarian LHR cDNA. Furthermore, primer extension and reporter gene analyses indicate that both the Gene II promoter and the human ovarian LHR mRNA transcriptional start sites are located within the -176 bp TATA-less 5' flanking domain. Additional upstream transcriptional start sites (> -176 bp) were identified in human testicular mRNA and the human choriocarcinoma JAR cell. Restriction enzyme analysis verifies that both LHR genes are present in the human genome, and gene dosing reveals four copies of the human LHR in contrast to a single copy in the rat genome. Chromosomal mapping localizes all copies of the human LHR to the chromosome 2p16-21 loci. These studies suggest that tissue-specific LHR promoter utilization and LHR gene expression may be correlated with gene diversity.

The expression of the prolactin receptor is under the control of two putative tissue-specific (PI, gonads; PII, liver) and one common (PIII) promoters (Hu, Z. Z., Zhuang, L., and Dufau, M. L. (1996) J. Biol. Chem. 271, 10242-10246). The three promoter regions were co-localized to the rat chromosomal locus 2ql6, in the order 5'-PIII-PI-PII-3'. To investigate the mechanisms of gonad-specific utilization of PI, the promoter domain, regulatory cis-elements, and trans-factors were identified in gonadal cells. The promoter domain localized to the 152-base pair 5' of the transcriptional start site at -549 is highly active in gonadal cells but has minimal activity in hepatoma cells. It contains a steroidogenic factor 1 (SF-1) element (-668) that binds the SF-1 protein of nuclear extracts from gonadal cells and is essential for promoter activation. A CCAAT box (-623) contributes minimally to basal activity in the absence of the SF-1 element, and two adjacent TATA-like sequences act as inhibitory elements. Thus, PI belongs to a class of TATA-less/non-initiator gene promoters. These findings demonstrate an essential role for SF-1 in transcriptional activation of promoter I of the prolactin receptor gene, which may explain the tissue-specific expression of PI in the gonads but not in the liver and the mammary gland.

The forskolin-induced steroidogenic block of testosterone production residing beyond pregnenolone synthesis in rat Leydig cells was localized to the level of the 17beta-hydroxysteroid dehydrogenase (17betaHSD) reaction in this study. The use of forskolin analogs that discriminate between the diterpene's inhibitory effect on the glucose transporter(s) (1,9-dideoxyforskolin) and its activation of adenylate cyclase (6-aminoethyl carbamyl forskolin) revealed that the block is related to inhibition of glucose transporter(s). 1,9-Dideoxyforskolin, but not 6-aminoethyl carbamyl forskolin, caused a significant inhibition of basal and hCG-stimulated testosterone production with accumulation of androstenedione. Glucose-deficient media produced the same metabolic block in the absence of forskolin, with a significant reduction in 17betaHSD activity and increases in the apparent Km for androstenedione. In contrast, metabolic steps before testosterone formation were not affected. Glucose-induced 17betaHSD activation was mimicked by the addition of ATP or GTP in glucose-deficient media, but not by nonhydrolyzable triphosphate analogs or NADPH. A decrease in 17betaHSD activity caused by KT-5720, a specific inhibitor of protein kinase A and the calmodulin antagonist W-7, indicates that the ATP requirement may be related to the participation of protein kinases in the activation of 17betaHSD. ATP levels derived from alternative (nonglycolytic) pathways are adequate to support basal and hormone-stimulated enzymatic activities in the metabolism of cholesterol to androstenedione. However, the integrity of the glucose transport system with subsequent ATP generation is required for activation of 17betaHSD in the final step of androgen biosynthesis. In conclusion, the conversion of androstenedione to testosterone requires the contribution of the glycolytic pathway to meet ATP requirements for 17betaHSD activity.

Isolation and structural characterization of the rat corticotropin releasing factor receptor (CRFR) gene was performed to determine the exon/intron organization of the coding region and the potential for splice variants. The CRFR gene contains 13 exons and 12 introns, and the positions of the exon/intron junctions are similar to those of other Class II G protein-coupled receptor genes including the parathyroid hormone and glucagon receptors. The promoter resides within 593 base pairs of the initiation codon and the major transcriptional start site at nucleotide −238. This domain does not possess a TATA box but contains multiple Sp1 and AP-2 sites upstream and downstream of the major transcriptional start site. Intron junctions were identified in the extracellular, transmembrane (TM), and cytoplasmic (C) domains of the CRFR, giving the potential for differential signal transduction by splice variants. CRFR cDNAs derived from rat Leydig cell mRNA included the pituitary Form A, which spans exons 1–13, and two splice variants with deletion of exon 3 or exons 7, 11, and 12. An evolutionary link between the intronless TM/C module of the glycoprotein hormone receptors and the intron-containing TM/C module of the CRFR is suggested by the common position of the luteinizing hormone receptor Form D alternate acceptor splice site and the CRFR intron 12.

