Glycoprotein Hormone Alpha-subunit Research Articles

Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies – A Cautionary Reminder

Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. IN VITRO models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50 ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to -346, but eliminated in the promoter truncated upstream of -244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between -244 and -346. Enhancement was maintained with the addition of a second constitutive reporter (bos beta-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos beta-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site.

Expression of MSX1 in Human Normal Pituitaries and Pituitary Adenomas

Transcription factors play specific roles in the development and differentiation of normal pituitary tissues and pituitary adenoma. The transcription factor, muscle segment homeobox 1 (MSX1), which belongs to the homeobox gene family, binds the promoter region of the glycoprotein hormone alpha-subunit (SU) in TSH-producing cells in the mouse pituitary and regulates alpha-SU expression. The present study investigated MSX1 expression in the normal human pituitary. In addition, 50 pituitary adenomas were examined using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) to clarify the role of MSX1 in the development and functional differentiation of pituitary adenoma cells. In the normal pituitary, MSX1 was predominantly expressed in the cytoplasm of GH-producing cells. Furthermore, MSX1 immunoreactivity was observed in the cytoplasm of some alpha-SU-producing cells. It is interesting to note that, in the pituitary adenoma, MSX1 was expressed in the nucleus of GH- and TSH-producing adenomas. RT-PCR using RNA extracted and purified from formalin-fixed paraffin-embedded pituitary adenoma specimens revealed MSX1 mRNA expressed in GH- and TSH-producing adenomas. Immunoelectron microscopy demonstrated MSX1 localized at intranuclear heterochromatin and euchromatin, which suggests transcriptional activity. These results suggest that MSX1 plays a specific role in human pituitary adenoma.

Ovarian hyperstimulation syndrome caused by an FSH-secreting pituitary adenoma

A 40-year-old woman presented with galactorrhea and oligomenorrhea. She had a history of multiple ovarian cysts and pelvic pain. Laboratory evaluation included measurements of the levels of estradiol, follicle-stimulating hormone, luteinizing hormone, prolactin, thyroid-stimulating hormone, free endogenous T4, the glycoprotein hormone alpha subunit, cortisol, adrenocorticotropic hormone, and insulin-like growth factor I. Radiological studies included MRI of the pituitary. Ovarian hyperstimulation syndrome caused by a pituitary adenoma, secreting follicle-stimulating hormone. The patient underwent trans-sphenoidal resection of the adenoma, with subsequent normalization of hormonal values and symptoms.

Non-Gonadotropin-Releasing Hormone-Mediated Transcription and Secretion of Large Human Glycoprotein Hormone α-Subunit in Human Embryonic Kidney-293 Cells

To identify genes that are most responsive to a sustained activation of a G(s) protein-coupled receptor, HEK293 cells were stably transfected with the beta(2)-adrenergic receptor and stimulated with agonist isoproterenol (1 mum). A microarray study indicated that the gene with the highest stimulation index (500-fold) encoded the common alpha-subunit of human glycoprotein hormones (GPHalpha). Induction of GPHalpha transcription in response to cAMP elevations resulted in a dramatic increase (600-fold) of protein secretion as shown by RT-PCR and a highly specific time-resolved immunofluorometric assay. Cloning and sequencing of the GPHalpha cDNA and mass spectrometric analysis of HPLC-purified GPHalpha derived from serum-free HEK293-beta(2)-adrenergic receptor-stimulated cells verified the nature of the molecule. Enzymatic deglycosylation with subsequent Western blots revealed that this was a large hyperglycosylated form of GPHalpha that had not been associated with a beta-subunit previously. This uncombined variant is known to be either cosecreted with GPHs from the pituitary, the placenta, and a variety of tumors or secreted without GPHs from APUD cells and rare tumors. Moreover, it is similar to GPHalpha found at high concentrations in seminal plasma. As shown by a panel of endogenous or transfected G protein-coupled receptors in HEK293 cells, the expression of large GPHalpha was controlled by G(s)- and G(q)- but not G(i)-dependent receptors and mediated via cAMP and Ca(++) release. This suggests that Gq- or G(s)-coupled receptors other than the classical GnRH receptor may play a role in the regulation of nonpituitary, nonplacental GPHalpha secretion under physiological and pathological conditions.

