Receptors and Actions of Peptide Hormones and Regulatory Proteins in Endocrine Mechanisms

Maria L. Dufau, MD, PhD, Head, Section on Molecular Endocrinology
Chon-Hwa Tsai-Morris, PhD, Staff Scientist
Juying Dong, PhD, Postdoctoral Fellow¹
Ravi K. Gutti, PhD, Postdoctoral Fellow¹
Junghoon Kang, PhD, Postdoctoral Fellow
Mingjuan Liao, PhD, Postdoctoral Fellow
Yuji Maeda, MD, PhD, Postdoctoral Fellow¹
Aamer Qazi, PhD, Postdoctoral Fellow¹
Hisashi Sato, MD, PhD, Postdoctoral Fellow
Yili Xie, PhD, Postdoctoral Fellow
Masato Fukushima, MD, PhD, Adjunct Scientist
Ying Zhang, PhD, Research Associate¹

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

Epigenetic control of LHR transcription

Liao, Zhang, Dufau

Our studies have revealed that transcription of LHR is subject to repression and derepression through complex modulation at both genetic and epigenetic levels. In breast (MCF-7) and placenta (JAR) cancer cells that express LHR, we have identified several modes of regulation centered on the Sp1-/Sp3-binding domains of the LHR promoter. Furthermore, our studies have determined signal transduction pathways involved in the regulation of LHR.

LHR transcription is subject to an epigenetic regulatory mode whereby the proximal Sp1 site acts as an anchor to recruit HDAC 1/2 and the mSin3A co-repressor complex, resulting in promoter-localized histone hypoacetylation and limited Pol II recruitment, which partly contribute to the gene silencing observed in MCF-7 and JAR cells. Site-specific methylation of histones H3 and H4 acts in concert with changes in histone acetylation levels to regulate the expression of LHR. The methylation status of the LHR promoter provides another layer of modulation in the control of LHR expression in a cell-specific manner. Maximal derepression of LHR is dependent on complete DNA demethylation of the promoter in conjunction with histone hyperacetylation. We have shown that the phosphadildyl inositol 3-kinase pathway (PI3K), protein kinase C-zeta (PKC-zeta) signaling pathways, and Ser/Thr phosphatases PP1 and PP2A are key participants in derepression of LHR in cancer cells induced by Trichostatin A (TSA). Phosphorylation of Sp1 at Ser 641 by PI3K/PKC-zeta causes dissociation of repressor protein p107 from the LHR promoter, inducing gene activation. On the other hand, TSA-mediated chromatin changes cause PP1 and PP2A release from the promoter and function as an “on” mechanism for Sp1 phosphorylation by PI3K/PKC-zeta, leading to p107 derecruitment and LHR activation. The coordinated balance between PI3K-PKC-zeta (constitutive or induced activity) and phosphatase(s) is critical for up- or downregulation of LHR expression through their effects on Sp1 phosphorylation status (Figure 4.1A). We have also demonstrated the participation of the PKC-alpha/Erk pathway, where its activation by phorbol esters enhances LHR expression by inducing Sp1 phosphorylation by PKC-alpha/Erk at serine residue(s), thus causing dissociation of the HDAC1/mSin3A complex from the promoter and transcriptional activation (Figure 4.1B). Dissociation was independent of promoter-associated chromatin changes. Collectively, our findings indicate that complex and diverse networks regulate LHR expression at the transcriptional level and that coordination and interaction among diverse regulatory effectors are crucial for silencing/activation of LHR expression.

Dufau ML, Tsai-Morris C-H. The luteinizing hormone receptor. In: Payne A, Hardy M, eds. Contemporary Endocrinology: The Leydig Cell in Health and Disease. Humana Press, 2007;227-252.
Liao M, Zhang Y, Dufau ML. Protein kinase C alpha-induced derepression of the human luteinizing hormone receptor gene transcription through ERK-mediated release of HDAC1/Sin3A repressor complex from Sp1 sites. Mol Cell Endocrinol 2008;22:1449-1463.
Zhang Y, Liao M, Dufau ML. Phosphatidylinositol 3-kinase/protein kinase C zeta-induced phosphorylation of Sp1 and p107 repressor release have a critical role in histone deacetylase inhibitor-mediated derepression of transcription of the luteinizing hormone receptor gene. Mol Cell Biol 2006;26:6748-6761.
Zhang Y, Liao M, Dufau ML. Unlocking repression of the human LH receptor gene by trichostatin A-induced cell-specific phosphatase release. J Biol Chem 2008;283:24039-24046.