The functional importance of cysteine residues in the extracellular domain and the extracellular loops (EL1 and EL2) to hormone binding of the rat luteinizing hormone receptor (LHR) was investigated. For this purpose, cysteines in the seven-transmembrane holoreceptor (Form A) and its hormone-binding splice variant (Form B) were replaced by serine residues, and mutant receptors were expressed in COS1 and/or insect cells. Within the extracellular domain, individual replacement of all four cysteines from Exon 1 abolished hormone binding activity, and replacement of Cys-109 and Cys-134 from exons 5 and 6 caused a 75% decrease in both cell surface and total cellular solubilized LHR hormone binding activity. Mutations of Cys-257 and -258 (Exon 9), Cys-321 and -331, and Cys-417 and -492 of EL1 and EL2, respectively (Exon 11), showed no surface hormone binding activity on intact cells, but exhibited wild type levels of total hormone binding activity when recovered from detergent-solubilized cellular extracts. This finding indicated that expression of high affinity LHR binding activity at the cell surface is independent of the acquisition of the high affinity binding conformation. Other cysteine residues, including Cys-282 (exon 10), and Cys-314 (exon 11) were not essential for hormone binding activity or plasma membrane insertion. This study demonstrates that the functional hormone binding domain utilizes all cysteines N-terminal to exon 7 and localizes the binding site to this N-terminal region of the extracellular domain.

Both major and incremental advances in the past decade have provided important insights into the structure, function, and regulation of the luteinizing hormone receptor (LHR) and its gene. The availability of the crystal structure of the extracellular domain of the follicular stimulating hormone receptor-hormone complex, and its applicability to other members of the gonadotropin receptor family, has facilitated prediction of the localized areas of specific interaction between the LHR and its cognate hormone. This has been supported by the incorporation of information derived from mutational and chimeric studies. The nature of the structural and molecular changes involved in the initiation and propagation of signaling is beginning to be revealed by the various models. It is likely that components of each domain of the LHR, the hinge region of the extracellular domain, and the LH/human chorionic gonadotrophin α-1,3 loops participate in the receptor activation. Naturally occurring and created mutations have provided fertile ground for modeling, leading to plausible proposals for the regions of the receptor that participate in receptor coupling functions. The knockout mouse model has demonstrated the lack of participation of the hormone/receptor in fetal gonadal development, in contrast with the situation in the human, where the essentiality of the LHR is exemplified by Leydig cell hypoplasia. The association of an LHR polymorphism with breast cancer age of onset and severity has been documented. Significant advances have been achieved in studies on LHR gene structure and regulation of transcription. Two independent mechanism of repression/derepression have been identified. Studies on the epigenetic control of LHR transcription have revealed the combined importance of promoter methylation status; and changes in histone acetylation/methylation in the association/release of inhibitory complexes from the promoter, and recruitment of the preinitiation complex and polymerase II. Furthermore, major advances await elucidation of the participation and molecular mechanisms of signaling pathways involved in the transcription of the LHR.

The contribution of N-linked glycosylation to the ligand binding activity of the rat luteinizing hormone receptor (LHR) was studied in wild-type and mutant LHR expressed in mammalian (COS1) cells and overexpressed in insect (Sf9) cells. The binding affinities of the holoreceptor and its truncated splice variant (form B) lacking the transmembrane domain were equivalent in both cell lines. Tunicamycin-treated transfected Sf9 cells expressed a carbohydrate-free LH receptor that lacked hormone binding activity. Functional carbohydrate chains essential for binding activity were localized to glycosylation sites at Asn-173 and Asn-152. Glycosidase treatment of the double mutant N173Q/N152Q revealed the presence of at least one additional carbohydrate chain at Asn-269, Asn-277, or Asn-291 that does not contribute to hormone binding. Asn-77 was not glycosylated, but its mutation to Gln reduced hormone binding. LHR expressed in insect cells contained only high mannose carbohydrate chains, and those located at Asn-173 and Asn-152 were sufficient for high-affinity hormone binding. Enzymatic cleavage of glycosyl chains indicated that only the proximal N-acetylglucosamine residue, which is common to high mannose and complex carbohydrate forms, is necessary for acquisition of the high affinity conformation of the receptor. The carbohydrate chains of the LHR appear to be involved in intramolecular folding of the nascent receptor rather than in its interaction with the hormone.

Studies of the mechanisms controlling the expression of the rat luteinizing hormone receptor gene were pursued by characterization of the gene structure and identification of regulatory protein binding domains in the 5'-non-coding region of the gene and of 3' non-coding functional domains responsible for generation of the major mRNA forms. The coding region of the rat LHR gene contains 10 introns and 11 exons, of which the first 10 exons comprise the hormone binding extracellular domain and exon 11, the seven transmembrane/G protein coupling module. Several alternative spliced variants of the LHR were identified that conform to deletions of complete and/or partial exons. Within the 6.2 kb of the 3'-non-coding region, two functional LHR pA domains (H1) and (H2) produce two sets of major mRNA transcripts, each coding for both holoreceptor and the form B splice variant. The H1 pA domain is unique to LHR and may represent a recombinant insertion domain. The functional efficiency of each pA domain is related to the specific pA signals, distal downstream elements, and tissue-specific factors. A TATA-less promoter region is present within the 173 bp 5'flanking region of the gene, with Initiator (Inr) elements at transcriptional start sites. Transcription is dependent on the binding of the Sp1 protein at two Sp1 domains that each contribute equally to transcript initiation. Promoter activity is regulated by at least three additional DNA domains, R (-1266 to -1307 bp), C-box (-42 to -73 bp) and M1 (-24 to -42 bp) that bind multiple trans-factors in a tissue-specific manner. Basal promoter activity is enhanced by a functional M1 domain in LHR-expressing mouse Leydig tumor cells (MLTC) but not in non-expressing CHO cells. C-box binding factors either inhibit promoter activity or block inhibition through overlapping but not identical DNA binding domains that carry AP-2 and NF-1 elements. Removal of the AP-2 element within the C-box results in MLTC-specific transcriptional activation that may involve an MTLC M1/C-box interaction. In addition, competition for C-box factors by an upstream regulatory element (R) that is only inhibitory in CHO cells, indicates that both C-box binding factors compete for this upstream (R) domain in a tissue-specific manner. Competition between the inhibitory and neutral DNA binding factors within both upstream (R) and promoter domains (C-box) could provide a mechanism for the control of LH receptor gene expression in gonadal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In previous studies we found that GHRH is secreted from rat adult Leydig cells, in which it stimulates basal and LH-induced cAMP formation and steroidogenesis. In other studies cAMP production in Sertoli cells was found to be stimulated by GHRH. In the present report, we describe a potential paracrine action of GHRH in the Sertoli cell, with stimulation of cAMP formation in cultured adult and pubertal Sertoli cells. GHRH increased FSH-stimulated cAMP production in adult and pubertal cultures in a time-dependent manner. GHRH stimulation of basal and FSH-induced extracellular cAMP formation was more prominent in pubertal than in adult cultures. Immunocytochemical studies demonstrated the presence of GHRH-like immunoreactivity in rat interstitial cells from day 4 to adult life and in the acrosomal region of early and intermediate spermatids at stages III-VI of the seminiferous epithelium cycle. Immunoreactive GHRH was not observed in late spermatids and mature sperm or in Sertoli cells at any age. These results indicate that GHRH acts synergistically with FSH to promote cAMP production in Sertoli cells in culture. Testicular GHRH of Leydig and germ cell origin may be an important paracrine regulator of Sertoli cell function. Alternatively, GHRH present in germ cells may exert stage-specific intracrine functions.