Single-Stranded DNA-Binding Proteins (SSBPs) Regulate the Abundance of the LIM-Homeodomain Protein LHX2 and Augment Its Transcriptional Activity.

A small family of proteins with putative single-stranded DNA-binding activity has been shown to augment the biological actions of LIM-homeodomain (LIM-HD) transcription factors through the mediation of the LIM domain-binding protein LDB1. We recently established that two of these SSBPs, Ssbp2 and Ssbp3, were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing transcription factors Tal1, E2A, and Gata-1 and LIM-only protein Lmo2 and showed that Ssbp2 stimulated E box-GATA DNA-binding activity by inhibiting Ldb1 ubiquitination and Ldb1 and Lmo2 turnover (Genes & Dev. 21:942–955, 2007). Since LIM-HD proteins are substrates of different E3 ubiquitin ligases than LIM-only proteins and have the additional property of binding DNA, we sought to determine the effect of SSBPs on LIM-HD expression and function. Using the prototype LIM-HD protein Lhx2 and one of its best-characterized target genes, Cga, for analysis, we found that an Ssbp3-, Ldb1-, and Lhx2-containing complex associated with an Lhx2 binding element in the Cga promoter in vitro and in mouse pituitary cells (alphaT3-1 cell line) in vivo. We then showed that enforced expression of Ssbp2 and Ssbp3 in alphaT3-1 cells increased Lhx2 and Ldb1 protein abundance, Lhx2 DNA-binding activity, and Cga expression and augmented Lhx2 transcriptional activity in an Ldb1-dependent fashion. While Lhx2-Ldb1-Ssbp3 DNA-binding activity increased in Ssbp3- relative to vector-transfected cells, the affinity of this complex for DNA was unaltered. Similar to the effect of Ssbp2 on Lmo2 in murine erythroleukemia (MEL) cells, overexpressed Ssbp3 reduced Lhx2 protein turnover in cycloheximide-treated alphaT3-1 cells without affecting Lhx2 RNA levels. In contrast, knockdown of endogenous Ssbp3, but not Ssbp2 which is expressed at much lower levels in these cells, reduced Lhx2 and Ldb1 abundance, Lhx2 DNA-binding activity, Lhx2, Ldb1, and Ssbp3 loading onto the Cga promoter, Cga promoter activity, and endogenous Cga gene expression. Significantly, neither overexpression nor knockdown of Ssbp2 in MEL cells, which express both the LIM-only protein Lmo2 and LIM-HD protein Lhx2, affected Lhx2 protein abundance, and Lhx2 DNA-binding activity was undetectable in nuclear extracts from these cells despite the presence of immunoreactive Lhx2. These studies indicate that SSBP augmentation of LIM-HD function results from Ldb1-mediated inhibition of LIM-HD protein turnover and increased assembly of a LIM-HD/LDB1/SSBP DNA-binding complex. The much greater affinity for LDB1 of LIM-only compared to LIM-HD proteins is likely a major determinant of the SSBP effect on LIM-HD protein abundance. Finally, these findings are consistent with cell type-specific contributions of different SSBPs, even for similar LDB1-dependent actions.

