Pituitary-specific Transcription Factor Research Articles

Growth hormone and ghrelin receptor genes are differentially expressed between genetically lean and fat selection lines of sheep

The objective of this study was to determine whether differences in mRNA levels of key pituitary genes that regulate GH production, pituitary development, and growth were present and/or associated with divergent body composition phenotypes observed between sheep from genetically divergent lean and fat selection lines. Real-time PCR transcription profiles for pituitary specific transcription factor 1, prophet of pit1, GH, GH receptor, GH secretagogue receptor, GHRH receptor, leptin receptor, and somatostatin receptors 1 and 2 were determined in pituitary tissue. There was a difference in the amount of both GH (P < 0.001) and GH secretagogue receptor (P < 0.001) mRNA between the selection lines (5 females and 5 males per line; 20 wk of age); the lean line had greater abundance than the fat line, irrespective of which endogenous control gene was used. The results obtained for GHRH receptor were equivocal but suggestive; there were greater GHRH receptor mRNA levels (P < 0.001) in the lean line using beta-2-microglobulin as the endogenous control but not when hypoxanthine phosphoribosyltransferase and glyceraldehyde-3-phosphate dehydrogenase were used. No difference in pituitary specific transcription factor 1, prophet of pit1, GH receptor, leptin receptor, or somatostatin receptors 1 and 2 mRNA concentration was observed between the lines. The greater abundance of GH mRNA in the pituitary somatotropes from genetically lean animals appears to be associated with increased levels of GH secretagogue receptor mRNA and possibly GHRH receptor mRNA. This suggests that the difference in GH secretion between the lines may be due to differences in the afferent signals, such as ghrelin and/or GHRH, arising from the hypothalamus, or as a result of differential pituitary sensitivity to these hormones.

The role of pit-1 in the regulation of the rat growth hormone-releasing hormone receptor gene transcription by glucocorticoids

Glucocorticoids are involved in the regulation of the rat growth hormone-releasing hormone (GHRH) receptor gene expression, but they act only in the presence of the pituitary specific transcription factor, pit-1. In this study, the role of pit-1 in the glucocorticoid stimulation of the GHRH-receptor gene transcription was examined. The results suggest the presence of a silencer element in the promoter and it is postulated that pit-1 permits glucocorticoid action through suppressing the inhibitory effect of an as yet unknown factor that binds to this element. The present results also suggest that the synergistic activation of the rat GHRH-receptor gene transcription depends on the proper distance between the proximal glucocorticoid response element and the pit-1 binding site.

The Role of CBP/p300 Interactions and Pit-1 Dimerization in the Pathophysiological Mechanism of Combined Pituitary Hormone Deficiency

Combined pituitary hormone deficiency (CPHD) in humans is caused by mutations of pituitary-specific transcription factors such as Pit-1. Although many patients with CPHD have an autosomal recessive disorder caused by a Pit-1 DNA-binding mutation, there are a number of reports of mutant Pit-1 molecules that either by prediction or through experimentation bind normally to DNA. The objective of this study was to understand the pathophysiological mechanisms of mutant Pit-1 molecules with intact DNA binding. DNA-binding and functional studies were used to assess five Pit-1 mutations: F135C, R143Q, A158P, K216E, and R271W. In gel-shift studies using well-characterized DNA-binding elements from the GH and prolactin genes, the K126E mutant displayed markedly enhanced Pit-1 dimer binding to either element, whereas the R271W mutant bound with high avidity, but only as a monomer. In contrast, the R143Q mutant was unable to bind these elements, and the F135C and A158P mutants displayed near-normal DNA-binding characteristics. We observed that CBP/p300 bound poorly to the A158P and K216E mutant Pit-1 molecules, but bound normally to the F135C, R143Q, and R271W mutants. In functional assays, CBP/p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of either the GH or prolactin reporter genes. From these studies, we suggest that CBP/p300 recruitment and Pit-1 dimerization are necessary for Pit-1 target gene activation and are important in the pathogenesis of CPHD.

Regulation of bone sialoprotein (BSP) gene transcription by lipopolysaccharide

Lipopolysaccharide (LPS) is a major mediator of inflammatory responses in periodontal disease that inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the suppression of bone formation, we have analyzed the effects of LPS on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone. Treatment of osteoblast-like ROS 17/2.8 cells with LPS (1 microg/ml) for 12 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N-acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with LPS attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, revealed that LPS (1 microg/ml) suppressed expression of luciferase construct, encompassing BSP promoter nucleotides -108 to +60, transfected into ROS17/2.8 cells. The effects of LPS were inhibited by protein kinase A (PKA) inhibitor, H89 and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2 bp mutations in the inverted CCAAT box (ATTGG; nts -50 and -46), a cAMP response element (CRE; nts -75 to -68), a FGF response element (FRE; nts -92 to -85), and a pituitary specific transcription factor binding element (Pit-1; nts -111 to -105) showed that the LPS effects were mediated by the CRE and FRE. Whereas the FRE and 3'-FRE DNA-protein complexes were decreased by LPS, CRE DNA-protein complex did not change after LPS treatment. These studies, therefore, show that LPS suppresses BSP gene transcription through PKA and tyrosine kinase-dependent pathways and that the LPS effects are mediated through CRE and FRE elements in the proximal BSP gene promoter.