A homology model of the rat 17 alpha-hydroxylase-17,20 desmolase (CYP17) steroid binding domain was derived from the alpha/beta F supersecondary structural element of the 3 alpha/20 beta hydroxysteroid dehydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity. A CYP17 arginine-rich domain, including Arg346, Arg361 and Arg363, that has previously been shown to be important to CYP17 catalytic activity, is conserved in this HSD structural element between two HSD domains known to be important to C19 steroid binding. These two HSD motifs, in addition to a C-terminal domain at the apex of the steroid binding cavity, are also present in similar though not identical forms in the rat CYP17 sequence. The model was evaluated in terms of both hydroxylase/lyase activity and stability of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358, and Tyr431. Those amino acids located at folding junctions in the model steroid binding domain (Glu358, Arg361, and Tyr431) are each individually required to prevent degradation of the nascent protein, as well as for basic hydroxylase/lyase activity. Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a correlation exists between the length of exon 6 and the boundaries of the HSD supersecondary element. These studies demonstrate that exon 6 of the rat CYP17 is essential for CYP17 activity, and may be structurally related to the NAD-linked prokaryote alpha/beta F supersecondary element.

Review on DDX25 (DEAD (Asp-Glu-Ala-Asp) box helicase 25), with data on DNA, on the protein encoded, and where the gene is implicated.

To investigate the responsiveness of the healthy aging male hypothalamo-pituitary-gonadal axis to short term interruption of androgen negative feedback, we administered a selective nonsteroidal competitive antagonist of the androgen receptor, flutamide hydrochloride (250 mg, orally, three times daily for 3.5 days), to five older (aged 63-72 yr) and eight young (aged 21-30 yr) men. Pulsatile bioactive LH release was assessed by the rat interstitial cell testosterone bioassay in plasma sampled at 10-min intervals for 8 h overnight at baseline and after flutamide administration. Pituitary responsiveness was evaluated after two successive i.v. injections of 10 micrograms GnRH. Deconvolution analysis was used to estimate the number, amplitude, duration, and mass of bioactive LH secretory bursts and the half-life of biologically active hormone. At baseline, older men exhibited a significantly lower spontaneous bioactive LH secretory burst frequency than young men, with a median of 5 events/8 h (older) vs. 7.5 bursts/8 h (younger, P0.05). In older men, mean 8-h plasma bioactive LH concentrations increased significantly in response to flutamide (P = 0.006), and the 8-h calculated secretion rate of bioactive LH rose concomitantly. These increases were similar to responses in young men. However, during antiandrogen administration, the frequency of bioactive LH secretory bursts failed to rise in older men to the baseline value seen in young men. Moreover, older (but not young) men showed a significant prolongation of the LH secretory burst duration in response to flutamide treatment. On the other hand, the estimated half-life of endogenous bioactive LH increased significantly after flutamide ingestion in young compared to older individuals. After GnRH injections, older and young men secreted similar amounts of LH before flutamide administration, but during flutamide treatment, older men released more biologically active LH after the first GnRH stimulus [older men, 104 +/- 11 IU/L (median, 110); younger men, 44 +/- 8.4 IU/L (median, 40); P0.05]. Serum free testosterone concentrations rose significantly during flutamide exposure in both young and older men, but estradiol concentrations increased significantly only in young men. In summary, healthy older men exhibit a reduced (overnight) spontaneous bioactive LH secretory burst frequency. Pharmacological attenuation of androgen-mediated negative feedback increases mean serum free testosterone concentrations and plasma bioactive LH concentrations to a similar degree in older and young individuals, but different mechanisms operate in the two age groups. In older men, flutamide treatment amplifies the mass of bioactive LH secreted per burst by prolonging the LH secretory burst duration, whereas in young men, flutamide administration increases the apparent half-life of biologically active LHG significantly relative to values in older men. We conclude that competitive nonsteroidal blockade of the androgen receptor unmasks qualitatively altered mechanisms of increased bioactive LH release in healthy older men.