Nonassembled Human Chorionic Gonadotropin Subunits and αα-Homodimers Use Fast-Track Processing in the Secretory Pathway in Contrast to αβ-Heterodimers

In multimeric glycoproteins, like glycoprotein hormones, mutual subunit interactions are required for correct folding, assembly, and transport in the secretory pathway. However, character and time course of these interactions need further elucidation. The influence of the glycoprotein hormone alpha-subunit (GPHalpha) on the folding of the human chorionic gonadotropin (hCG) beta-subunit (hCGbeta) in hCG alphabeta-heterodimers was investigated in [(35)S]Met/Cys-labeled JEG-3 cells. Completeness of disulfide bridge formation during the time course of folding was estimated by labeling with [(3)H]N-ethylmaleinimide of free thiol groups not yet consumed. Subunit association took place between immature hCGbeta (high (3)H/(35)S ratio) and almost completely folded GPHalpha. Analysis revealed a highly dynamic maturation process comprising of at least eight main hCGbeta folding intermediates (molecular masses from 107 to 28 kDa) that could be micro-preparatively isolated and characterized. These hCGbeta variants developed while being associated with GPHalpha. The 107-kDa variant was identified as a complex with calnexin. In contrast to hCG alphabeta-heterodimers, free nonassociated hCGbeta, free large GPHalpha, and GPHalphaalpha homodimers showed a fast-track-like processing in the secretory pathway. At 10 min before hCG secretion, sialylation of these variants had already been completed in the late Golgi, whereas hCG alphabeta-heterodimers had still not arrived medial Golgi. This shows that the GPHalpha in the hCG alphabeta-heterodimers decelerates the maturation of the hCGbeta portion in the heterodimer complex. This results in a postponed approval of hCG alphabeta-heterodimers by the endoplasmic reticulum quality control unlike GPHalphaalpha homodimers, free hCGbeta, and GPHalpha subunits.

TSH-induced hyperthyroidism caused by a pituitary tumor

A 45-year-old man presented with frontal headache and visual disturbances to our clinic. For the previous 5 years, he had been receiving treatment for long-lasting mild hyperthyroidism with antithyroid therapy, but therapy had not been carefully followed. During the last 2 years he had also complained of erectile dysfunction and loss of libido. On physical examination, he had a small goiter, normal skin, no Graves' ophthalmopathy, normal BMI, and reduced testis volume and pubic hair. Serum levels of free T3 and T4, serum prolactin, testosterone, serum gonadotropins, insulin-like growth factor 1, adrenocorticotropic hormone, and cortisol were measured. MRI scan, TSH-releasing hormone test, and T3 suppression test were carried out. Levels of pituitary glycoprotein hormone alpha-subunit and sex-hormone-binding protein were also measured. Hyperthyroidism caused by a mixed pituitary adenoma that secretes prolactin and TSH. Trans-sphenoidal resection of the pituitary tumor. After surgery, T3 suppression test failed to completely suppress TSH secretion, which suggested a persistence of residual adenomatous cells. Hyperthyroidism and hypogonadism recurred after 5 years, therefore, treatment with lanreotide was initiated, and resulted in complete resolution of signs and symptoms of the disease.

FOXL2 in the Pituitary: Molecular, Genetic, and Developmental Analysis

FOXL2 is a forkhead transcription factor expressed in the eye, ovary, and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 colocalizes with the glycoprotein hormone alpha-subunit (alphaGSU) in quiescent cells of the mouse pituitary from embryonic d 11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and alphaGSU suggested that the alphaGSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived (alphaT3-1, LbetaT2), thyrotrope-derived (alphaTSH) and heterologous (CV-1) cells in a context-dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression.

A Pituitary Cell Type Coexpressing Messenger Ribonucleic Acid of Proopiomelanocortin and the Glycoprotein Hormone α-Subunit in Neonatal Rat and Chicken: Rapid Decline with Age and Reappearancein Vitrounder Regulatory Pressure of Corticotropin-Releasing Hormone in the Rat