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.

Oestrogen Replacement in vivo Rescues the Dysfunction of Pituitary Somatotropes in Ovariectomised Aromatase Knockout Mice

The mechanism of regulation of growth hormone (GH) secretion by oestrogens and androgens is still controversial. Available data on the action of oestrogens on GH expression and secretion in somatotropes is poorly understood. We previously reported that the aromatase knockout (ArKO) mouse with oestrogen deficiency and excessive androgen levels had dysfunctional somatotropes. In order to clarify the influence of androgen and oestrogen, we investigated the in vivo treatment of ovariectomised (OVX) ArKO mice with exogenous oestradiol (E₂) on the mRNA expression of GH, GH-secretagogue receptor (GHS-R), GH-releasing hormone receptor (GHRH-R), pituitary-specific transcription factor (Pit-1), and somatostatin receptors (sst1–5) in pituitary glands. Circulating plasma GH levels were also evaluated. The results showed that ArKO/OVX mice have a low expression of pituitary GH, GHRH-R, GHS-R and Pit-1, and significantly reduced GH levels. Treatment of female ArKO/OVX (E₂-deficient without excessive androgen) mice with E₂ for 21 days enhanced expression of pituitary GHRH-R and Pit-1 to 151 and 168%, respectively, of that in mice without treatment. E₂ treatment increased GH expression and plasma levels in ArKO/OVX mice to levels comparable with those in wild-type female mice. We conclude therefore that long-term E₂ replacement rescues the dysfunction of somatotropes in ArKO/OVX mice through increases in expression of GH, GHRH-R, and Pit-1 in the pituitary somatotropes, whereas the level of androgen in this oestrogen-deficient female mouse does not significantly influence the function of somatotropes.

Novel Mutations within the POU1F1 Gene Associated with Variable Combined Pituitary Hormone Deficiency

Mutations within the gene encoding the pituitary-specific transcription factor POU1F1 are associated with combined pituitary hormone deficiency (CPHD). Most of the affected individuals manifest GH, prolactin, and TSH deficiency. We have now screened 129 individuals with CPHD and isolated GH deficiency for mutations within POU1F1. Causative mutations were identified in 10 of 129 individuals (7.8%). Of these, five patients harbored the dominant negative R271W mutation, which is a well-recognized mutational hot spot. We have also identified a second frequently occurring mutation, E230K, which appears to be common in Maltese patients. Additionally, we describe two novel mutations within POU1F1, an insertion of a single base pair (ins778A) and a missense mutation (R172Q). Functional studies have revealed that POU1F1 (E230K) is associated with a reduction in transactivation, although DNA-binding affinity is similar to the wild-type protein. On the other hand, POU1F1 (R172Q) is associated with a reduction in DNA binding and transactivation, whereas POU1F1 (ins778A) is associated with loss of DNA binding and a reduction in transactivation. Our data suggest that the phenotype associated with POU1F1 mutations may be more variable, with the occasional preservation of TSH secretion. Additionally, our data revealed POU1F1 mutations in three patients who were diagnosed as having ACTH deficiency but who, on further evaluation, were found to have normal cortisol secretion. Hence, elucidation of the genotype led to further evaluation of the phenotype, with the cessation of cortisol replacement that had been commenced unnecessarily. These data reflect the importance of mutational analysis in patients with CPHD.

Effects of salmon GnRH and sex steroid hormones on expression of genes encoding growth hormone/prolactin/somatolactin family hormones and a pituitary-specific transcription factor in masu salmon pituitary cells in vitro