The functional importance of specific protein binding domains on transcription within the GC-rich 173-base pair promoter of the luteinizing hormone receptor gene was studied by mutagenesis and gel retardation analysis. Transcription was dependent on the presence of two Sp1 elements in the promoter domain of transfected expressing mouse Leydig tumor cells (mLTC) and nonexpressing Chinese hamster ovary cells. Mutation of two protein binding domains located downstream of the Sp1 elements (M1 and C-box) revealed tissue-specific regulation of promoter activity by each domain. Also, gel retardation studies indicated the presence of multiple trans factors that bind to the C-box and M1 domains. Removal of the AP-2 element from the C-box resulted in mLTC-specific transcriptional activation that may involve an M1/C-box interaction. In addition, competition by overlapping NF-1 and AP-2 elements was demonstrable in both the C-box and upstream R domain for separate trans factors that exhibit neutral or inhibitory functions, respectively. Competition between the inhibitory and neutral DNA binding factors within both upstream and promoter domains may be responsible for a mechanism that controls the on/off state of luteinizing hormone receptor gene expression in gonadal cells. These studies reveal a complex pattern of transcriptional regulation that may reflect targeted mechanisms for the control of luteinizing hormone receptor gene expression.

The protein association of estrogen receptor α ERα with DNA-bound SP1 and C/EBPβ is essential for the 17β-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/EBPβ, 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/EBPβ 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/EBPβ 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/EBPβ by E2 has an essential role in the transcriptional activation of the hPRLR gene.

Review on PRLR (prolactin receptor), with data on DNA, on the protein encoded, and where the gene is implicated.

Review on LHCGR (luteinizing hormone/choriogonadotropin receptor), with data on DNA, on the protein encoded, and where the gene is implicated.

In rat Leydig cells, serotonin (5HT) binds to 5HT2 receptors and stimulates the secretion of CRF which in turn acts as an inhibitor of gonadotropin-induced cAMP formation and androgen production. In the present study we defined the regulation of 5HT secretion in cultured Leydig cells. Adult Leydig cells secreted considerable quantities of 5HT (100-150 pg/10(6) cells per 10 min). The release of 5HT was acutely stimulated by hCG (ED50, 1.1 pM) with maximal stimulation at 10 pM hCG (160%). Forskolin also increased (+220%) 5HT release from cultures (ED50, 50 nM) while TPA was much less effective (+20%), indicating a major role for cAMP in gonadotropin-induced 5HT release. This was confirmed by the finding that 8-Br cAMP (1 mM) was an effective stimulus of 5HT release (+360%). Similar increases of 5HT release by hCG were observed in the absence of extracellular Ca2+. However, ionomycin was a potent stimulus of 5HT release, indicating that elevation of cytoplasmic [Ca2+] could also induce amine secretion. The 5HT content of Leydig cells ranged from 300 to 350 pg/10(6) cells, and decreased during stimulation of 5HT release. Also, immunohistochemical studies revealed specific staining of 5 HT in interstitial cells of the adult rat testis. These studies demonstrated that rat Leydig cells contain and secrete 5HT, and that 5HT release is stimulated by gonadotropin acting primarily through a cAMP-mediated mechanism.

The biological activity of circulating luteinizing hormone (LH) molecules can be assessed with high sensitivity, specificity, and precision by the in-vitro rat interstitial (Leydig)-cell testosterone (RICT) bioassay. Compared to immunoreactive estimates of the LH content of plasma, the living Leydig-cell bioassay integrates the functional in-vitro potency of all circulating LH isoforms, while simultaneously reflecting the effects of otherwise potentially confounding in-vivo antagonists of LH action on steroidogenesis. Here, we review the pathophysiological regulation by steroid hormones of bioactive LH secretion and clearance in men and women. Our investigations and those in the available literature indicate that: (i) exogenous (non-aromatizable) 5-alpha-dihydrotestosterone infusion suppresses LH bioactivity, whereas blockade of the endogenous androgen receptor with flutamide increases secretion of biopotent LH in men; (ii) endogenous androgen excess due to a masculinizing testosterone-secreting adrenal tumour in a post-menopausal woman reversibly suppressed plasma LH bioactivity markedly; (iii) exogenous oestradiol infusion will reduce whereas the antioestrogen, tamoxifen-HCl, can increase bioactive LH secretion in young men; (iv) the effect of anti-oestrogen is blunted in healthy older men; (v) endogenous hyperoestrogenism due to an adrenal oestrogen-secreting tumour in a middle-aged man with gynaecomastia profoundly but reversibly inhibited bioactive LH secretion; (vi) in post-menopausal women, exogenous oestrogen administration, whether oral (diethylstilbestrol), percutaneous (oestradiol), or intravaginal (oestradiol-impregnated silastic ring), exerts a temporally biphasic effect on basal bioactive LH secretion, i.e. acute suppression (within 24 h), subacute escape (at 5-10 days), and longer-term inhibition (30 days); (vii) during the normal menstrual cycle, bioactive LH secretion is pulsatile and both frequency and amplitude regulated; and (viii) oestrogen exposure can enhance gonadotrophin releasing hormone (GnRH)'s stimulatory action on biologically active LH secretion, resulting in so-called GnRH self-priming. Oestrogen's facilitative effects are achieved by a novel mechanism, in which oestrogen augments LH secretory burst mass and duration. Moreover, GnRH dose-LH secretory response studies show that oestrogen promotes an increase in GnRH efficacy (maximal effects) but not GnRH sensitivity (potency or half-maximally effective doses of GnRH). We conclude that steroid hormones are primary regulators of physiologically pulsatile bioactive LH secretion in healthy men and women. Moreover, steroids exert potent pathological effects on biologically active LH release in conditions of selective hyperandrogenism or hyperoestrogenism due to steroid-secreting adrenal or gonadal tumours.