Promiscuous hormone mRNA expression in the pituitary remains poorly understood. We examined by means of RT-PCR and immunostaining whether glycoprotein hormone alpha-subunit (alphaGSU) could be coexpressed with proopiomelanocortin (POMC) in vivo and under pressure of CRH in vitro. Cells coexpressing alphaGSU and POMC mRNA amounted to 2.6% of the cells in ex vivo rat pituitary at birth [postnatal d 1 (P1)], fell to much lower level at P14, and were undetectable in adulthood. In cultured pituitary aggregates of P14 rats, alphaGSU/POMC cells remained scarce but represented up to 6.6% after chronic treatment with CRH but not leukemia inhibitory factor. CRH was less effective in aggregates from P1 and adult rats. The total alphaGSU population ex vivo at P1 was two times smaller than at P14, but in culture it expanded 2.5 times, concomitantly with a reciprocal change in POMC cell abundance. Tpit transcripts were detected in POMC-only and alphaGSU/POMC cells but not in alphaGSU-only cells. Cells coexpressing alphaGSU and POMC mRNA were relatively abundant in P14 chicken pituitary and aggregate cultures, but occurrence was not affected by CRH. Immunostaining showed alphaGSU and POMC colocalization in sporadic cells in intact rat pituitary and CRH-treated cultures at P1 but not at P14 and adult age. The data demonstrate the occurrence of cells coexpressing alphaGSU and POMC in rat and chicken pituitary. The developmental dynamics of this cell population and its response to CRH in vitro in the rat suggest a relationship of these cells with the embryonic branching of the POMC and alphaGSU cell lineages and their mutually opposite developmental course during early postnatal life.

Biological Properties of a Novel Follicle-Stimulating Hormone/Human Chorionic Gonadotropin Chimeric Gonadotropin

A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The alpha-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone alpha-subunit. The beta-subunit DNA construct for C3 encodes residues 1-145 from human chorionic gonadotropin (hCG)-beta with the exceptions that FSH beta amino acid 88 (D) is substituted for hCG beta amino acid 94 (R) and FSH beta amino acids 95-108 (TVRGLGPSYCSFGE) are substituted for hCG beta amino acids 101-114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.

The forkhead factor FOXL2 regulates expression of the glycoprotein hormone alpha-subunit

The forkhead factor FOXL2 regulates expression of the glycoprotein hormone alpha-subunit

Pituitary tumors arising from glycoprotein hormone α-subunit-deficient mice contain transcription factors and receptors present in thyrotropes

Glycoprotein-hormone alpha-subunit deficient (alphaSUnull) mice are hypothyroid and hypogonadal due to the absence of functional TSH, LH and FSH, despite normal production of the corresponding beta subunits. Pituitary tumors spontaneously developing in alphaSUnull mice were propagated in hypothyroid mice. The purpose of the current studies was to compare the gene expression profile of these alphaSUnull tumors with previously characterized TtT-97 thyrotropic tumors. A group of animals bearing each tumor type was treated with thyroid hormone (T4) prior to tumor removal. Both tumor types equally expressed TSHbeta mRNA, which significantly decreased when exposed to T4, whereas alpha-subunit mRNA was absent in alphaSUnull tumors. Northern blot analysis was performed using cDNA probes for the following transcription factors: Pit1, GATA2, pLIM, Msx1, Ptx1 and Ptx2. Both tumors were found to contain identical transcripts with similar responses to T4, with the exception of Pit1. In contrast to the signal pattern seen in TtT-97, only two bands were seen in alphaSUnull tumors, which were similar in size to those in alphaTSH cells, a thyrotropic cell line that lacks TSHbeta-subunit expression and Pit1 protein. However, western blot analysis revealed a protein band in the alphaSUnull tumors consistent with Pit1, while this signal was absent in alphaTSH cells. Northern blot analysis was also performed with specific cDNA probes for the following receptors: TRbeta1, TRbeta2, TRalpha1, non-T3 binding alpha2, RXRgamma and Sst5. Similarly-sized transcripts were found in both types of tumor, although the signal for Sst5 was seen in T4-treated alphaSUnull tumors only with a more sensitive RT-PCR analysis. The overall similarity between the two tumor types renders the alphaSUnull tumor as a suitable thyrotropic tumor model.