Figure 4.1 Transcriptional regulation of the LH receptor
A. Schematic representation of functional roles of PP1 and PP2A and signaling events participating (PI3K/PKCζ) in TSA-induced LHR activation

The TSA-induced changes of the chromatin modification status at the LHR promoter favor cell-specific release of PP1 (MCF-7 cells) or PP2A (JAR cells) from the promoter. This in turn facilitates Sp1 phosphorylation by PI3K/PKCζ, which triggers the dissociation of p107 from the promoter and LHR activation.

B. Participation of PKCα/ERK pathway in transcriptional activation of the LH receptor

The transcriptional activator Sp1 is the anchor site of the HDAC1/2/mSin3A repressor complex within the LHR promoter. Endogenous PKCα activation by PMA (phorbol-12-myristate-13-acetate) raises LHR promoter activity and transcript levels through the ERK downstream pathway. Phospho-ERK (pERK) interacts with and phosphorylates Sp1 at serine residue(s), causing the release of the HDAC1/mSin3A repressor complex from the promoter. Such release facilitates histone acetylation, recruitment of TFIIB and Pol II, and derepression of LHR. mSin3A functions as a co-repressor for HDAC1 but not for HDAC2 in the regulation of LHR expression.

Gonadotropin-regulated testicular genes

Tsai-Morris, Gutti, Sato, Fukushima, Maeda, Dufau

We previously identified a novel gonadotropin-regulated testicular helicase (GRTH/Ddx25) present in the nucleus and cytoplasm of pachytene spermatocytes and round spermatids (Sheng Y, et al., J Biol Chem 2006;281:35048). GRTH is a component of messenger ribonucleoprotein particles, which transport target mRNAs for storage in chromatoid bodies in the cytoplasm of spermatids. These messages are released for translation during spermatogenesis. GRTH is also found in polyribosomes, where it regulates the translation of mRNAs encoding spermatogenic factors. GRTH^−/− male mice are sterile owing to spermatid arrest (step 8 of spermatogenesis), with marked diminution of chromatoid bodies and failure of spermatids to elongate. We found that the transcription of messages in spermatids of these mice was not altered but that their translation was selectively abrogated. We identified two GRTH species: 56 kDa and phosphorylated 61 kDa forms. The 56-kDa nuclear species interacted with CRM1 and participated in nuclear export of specific messages. The phosphorylated cytoplasmic 61-kDa species was associated with polyribosomes and regulated the translation of genes that govern spermatogenesis. GRTH/Ddx25 is thus a multifunctional RNA helicase that is an essential regulator of sperm maturation.

Apoptosis is a major event in prepubertal testis but occurs only sporadically in adult testis. In GRTH^−/− mice, major apoptosis occurred in spermatocytes entering the metaphase of meiosis before the appearance of round spermatids and was dose-related, with apoptotic cells observed in GRTH^+/− mice. In GRTH^−/− mice, activation of survival mechanisms and/or the participation of other helicases might permit progression of the remaining viable meiotic cells to the point of arrest in haploid germ cells with no evidence of ultrastructural changes. The striking apoptosis observed in spermatocytes of adult GRTH^−/− mice underscored the important role of GRTH in determining the survival and apoptotic fate of adult germ cells. In comparative studies of spermatocytes from wild-type and GRTH^–/– mice, we found pro- and anti-apoptotic factors to be under GRTH regulation. Knockout mice displayed lower levels of Bcl-2 and Bcl-xL (anti-apoptotic factors), and phospho-Bad, higher levels of Bid, Bak, Bad (pro-apoptotic), as well as release of cytochrome c. We also observed an increase in Smac, a competitor of inhibitor apoptotic proteins that release caspases. These changes caused more cleavage of caspases-9 and caspase-3, activation of caspase-3, and increases in cleavage products of PARP [poly (ADP-ribose) polymerase]. The half-life of caspase-3 transcripts was markedly elevated in GRTH^–/–, indicating that GRTH had a negative effect on the caspase’s mRNA stability. GRTH-mediated apoptotic regulation was further supported by its selective association with mRNAs of certain pro- and anti-apoptotic factors. GRTH also associated with caspases-9 and -3 and PARP mRNAs, the NF-κB cytoplasmic inhibitor IκBα, and the nuclear regulators p300 and HDAC1. IκBα, which sequesters NF-κB from its transcriptional activation of pro-apoptotic genes, was highly elevated in GRTH^–/– mice, and its phospho-form, which promotes its dissociation, was reduced. The increase of HDAC1 and abolition of p300 expression in GRTH^–/– indicated a nuclear action of GRTH on the NF-κB–mediated transcription of anti-apoptotic genes. GRTH also regulated the associated death-domain pathway and caspase-8–mediated events. Our studies demonstrate that, as a component of mRNP particles, GRTH acts as a negative regulator of the tumor necrosis factor receptor 1 and caspase pathways and promotes NF-κB function to control apoptosis in spermatocytes of adult mice (see Figure 4.2).