Expression of genes encoding growth hormone (GH), prolactin (PRL), and somatolactin (SL) in growing and maturing salmon was stimulated by gonadotropin-releasing hormone (GnRH) analog during particular periods of the life cycle. GnRH therefore appears to directly and/or indirectly regulate gene expression for GH, PRL, and SL in combination with the pituitary–gonadal axis, such as sex steroid hormones. Direct effects of salmon GnRH (sGnRH), estradiol-17β (E2), testosterone, and 11-ketotestosterone (11KT) on the amounts of GH, PRL, and SL mRNAs were thus examined using primary pituitary cell cultures of masu salmon at the four reproductive stages. We also determined the amounts of mRNA encoding pituitary specific POU homeodomain transcription factor (Pit-1) by real-time polymerase chain reactions. The amounts of GH, PRL, and SL mRNAs in the control cells elevated with gonadal maturation, coincidently with those of Pit-1 mRNA. sGnRH at 1.0 nM elevated the amounts of all mRNAs examined in the pre-spawning females, whereas significant effects were not observed with 100 nM sGnRH at any reproductive stages. Sex steroid hormones had no significant effects before initiation of gonadal maturation and at the maturing stage. In the males, E2 tended to decrease the amounts of SL mRNA in the pre-spawning stage. In the females, E2 and 11KT increased the amounts of PRL and SL mRNAs in the pre-spawning stage, but halved those of PRL mRNA in the spawning stage. The amounts of Pit-1 mRNA changed coincidently with those of PRL and SL mRNAs at all examined stages. The effects of E2 alone were abolished by 100 nM sGnRH. The present results indicated that both sGnRH and steroid hormones directly modulate synthesis of Pit-1, and further expression of PRL and SL genes. sGnRH may indirectly regulate GH/PRL/SL family hormone genes through the pituitary–gonadal axis, particularly in the late stage of gametogenesis.

Clinical and Molecular Features of a TSH-Secreting Pituitary Microadenoma

We describe a case of a thyroid stimulating hormone (TSH)-secreting pituitary microadenoma, and report the systematic gene expression profile of the surgically- removed tumor. A 50-year-old woman was referred to our hospital because she had high TSH, free-T4, and free-T3 levels, and a pituitary tumor that was visualized with magnetic resonance imaging. Her basal TSH level was high even after a high T3 loading dose, and increased following administration of thyroid releasing hormone (TRH) even after administration of a high dose of exogenous T3. Her clinical symptoms and peripheral markers for T3 were responsive to exogenous T3. There was no thyroid hormone receptor (TR) beta gene mutation. The patient was diagnosed with a TSH-secreting pituitary adenoma, and trans-sphenoid surgery was performed. The histologic features and immunophenotype were consistent with a TSH-secreting pituitary adenoma. Reverse transcription-polymerase chain reaction analysis of pituitary hormones, pituitary-specific transcription factors, receptors, and transcriptional cofactors of clinical significance was performed on the removed tumor. The tumor expressed TSH, growth hormone, prolactin, alpha-subunit, pituitary transcription factor-1 (pit-1) but not proopiomelanocortin (POMC), prophet of pit-1 (prop-1) and pituitary cell-restricted T box factor (Tpit). TRbeta and TRH-receptor gene expression was normal. Three steroid receptor coactivators (SRC)-1, SRC-2, and SRC-3 were expressed. Nuclear receptor corepressor (N-CoR)2 was absent in the tumor, whereas nuclear receptor corepressor (N-CoR1) was expressed. Somatostatin receptor type 1 expression was significantly decreased, whereas type 4 receptor was expressed, which are unusual characteristics for pituitary tumors. The gene expression pattern in the tumor might have a role in the clinical features of this case.

Identification and characterization of single nucleotide polymorphisms in 12 chicken growth-correlated genes by denaturing high performance liquid chromatography

The genes that are part of the somatotropic axis play a crucial role in the regulation of growth and development of chickens. The identification of genetic polymorphisms in these genes will enable the scientist to evaluate the biological relevance of such polymorphisms and to gain a better understanding of quantitative traits like growth. In the present study, 75 pairs of primers were designed and four chicken breeds, significantly differing in growth and reproduction characteristics, were used to identify single nucleotide polymorphisms (SNP) using the denaturing high performance liquid chromatography (DHPLC) technology. A total of 283 SNP were discovered in 31 897 base pairs (bp) from 12 genes of the growth hormone (GH), growth hormone receptor (GHR), ghrelin, growth hormone secretagogue receptor (GHSR), insulin-like growth factor I and II (IGF-I and -II), insulin-like growth factor binding protein 2 (IGFBP-2), insulin, leptin receptor (LEPR), pituitary-specific transcription factor-1 (PIT-1), somatostatin (SS), thyroid-stimulating hormone beta subunit (TSH-β). The observed average distances in bp between the SNP in the 5'UTR, coding regions (non- and synonymous), introns and 3'UTR were 172, 151 (473 and 222), 89 and 141 respectively. Fifteen non-synonymous SNP altered the translated precursors or mature proteins of GH, GHR, ghrelin, IGFBP-2, PIT-1 and SS. Fifteen indels of no less than 2 bps and 2 poly (A) polymorphisms were also observed in 9 genes. Fifty-nine PCR-RFLP markers were found in 11 genes. The SNP discovered in this study provided suitable markers for association studies of candidate genes for growth related traits in chickens.