Cloning and sequencing of feline thyrotropin (fTSH): Heterodimeric and yoked constructs

The genes encoding the mature common glycoprotein α (CGA) and hormone-specific β subunits of feline thyroid stimulating hormone (fTSH) were cloned and sequenced. The feline CGA gene was cloned from RNA extracted from the feline pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR). The gene fragment that encodes mature TSHβ was cloned from feline genomic DNA after direct polymerase chain reaction (PCR). In both cases, primers were based on the consensus sequences from TSH in other species. The resulting 510 bp PCR product for the CGA-subunit included the full coding sequence for the 96 amino acid mature subunit preceded by a 24 amino acid signal peptide. The 850 bp sequence of fTSHβ genomic DNA consisted of two coding exons, an intron of 418 bp, and a 60 bp signal sequence. The octapeptide immunoaffinity tag FLAG was added to 3′ end of the α gene to facilitate detection and purification. Both genes were cloned independently downstream from the EF1α promoter of the PEAK™ transfer vector to facilitate co-expression studies in PEAK™ cells (modified human embryonic kidney (HEK) cells). A single-chain analogue of fTSH termed yoked fTSH (yfTSH) was developed by fusing the nucleotides encoding the C-terminus of the β-subunit fused to the N-terminus of the α-subunit with DNA encoding the C-terminal peptide (CTP) of human chorionic gonadotropin β-subunit as a linker peptide. The resulting single-chain analogue encoded from N-terminus to C-terminus: β-CTP-α-FLAG. The resulting DNA sequence was cloned, sequenced, ligated and recloned into expression vector PEAK™. This report constitutes the first cloning and sequencing of the genes encoding the subunits of feline thyrotropin.

Induction of GH, PRL, and TSHβ mRNA by transfection of Pit-1 in a human pituitary adenoma-derived cell line

The functional development of pituitary cells depends on the expression of a combination of transcription factors and co-factors. Pituitary-specific transcription factor-1 (Pit-1) is required for the expression of growth hormone (GH), prolactin (PRL), and the thyroid-stimulating hormone beta subunit (TSH beta) and acts synergistically with the estrogen receptor (ER) and GATA-binding protein 2 (GATA-2) to induce PRL and TSH beta expression, respectively. The glycoprotein hormone alpha subunit (alpha SU) is the first hormone to be expressed during pituitary development. In addition to being expressed in follicle-stimulating hormone, luteinizing hormone (LH), and TSH cells, alpha SU is reported to co-localize with GH in pituitary cells. These findings have led to the suggestion that the expression of Pit-1 in cells of the alpha SU-based gonadotropin cell lineage might also lead to the expression of GH. In this study, we transfected HP 75 cells (derived from a human non-functioning pituitary adenoma that expressed alpha SU and LH beta) with Pit-1 by using an adenovirus FLAG-Pit-1 construct. Most of the transfected cells expressed GH mRNA, with fewer cells expressing PRL and TSH beta mRNA. The HP 75 cells expressed the genes for ER and GATA-2, thus allowing their expression of GH, PRL, and TSH beta mRNA in response to Pit-1. These results support the hypothesis that GH can be induced in cells that possess an active alpha SU gene and shed light on the basic molecular mechanism that drives the development of GH, PRL, and TSH beta expression in the alpha SU-based gonadotroph lineage.

Transcript Profiling of Immediate Early Genes Reveals a Unique Role for Activating Transcription Factor 3 in Mediating Activation of the Glycoprotein Hormone α-Subunit Promoter by Gonadotropin-Releasing Hormone