Site-directed mutagenesis of a domain (amino acids 343-348) within the conserved region of rat CYP17 was performed to investigate species-specific differences between rat and human/bovine delta 4-versus delta 5-lyase activity. This domain displays substantial deviations between the rat and human/bovine/pig sequences and includes Arg346, which is known to be essential for delta 4-lyase (Kitamura, M., Buczko, E., and Dufau, M. L. (1991) Mol. Endocrinol. 5, 1373-1380) and delta 5-lyase activities. Analysis of the delta 4-lyase activity of mutant rat CYP17 cDNA expressed in nonsteroidogenic COS-1 cells revealed that substitution of Phe at position 343 in the rat with Ile of the human/bovine sequence resulted in a reduction in delta 4-lyase activity to levels in the range of the delta 5-supported reaction. This Phe343-->Ile mutant CYP17 did not exhibit changes either in delta 5-supported lyase activity or in delta 4- and delta 5-hydroxylase activities. Substitution of Asn344, Ser347, and His348 in rat CYP17 with the corresponding bovine amino acids Ser, Asn, and Arg did not enhance this effect. Thus, the reduced activity of the bovine CYP17 delta 4-lyase reaction can be mimicked in part in the rat polypeptide by the substitution of Phe343 with the bovine counterpart, Ile. Unlike the bovine CYP17-catalyzed reaction, the rat Phe343-->Ile mutant exhibited a low level lyase activity (kcat) that did not discriminate between delta 4- and delta 5-substrates. These results suggest that the presence of Phe343 enhances the delta 4-supported lyase activity possibly through stabilization of a delta 4-specific interaction.

The beta-adrenergic antagonist propranolol binds to serotonin (5HT) receptors (5HT1B > 5HT1A > 5HT2) in brain membranes. We have recently demonstrated that 5HT acts through 5HT2 receptors in rat Leydig cells to release CRF, which, in turn, inhibits hCG-stimulated cAMP production and steroidogenesis. These observations prompted us to study the effects of propranolol on CRF secretion and cAMP and testosterone production in cultured rat Leydig cells. Treatment with (-)propranolol increased CRF release and inhibited basal and hCG-stimulated cAMP and steroidogenesis, with effects evident at 0.1 microM, an IC50 of 6 microM, and reduction of stimulated levels to near basal at 100 microM. These effects of the drug were prevented by pretreatment of cultures with the 5HT2 receptor antagonist ketanserin or a CRF antagonist or antiserum. Furthermore, propranolol increased the level of 5HT in the incubation medium of cultured Leydig cells. The (+)isomer of propranolol had minor effects on these parameters. Increasing concentrations of (-)propranolol displaced the binding of [125I] +/- 1-[2,5-dimethoxy-4-iodophyryl]-2-amino propane hydrochloride (DOI), a selective 5HT2 ligand, to Leydig cell membranes (IC50, 0.2 microM), and (+)propanolol showed weaker potency. The inhibitory actions of propranolol were exerted through its blockade of the Leydig cell 5HT2 low affinity receptor, which has functional properties of an autoreceptor, with consequent increases in 5HT and stimulation of CRF release through 5HT action at the high affinity site. The serotonergic actions of propranolol were prevented by DOI, an inhibitor of 5HT actions at the high affinity site. In addition, the propranolol-induced blockade of the low affinity site further increased the cAMP and steroidogenic responses to gonadotropin over those observed with DOI treatment alone. These studies demonstrate that propranolol acts as an antagonist at the Leydig cell low affinity 5HT2 receptor and stimulates CRF release via a serotonergic mechanism, with consequent inhibition of cAMP generation and steroidogenesis. This serotonergic action of the drug could contribute to the impairment of sexual function reported during propranolol treatment in man.

Gonadotropin-regulated testicular RNA helicase is a multifunctional enzyme present in both Leydig and germ cells that is essential for the progress of spermatogenesis. GRTH gene expression is transcriptionally upregulated by human chorionic gonadotropin (hCG) via second messenger (cAMP) and androgen in Leydig cells. The regulatory region(s) in the GRTH gene that is/are required for its cell-specific expression in the testis and hCG/androgen dependent expression were investigated in transgenic mice carrying sequential deletions of 5' flanking sequences of the GRTH gene. GFP-reporter gene expression directed by the GRTH 5' flanking sequences extending to -3.6 kb was specifically located in Leydig cells and the 205 bp minimal promoter domain was sufficient for this cell-specific expression. The 1 kb (5' to the ATG codon) transgene-directed expression was markedly increased by in vivo hCG treatment. Administration of the androgen receptor inhibitor Flutamide blocked the basal and hCG stimulated GFP expression in Leydig cells. We conclude that the expression of GRTH in testicular cells is differentially regulated by its 5' flanking sequence and that the 1 kb fragment of GRTH gene contains sequences for androgen regulation of its expression in Leydig cells.