Figure 4.2 Summary of GRTH impact in apoptotic pathways in germ cells
GRTH regulates subsets of pro-apoptotic and anti-apoptotic factors involved in mitochondria, death receptor, and NF-κB signaling pathways in germ cell apoptosis. Left, GRTH acts as a negative regulator to prevent anti-apoptotic factor expression (x) during spermatogenesis. In the absence of GRTH, expression of anti-apoptotic factors increased (solid oval) while that of pro-apoptotic factors was suppressed (stippled oval) in the mitochondria-controlled apoptotic pathway (and the subsequent cytochrome c release) and downstream caspase-3 and caspase-9 were activated (middle). Regarding the TNFα/TNF-R1 signaling pathway, the lack of GRTH increased expression of the TNF-R1–associated adaptor protein TRADD, which could cause downstream caspase-8 activation (solid oval). Other cytoplasmic adaptor proteins are apparently not involved in GRTH’s regulatory action (open oval). Right, NF-κB pathway. GRTH could have an impact on NF-κB action at both the cytoplasmic and nuclear levels. In the cytoplasm, lack of GRTH (–) caused a decrease in IKKα/β kinase complex expression and IκB phosphorylation; consequently IκB’s proteasomal-dependent degradation is curtailed. This favors non-phosphorylated IκBα/β association with NF-κB, which reduces NF-κB’s import into the nucleus and presumably the transcriptional activation of anti-apoptotic genes. Lack of GRTH could also alter the acetylation status of NF-κB in the nucleus by increasing HDAC and decreasing acetylase p300 expression, resulting in a reduction in anti-apoptotic gene transcription and apoptosis. The presence of GRTH (+) promotes cell survival. P denotes phosphorylation.

Given that GRTH is essential for spermatogenesis in mice, we are investigating its potential role in male infertility. In a Japanese population, an SNP- (single-nucleotide polymorphism) scanning study identified a polymorphic mis-sense mutation at exon 8 (nt 725G→C, aa242, R→H) and a silent mutation at exon 11 (nt 1245, C→G, aa 415, L→L) that might be associated with non-obstructive azoospermia. The unique heterozygous mis-sense mutation Arg²⁴²His in exon 8 could affect the post-translational modification of the expressed protein as it cannot undergo phosphorylation and consequently could affect translational regulation of essential genes during germ cell development. In a population from Western China, infertility has been associated with a silent mutation (nt 1194 C→T) of the GRTH gene that might alter RNA splicing and increase the risk of impaired spermatogenesis. In contrast to this finding, we found no statistically significant change in this allele in Japanese men with non-obstructive azoospermia. Interestingly, we also noted that the SNPs of exons 8 and 11 in Japanese patients were not evident in Chinese patients.

Dufau ML, Tsai Morris C-H. Gonadotropin-regulated testicular helicase (GRTH/DDX25) an essential regulator of spermatogenesis. Trends Endocrinol Metab 2007;18:314-320.
Gutti R, Tsai-Morris C-H, Dufau ML. Gonadotropin regulated testicular helicase (DDX25) an essential regulator of spermatogenesis, prevents testicular germ cell apoptosis. J Biol Chem 2008;283:17055-17064.
Li J, Sheng Y, Tang P-Z, Tsai-Morris C-H, Dufau ML. Tissue-cell- and species-specific expression of gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) in gonads, adrenal and brain. Identification of novel forms in the brain. J Steroid Biochem Mol Biol 2006;98:207-217.
Tsai-Morris C-H, Koh E, Dufau ML. Differences in gonadotropin-regulated testicular helicase (GRTH/DDX25) single nucleotide polymorphism between Japanese and Chinese populations. Hum Reprod 2008;23:2611-2613.
Tsai-Morris C-H, Koh E, Sheng Y, Maeda Y, Gutti R, Namiki M, Dufau ML. Polymorphism of the GRTH/DDX25 gene in normal and infertile Japanese men: a missense mutation associated with loss of GRTH phosphorylation. Mol Human Reprod 2007;13:887-892.