Recent studies profiling immediate early gene responses to GnRH in the LbetaT2 gonadotrope cell model revealed increased expression of numerous genes including activating transcription factor (ATF) 3. The present studies demonstrate similar results with GnRH administration in vivo in ovariectomized mice. In this model, ATF3 mRNA was markedly up-regulated at 20, 40, and 60 min after in vivo administration of a GnRH analog. In alphaT3-1 gonadotrope cells, ATF3 mRNA and protein were induced by GnRH in a manner consistent with in vivo observations. Pharmacological studies implicated a combined role for the activities of protein kinase C isozymes, ERK and c-Jun N-terminal kinase, in modulating ATF3 expression. The role of ATF3 was further investigated in the activation of the human glycoprotein hormone alpha-subunit gene promoter. GnRH induced the alpha-subunit promoter-luciferase reporter approximately 16-fold, and this induction was completely abolished with mutations in the dual cAMP response elements (CREs) or the combined inhibition of GnRH-induced ERK and c-Jun N-terminal kinase. GnRH induced recruitment of ATF3, c-Jun, and c-Fos to the dual CREs. Overexpression and specific knockdown of ATF3 by small inhibitory RNA implicate a functional role for ATF3 in mediating activation of the alpha-subunit gene promoter. These studies provide clear evidence that ATF3 is a key immediate early gene induced by GnRH administration in vivo and in the alphaT3-1 gonadotrope cell model. These studies support the conclusion that the dual CREs of the human alpha-subunit promoter are the target of GnRH-induced MAPK regulation through ATF3.

Combinatorial Expression of Phenotypes of Different Cell Lineages in the Rat and Mouse Pituitary

As studied by single cell RT-PCR of pituitary hormones, we demonstrated that the pituitaries of rats and mice contain a subpopulation of cells that express two or more hormone phenotypes typically belonging to lineages that are branched separately early during embryonic development, such as glycoprotein hormone alpha-subunit (alphaGSU) mRNA + PRL mRNA, alphaGSU mRNA + POMC mRNA, and POMC mRNA + GH or PRL mRNA. GnRH in vitro selectively expands the population of cells coexpressing alphaGSU mRNA + PRL mRNA, and CRH selectively increases the proportion of cells coexpressing alphaGSU mRNA + POMC mRNA. Colocalization of alphaGSU + PRL or alphaGSU + POMC could not be detected by double immunofluorescence. This lineage promiscuity was also observed in the pituitary in vivo.

Early Multipotential Pituitary Focal Hyperplasia in the α-Subunit of Glycoprotein Hormone-Driven Pituitary Tumor-Transforming Gene Transgenic Mice

Pituitary tumor-transforming gene (PTTG), a securin protein isolated from pituitary tumor cell lines, is highly expressed in invasive tumors and exhibits characteristics of a transforming gene. To determine the role of PTTG in pituitary tumorigenesis, transgenic human PTTG1 was targeted to the mouse pituitary using the alpha-subunit of glycoprotein hormone. Males showed plurihormonal focal pituitary transgene expression with LH-, TSH-, and, unexpectedly, also GH-cell focal hyperplasia and adenoma, associated with increased serum LH, GH, testosterone, and/or IGF-I levels. MRI revealed both pituitary and prostate enlargement at 9-12 months. Urinary obstruction caused by prostatic hyperplasia and seminal vesicle hyperplasia, with renal tract inflammation, resulted in death by 10 months in some animals. Pituitary PTTG expression results in plurihormonal hyperplasia and hormone-secreting microadenomas with profound peripheral growth-stimulatory effects on the prostate and urinary tract. These results provide evidence for early pituitary plasticity, whereby PTTG overexpression results in a phenotype switch in early pituitary stem cells and promotes differentiated polyhormonal cell focal expansion.

Conservation of the Heterodimeric Glycoprotein Hormone Subunit Family Proteins and the LGR Signaling System from Nematodes to Humans