Rat GH-releasing hormone (GHRH), mainly contained in hypothalamic neurons, has also been identified in several extraneural tissues, including the gastrointestinal tract, placenta, ovary, and testis. In the testis, GHRH mRNA is ontogenically regulated, and GHRH immunoreactivity can be observed in interstitial cells and tubules, suggesting an intratesticular role for the peptide. Leydig cells in culture are able to produce hypothalamic releasing hormones, i.e. CRH, which acts as an autocrine negative regulator of Leydig cell function. In this study we investigated whether GHRH is present in Leydig cells and evaluated the role of the peptide in Leydig cell function. Adult Leydig cells in culture produced considerable amounts of immunoreactive GHRH [23.9 +/- 2.1 (+/- SE) pg/10(6) cells.30 min], and the release of the peptide was acutely stimulated by hCG. HPLC analysis of GHRH in media from basal and hCG-treated cultures showed the presence of a single peak eluting at the same retention time as that of hypothalamic rat GHRH. Radioligand binding and activation studies revealed a common receptor for vasoactive intestinal peptide (VIP) and rat GHRH in Leydig cell membrane. Specific binding of [125I]VIP to Leydig cell membranes showed the presence of a single site, with high affinity and low binding capacity. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIPrat GHRHsecretinhuman GHRH. In cultured Leydig cells, GHRH and VIP stimulated cAMP production, consistent with coupling of the receptor to the adenylate cyclase system. VIP displayed a lower ED50 than GHRH in stimulating cAMP production (P0.01), comparable with the higher binding potency of this peptide. No additive effects of VIP- and GHRH-stimulated cAMP generation were observed, suggesting that both peptides compete for the same receptor protein. GHRH and VIP had no effect on basal steroidogenesis, indicating a lack of tonic actions and compartmentalization of the peptides' effect. On the other hand, GHRH acted as a potentiator of the acute gonadotropin stimulation of testosterone production and cAMP generation. [125I]hCG binding to the Leydig cells in culture showed that GHRH was unable to affect the number or affinity of binding sites for hCG, indicating that the GHRH-sensitizing effect on LH action is beyond the level of gonadotropin binding and possibly is through the facilitation of LH receptor coupling functions.(ABSTRACT TRUNCATED AT 400 WORDS)

CRF is produced in the Leydig cells and acts as a negative autocrine regulator of Leydig cell function. To clarify the hormonal control of CRF secretion by Leydig cells, we evaluated the participation of serotonin (5HT) and serotonin agonists in the release of CRF from Leydig cells and their effects on hCG-induced cAMP generation and steroidogenesis. Serotonin stimulated CRF secretion up to 4-fold above basal levels and inhibited basal and hCG-stimulated cAMP generation and testosterone production (ID50, 1 nM). The inhibitory action of 5HT was prevented by a CRF antibody and the alpha-helical CRF-(9-41) antagonist. The selective 5HT2 receptor agonist (+-)1-[2,5-dimethoxy-4-iodophyryl]2-amino propane hydrochloride (DOI) also stimulated CRF secretion and inhibited hCG-stimulated cAMP generation and testosterone production to control levels (ID50, 7 microM). Serotonergic 5HT1A, 5HT1B/1C, 5HT1D, and 5HT3/5HT2 agonists were less effective inhibitors of hCG-stimulated cAMP and testosterone production, while agonists for the 5HT3 receptor had no effect. [125I]DOI binding studies in Leydig cells demonstrated two sets of receptors with Kd values in the nanomolar and micromolar range, with low and high capacities, respectively. The low affinity site differed from that of brain receptors (Kd, 4.2 nM) and displayed higher binding capacity (50-fold). The selective 5HT2 receptor antagonist ketanserin prevented CRF stimulation and blocked the inhibitory actions of 5HT and DOI, while the alpha 1-adrenergic antagonist prazosin had no effect. Also, treatment of cells with ketanserin increased sensitivity to hCG and raised maximal cAMP and testosterone production. 5HT was a more effective stimulus than hCG in stimulating CRF secretion, and gonadotropin-induced CRF release was inhibited by ketanserin. Inhibitory effects of exogenous CRF were demonstrable after blockade of 5HT action by ketanserin. The inhibitory actions of 5HT were unaffected by pertussis and cholera toxins and were reversed by the addition of 8-bromo-cAMP. These results demonstrate that 5HT acts on 5HT2 receptors in Leydig cells that are distinct from those in the brain to stimulate CRF secretion through a pertussis toxin-insensitive G-protein. This action of 5HT is predominantly mediated by the low affinity 5HT2-binding site and requires full occupancy for maximal CRF stimulation, indicating the absence of spare receptors. 5HT-stimulated CRF inhibits basal and hCG-induced cAMP generation and steroidogenesis. Furthermore, 5HT mediates the stimulatory action of LH/hCG on CRF secretion from Leydig cells and, thus, participates in a negative autoregulatory loop to limit the testosterone response to the gonadotropic stimulus.

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.