Prolactin receptor

Xie, Qazi, Dong, Kang, Tsai-Morris, Dufau; in collaboration with Hassan

In earlier studies, we demonstrated that short forms (SF) of the prolactin (PRL) receptor—S1a and S1b—that have an abbreviated cytoplasmic domain, can silence PRL-induced activation of gene transcription by the long form (LF). This results from LF/SF heterodimerization and the absence of cytoplasmic (C) sequences in the SF partner, which are essential for STAT (signal transducer and activator of transcription) activation. Compared with the LF, S1b does not contain the conserved cytoplasmic motif structure beyond the Box1 JAK2 site. Our studies also demonstrated that hetero- and homodimerization of hPRLR can occur independently of ligand and that PRL is a conformational modifier inducing JAK (Janus kinase) /STAT signaling. In recent studies, we provided evidence, derived from both functional analysis and computer simulations, that the four conserved Cys residues in the D1 subdomain of the extracellular domain (EC) of the hPRLR are essential for maintaining the correct conformation of dimerized PRLR and for the dominant negative action of the short form (S1b) on ligand-induced long-form–mediated STAT5-dependent transcriptional activation. Our studies have demonstrated that the inhibitory action of wild-type S1b on LF function was abolished upon disruption of these intramolecular disulfide bridges. The lack of inhibitory action of mutated S1b forms might result from their higher affinity to form homodimers than the wild type and from a significant reduction in their affinity to form heterodimers with the LF. The differential preference of mutated S1b forms to form homodimers rather than heterodimers contrasts with the wild type’s S1b propensity to form heterodimers with LF. This in turn facilitates the formation of LF homodimers competent to mediate PRL-induced downstream signaling. Thus, the EC Cys mutant lacks the inhibitory action normally observed in the wild-type SF.

We recently studied the impact of the Cys mutation on JAK2 phosphorylation. In the absence of hPRL, we observed a basal level of JAK2 phosphorylation in cells transiently transfected only with wild-type S1b but not with the S1b Cys mutant (S1b4x). Following hPRL treatment, we observed significantly higher JAK2 phosphorylation than control in cells transiently transfected with wild-type S1b but not with mutant S1b4x. Similarly, cells stably transfected with S1b displayed JAK2 basal phosphorylation that was stimulated (2–3 fold) upon exposure to PRL. In addition, we observed a significant amount of basal JAK2 phosphorylation in cells stably expressing LF transfected with S1b. In contrast, JAK phosphorylation was not present in control cells stably expressing LF or upon transfection of S1b4x. Co-immunoprecipitation (Co-IP) studies of cells transiently co-transfected with either S1b or S1b 4x with JAK2 followed by Western analysis showed loss of JAK2 association with S1b4x compared with wild-type S1b. It was of interest to observe basal levels of JAK2 phosphorylation with S1b in the absence of PRL stimulation in cells both transiently and stably expressing the short-form S1b. Thus, in the absence of hormone, the abbreviated length of the cytoplasmic domain seems to favor basal JAK2 phosphorylation, presumably via auto- and/or transphosphorylation. Alternatively, other associated kinase modules may be involved. The apparent loss of JAK2 binding to S1b4x compared with the S1b wild type observed in the Co-IP study revealed a functional link between the EC domain conformation and the JAK2 association with its docking site at Box 1. Our findings indicate an effect of the mutation (S1b4x) on the conformation of the SF homodimer and heterodimer with LF that compromised the JAK2 basal phosphorylation activity and on the JAK2 association observed in wild-type S1b. Overall, our studies demonstrate the relevance of the intramolecular disulfide bridges of the prolactin receptor for S1b-inhibitory action on PRL-induced LF-mediated STAT5-dependent action and for cytoplasmic events related to JAK2 association/activity.

Dong J, Tsai-Morris C-H, Dufau ML. A novel estradiol/estrogen receptor α-dependent transcriptional mechanism controls expression of the human prolactin receptor. J Biol Chem 2006;281:18825-18836.
Qazi AM, Tsai-Morris C-H, Dufau ML. Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization. Mol Endocrinol 2006;20:1912-1923.

¹ No longer at the NIH.

Collaborators

Sergio A. Hassan, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
Eitsu Koh, MD, PhD, Kanazawa University, Kanazawa, Japan
Mikio Namiki, MD, PhD, Kanazawa University, Kanazawa, Japan

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