Glycoprotein hormones, follicle-stimulating hormones (FSHs), luteinizing hormones (LHs), thyroid-stimulating hormones (TSHs), and chorionic gonadotropin (CG) are key endocrine hormones secreted from the pituitary gonadotrophs and thyrotrophs and the placenta in primates. These hormones, consisting of a common alpha subunit and a specific beta subunit, act through the FSH receptor (FSHR), the LH receptor (LHR), and the TSH receptor (TSHR) that are highly specific for their cognate hormones. These glycoprotein hormones are structurally and functionally conserved in various vertebrates and have been identified in most lineages of actinopterygians (bony fish) and sarcopterygians (tetra-pods). Of interest, recent genomic studies showed that vertebrate glycoprotein hormone receptors belong to an ancient subfamily of G protein-coupled receptors (GPCRs) named as leucine-rich repeat-containing GPCRs (LGRs). These findings have prompted the hypothesis that there could be additional glycoprotein hormones in vertebrate genomes. Indeed, searches of vertebrate genomes have led to the identification of two novel glycoprotein hormone subunits, glycoprotein alpha 2 (GPA2) and glycoprotein beta 5 (GPB5), as well as their homologs in invertebrates. Subsequently, it was demonstrated that GPA2 and GPB5 form a heterodimeric hormone, thyrostimulin/OGH, capable of activating TSHR in vivoand the thyroid axis in transgenic mice. However, the exact role of this novel glycoprotein hormone and its homolog in invertebrates is not clear. To gain a better understanding of the physiological role of the novel glycoprotein hormone subunits and their evolution, it is imperative to carry out systematic studies of these genes in representative model species. In the present report, we summarize our findings based on studies of genomes of model organisms from sea anemones to humans. We found that GPA2 and GPB5 represent the ancient forms of glycoprotein hormone alpha and beta subunits, respectively, and that vertebrate and invertebrate glycoprotein hormone subunit proteins shared common ancestors that evolved during early metazoan evolution. It is important to note that glycoprotein hormone alpha and beta subunit proteins from invertebrates formed a heterodimer with structural functional characteristics similar to that of vertebrate glycoprotein hormones. Taken together, both glycoprotein hormone alpha and beta subunits evolved before the evolution of nematodes, arthropods, and vertebrates.

Transcriptional Activity of the 5' Upstream Region of the Porcine Glycoprotein Hormone .ALPHA. Subunit Gene

Gene expression of the porcine glycoprotein hormone alpha subunit (p-alphaGSU) was examined in LbetaT2 cells, which were established from the anterior pituitary lobe of the immortalized transgenic mouse and produce alphaGSU, and in CHO cells cloned from Chinese hamster ovaries. Expression of the reporter gene fused with p-alphaGSU gene upstream in LbetaT2 cells showed that the distal regions -540/-240 and -798/-541 are important for the activation of gene expression. In contrast, the transcriptional activity of the distal region of p-alphaGSU gene was repressed in CHO cells. The region -540/-240 contains an adequate enhancer, called pituitary glycoprotein hormone basal element, whereas the region -798/-541 has no distinguished element. Transfection of the expression vector containing cDNA of a pan-pituitary activator, Ptx1, whose putative binding sites are present scatted in the distal region of the p-alphaGSU gene, revealed unexpectedly that this factor significantly suppressed the expression of p-alphaGSU gene in LbetaT2 cells, indicating that Ptx1 is unrelated to the upregulation in the region -798/-541. Thus, this study demonstrated for the first time that the distal region -798/-541of the p-alphaGSU gene is indispensable for prominent expression of this gene in which an as yet unidentified factor may participate.

Thyrotropin-releasing Hormone (TRH) specific interaction between amino terminus of P-Lim and CREB binding protein (CBP)

P-Lim (Lhx3a) is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. The Lhx3 gene encodes two isoforms, which differ in their amino (N) termini, Lhx3a and 3b. The P-Lim DNA binding site on the glycoprotein hormone alpha subunit (α-GSU) gene promoter is conserved in mammals. P-Lim plays a pivotal role in mediating thyrotropin-releasing hormone (TRH) signaling by binding CREB binding protein (CBP), as we have reported previously. Here, we demonstrate that P-Lim (Lhx3a) but not Lhx3b can mediate TRH signaling and bind to CBP. Moreover, TRH specifically induces P-Lim–CBP binding through the N-termini of P-Lim. We also found that the protein kinase C (PKC) phosphorylation site within the N-terminus of P-Lim is responsible for the P-Lim–CBP binding suggesting that the TRH signaling pathway phosphorylates P-Lim. These studies have elucidated the molecular mechanism by which TRH stimulates α-GSU gene expression.