The luteinizing hormone receptor (LHR) transcription is subject to an epigenetic regulatory mode whereby the proximal Sp1 site acts as an anchor to recruit histone deacetylases (HDAC)1/2 and the Sin3A co-repressor complex. This results in promoter-localized histone hypo-acetylation that contributes to the silencing of LHR transcriptional expression. Chromatin changes resulting from site-specific acetylation and methylation of histones regulate LHR gene expression. The HDAC inhibitor TSA-induced cell-specific phosphatase release from the promoter, which serves as an 'on' mechanism for Sp1 phosphorylation by phosphatidylinositol 3-kinase/protein kinase Czeta (PI3K/PKCzeta) at Ser641, leading to p107 repressor derecruitment and LHR transcriptional activation. The methylation status of the promoter provides another layer of modulation in a cell-specific manner. Maximal derepression of the LHR gene is dependent on complete DNA demethylation of the promoter in conjunction with histone hyperacetylation and release of repressors (p107 and HDAC/Sin3A). Independently, the PKC-alpha/Erk pathway, participates in LHR gene expression through induction of Sp1 phosphorylation at Ser site(s) other than Ser641. This causes dissociation of the HDAC1/mSin3A from the promoter, recruitment of TFIIB and Pol II, and transcriptional activation. Collectively, these findings demonstrate that LHR gene expression at the transcriptional level is regulated by complex and diverse networks, in which coordination and interactions between these regulatory effectors are crucial for silencing/activation of LHR expression.

Site directed mutagenesis of the rat ovarian luteinizing hormone (LH) receptor cDNA was performed at each of the six potential N-linked glycosylation sites to determine the effect of putative carbohydrate chains on the activity of the membrane receptor. The conversion of Asn173 to Gln resulted in the total loss of hormone binding to the surface of the transfected cell. Mutant receptors synthesized with substitutions at the remaining potential N-linked glycosylation positions of 77, 152, 269, 277 and 291 revealed no significant change in the hormone affinity. However Asn77Gln and Asn152Gln exhibited significant decreases (approximately 80%) in the number of high affinity hormone binding sites. The changes in hormone binding activity upon elimination of the potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.

The luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor gene was isolated from rat liver genomic libraries and spanned at least 75 kilobase pairs from nucleotide -2057 of the 5'-flanking region to the 3'-noncoding end. The structural configuration of the coding region of the LH receptor gene consists of 11 exons separated by 10 introns that are all located within the putative extracellular domain prior to the first transmembrane region. The 5'-noncoding region contains several potential TATA boxes and SP1 promoter binding sites, as well as six palindromic elements, potential intron/exon splice junctions, and two extended open reading frames in frame with the initiation codon. Primer extension studies indicate the presence of multiple transcriptional initiation sites. Truncated forms of the LH/hCG receptor conform to alternative splicing patterns that are consistent either with the deletion of complete exons or alternate acceptor sites within exons. All splice site junctions correspond to known donor and acceptor consensus sequences. Exons 2-8 approximate the regions of the 14 consecutive 20 amino acid repeated motifs present in the LH, thyrotropin-stimulating hormone, and follicle-stimulating hormone receptor cDNAs, with the exception of a six to eight amino acid shift in each motif. Exons 1, 9, 10, and 11, do not conform to this repetitive intronic motif. These exons, however, contain important structural elements including the conserved cysteines (exons 1, 9, 11), the soybean lectin motif (exon 9), the seven-transmembrane domain with cytoplasmic G protein coupling elements (exon 11), and three putative N-linked glycosylation sites (exon 10), consistent with preservation of significant functional domains within single exons. Exon 10 and the beginning of exon 11 along with the lectin motif are unique to the LH receptor and may be involved in specific association with the hormonal ligand. The homologous regions with other members of the glycoprotein receptor family encoded by exons 2-8, and the common amino acid motif that contains 3 conserved cysteines immediately prior to the first transmembrane region, may be involved in common hormonal interactions and in coupling functions, respectively.

Estrogen produces time-dependent bidirectional effects on the GnRH-stimulated release of immunoactive LH in various species. To examine estrogen's regulation of biologically active LH secretion in response to pulsatile stimulation by GnRH, we studied estrogen-deficient postmenopausal women basally and during treatment with diethlystilbesterol (DES; 1 mg, orally, daily). Basal and GnRH-stimulated plasma concentrations of bioactive LH were assayed by the in vitro rat interstitial cell testosterone bioassay. GnRH-promoted LH secretory bursts in response to two consecutive stimuli were quantitated by multiple parameter deconvolution analysis. Basal half-lives of LH averaged 171 +/- 17 min (immunoactive) and 223 +/- 10 min (bioactive). Analysis of variance revealed a significant decrease in mean basal plasma bioactive LH concentrations on days 10 and 30 of DES treatment. Mean serum immunoactive LH concentrations fell similarly. DES significantly increased the half-life of immunoactive LH (days 5 and 10), but did not change that of bioactive LH. GnRH self-priming of bioactive LH secretion (increased LH secretory peak 2 compared to peak 1) was demonstrated, with a maximal value on day 10 of DES treatment. In addition, the ratio of the mass of bioactive to immunoactive LH secreted in response to the first GnRH pulse was significantly enhanced by estrogen on day 5, whereas that after the second pulse of GnRH was significantly suppressed on day 30 of DES. The self-priming action of GnRH on bioactive LH release evident in the presence of oral DES was corroborated in a separate group of six women, who were treated for 30 days with 17 beta-estradiol via an intravaginally placed Silastic ring. In conclusion, we infer that estrogen exerts a highly selective effect on the gonadotroph secretory process, such that successive GnRH stimuli result in an increase in the maximal rate and mass of secretion of biologically active LH.

Gonadotropin-regulated long chain fatty acid Acyl-CoA synthetase (GR-LACS), is a member of the LACS family that is regulated by gonadotropin in the rat Leydig cell (LC). Its mouse/human homologs, lipidosin/bubblegum, have been suggested to participate in X-linked adrenoleukodystrophy (X-ALD), an adreno/neurodegenerative disorder with accumulation of very long chain fatty acids (VLCFA) in tissues and plasma. To further gain insights into its regulatory function, a GR-LACS/lipidosin null mouse was generated. No apparent phenotypic abnormalities were observed in the X-ALD target tissues (brain, testis, adrenal). Nuclear inclusions seen in mice >15 month-old, were present in LC of 9 month-old GR-LACS(-/-) mice. LC of the null mice showed refractoriness to the gonadotropin-induced desensitization of testosterone production that is observed in adult animals. LCFAs were moderately increased in the testis, ovary and brain, but not in the adrenal gland of GR-LACS(-/-) mice, with no major changes in VLCFA. No change in LACS activity was observed in these tissues, suggesting a compensatory mechanism exhibited by other LACS members. The GR-LACS(-/-) model did not support its association with X-ALD. These studies revealed a role of GR-LACS in reducing the aging process of the LC, and its participation in gonadotropin-induced testicular desensitization of testosterone production.

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 ζ (PKCζ) 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/PKCζ 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/PKCζ and phosphatases could be critical for up- and down-regulation of LHR gene expression in physiological and pathological settings.

The diverse functionality of prolactin and the wide expression of the prolactin receptor suggest a complex system regulated by this polypeptide hormone. Different hormone and receptor forms, as well as differential signal transduction pathways, contribute to the functional diversity of prolactin's actions. The heterogeneity of rat prolactin receptor gene transcripts in their 5′-untranslated region has led to the recognition of multiple and tissue-specific utilization of prolactin receptor gene promoters in gonadal and non-gonadal tissues. These findings have provided insights into the molecular bases for the diversity of prolactin's actions. It is now clear that cellular responsiveness to prolactin can be regulated through differential promoter control of the expression of the surface receptors for prolactin in different target tissues.

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.

In addition to well known direct stimulatory and potentiatory actions of forskolin, we have previously reported that low doses of this diterpene (10 −9 , 10 −12 M) markedly inhibit the production of cAMP and testosterone in rat Leydig cells through a pertussis toxin sensitive G-protein (A. Khanum and M. L. Dufau, J. Biol. Chem. 261 , 1986). A different type of inhibitory effect of forskolin is described in this study. Forskolin (10 −5 M) markedly stimulates basal adenylate cyclase activity (about 200%) in rat Leydig cell membranes and potentiates the stimulatory effect of gonadotropin (10 −9 , 10 −7 M) on adenylate cyclase in presence or in absence of GTP (10 −5 M). Similarly a time-dependent stimulation of forskolin (10 −5 M) alone is noted on all cAMP pools and testosterone production. Using a supramaximal steroidogenic dose of hCG (0.26 nM) or choleragen (0.1 μM), forskolin potentiates the gonadotrophin and toxin-induced responses of all cAMP pools significantly while inhibiting testosterone production. Moreover, forskolin also inhibits 8-Bromo-cAMP stimulated steroidogenesis. In contrast, pregnenolone synthesis was not altered by the diterpene. We have demonstrated in this study that the inhibitory effect of high doses of forskolin on steroidogenesis is distal to cAMP generation, and resulted from a steroidogenic block residing beyond pregnenolone synthesis.

A rat testicular luteinizing hormone (LH) receptor cDNA containing a 266-base pair deletion resulting in the omission of the 1st transmembrane region and truncation of the open reading frame was isolated using a rat ovarian LH receptor cDNA probe. Comparison of this clone with a restriction fragment from the LH receptor genomic DNA revealed potential alternative splice sites following the consensus sequence TTXCAG that is present at an intron acceptor splice site and also within the next exon, accounting for the specific deletion mutation observed in this cDNA. Expression of the testicular cDNA in COS1 cells resulted in synthesis and secretion of a soluble binding protein with high affinity and specificity for LH and human chorionic gonadotropin. These studies have demonstrated that the LH receptor gene contains intron(s) within the region coding for the extracellular domain of the molecule, which determine the nature and generation of LH receptor isoforms. Expression of the soluble form of the LH receptor has indicated that the amino-terminal extracellular region plays a major role in gonadotropin binding. These features of the LH receptor are distinct from those of most other G protein-coupled receptors, which are intronless and contain their binding sites within the transmembrane region rather than the extracellular domain.

Two novel lactogen receptor cDNA clones (2.1 and 1.2 kb) were isolated from a rat ovarian cDNA library. Nucleotide sequence of the 2.1 kb clone codes for a 610 aa receptor (nonglycosylated mol. wgt. 66,000 D) with an extracellular domain, a transmembrane region and an intracellular domain, and exhibited significant overall similarity with the rat liver receptor (310 aa) and both rabbit mammary and human hepatoma receptors (616 and 622 aa). However, the ovarian lactogen receptor sequence contains a unique cytoplasmic domain of 110 aa and consensus sequences for both a tyrosine phosphorylation site and an ATP/GTP type A binding site, and thus has potential for signal transduction and mitogenic activity. The 1.2 kb clone codes for a truncated binding form of 150 aa that is identical with the ovarian long form over only the first 130 residues, and lacks the transmembrane region. Differences between long and short forms of the ovarian lactogen receptors and the truncated liver species may result from alternative splicing. The prolactin holoreceptor gene(s) has the potential for producing several receptor subtypes that differ in tissue-specific expression, size, compartmentalization and mode of signal transduction, and may subserve the divergent functions of prolactin in its several target cells.

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.