Three Novel Mutations in POU1F1 in Israeli Patients with Combined Pituitary Hormone Deficiency

POU1F1, a pituitary-specific transcription factor of the class 1 POU family, is crucial for the development and differentiation of the anterior pituitary gland. Mutations in the POU1F1 gene have been shown to be responsible for a syndrome of combined pituitary hormone deficiency (CPHD), including prolactin, growth hormone and thyroid-stimulating hormone deficiencies. Five patients with CPHD from three families were evaluated. The clinical and biochemical data were taken from the medical records. DNA was analyzed by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and sequencing. Molecular analysis yielded three novel mutations in POU1F1: W193X, Q242R (-2 bp), and F262L. Three novel POU1F1 mutations were detected in Israeli patients with CPHD. Two of them, a W193X missense mutation and a deletion of two adenine bases at position 242Q, may lead to the production of a truncated protein that lacks the entire POU homeodomain or part of it, respectively. The third mutation, F262L, resides in the POU homeodomain and hence might change the activity of the protein.

Regulation of growth hormone expression by thyrotropin-releasing hormone through the pituitary-specific transcription factor Pit-1 in chicken pituitary

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone beta- (TSHbeta) encoding genes. Pit-1 has been identified in several mammalian and avian species. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and it stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully developed a competitive RT-PCR for the detection of Pit-1 expression in the chicken pituitary, that was sensitive enough to detect picogram levels of Pit-1 mRNA. Applying this method, the effect of TRH injections on Pit-1 mRNA expression was determined in the pituitary of chick embryos and growing chicks. In both 18-day-old embryos and 10-day-old male chicks the Pit-1 mRNA expression was significantly increased following TRH injection, thereby indicating that the stimulatory effects of TRH on several pituitary hormones is mediated via its effect on Pit-1 expression. Therefore, a semi-quantitative RT-PCR method was used to detect possible changes in GH levels. TRH affected the GH mRNA levels at both developmental stages. These results, combined with the data on Pit-1 mRNA expression, indicate that Pit-1 has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH.

The pituitary-specific transcription factor, Pit-1, can direct changes in the chromatin structure of the prolactin promoter.

The chromatin structure of a promoter is an important determinant of its transcriptional activity. Many promoters are assembled into repressive polynucleosomal arrays that are subsequently remodeled to allow for the activation of gene expression. This study addresses the contribution of a single transcription factor, Pit-1, in orchestrating the chromatin structure of the prolactin gene. Utilizing an in vivo reconstitution system, we found that Pit-1 can bind to multiple sites in the chromatin-assembled 5'-flanking region of the prolactin gene and activate transcription from the chromatin-assembled template. Interestingly, Pit-1 was able to substantially alter micrococcal nuclease digestion of the prolactin 5'-flanking region, and the results are consistent with presence of a translationally positioned nucleosome on the prolactin promoter. Changes in micrococcal nuclease digestion were also observed with a truncated Pit-1 mutant containing only the DNA-binding domain. As the truncation mutant was unable to activate transcription from the chromatin-assembled template, the ability of Pit-1 to alter chromatin structure of the prolactin gene is not dependent on transcriptional activation. We propose that Pit-1 likely plays a role in altering chromatin to facilitate recruitment and subsequent transcriptional activation by additional factors.

Static magnetic fields-induced bone sialoprotein (BSP) expression is mediated through FGF2 response element and pituitary-specific transcription factor-1 motif.

Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein found almost exclusively in mineralized connective tissues. Recent studies on the developmental expression of BSP mRNA and temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts, and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. Physical forces may play a fundamental role in the regulation of cell function in bone, but little is known about how cells are able to sense mechanical loads and signal transduction. Magnetic fields of sufficient magnitude have been shown to affect various biologic systems at organ, tissue, cellular, and subcellular levels. In the present study, rat osteosarcoma-derived osteoblast-like cells, UMR 106, were used to assess the effect of static magnetic fields (SMF) on gene transcription of BSP. In our culture system, application of 300 and 800 Gauss SMF increased BSP mRNA levels after 24 h stimulation. To determine the molecular basis of the transcriptional regulation of BSP gene transcription by SMF, we conducted transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase (LUC) reporter gene. SMF (300 and 800 Gauss) increased expression of the construct (pLUC3; -116 to +60) after 24 h treatment. Further deletion analysis of the BSP promoter showed that a region within nt -116 to -84 was targeted by SMF, the effect of which was inhibited by the tyrosine kinase inhibitor herbimycin A (HA). Mutations (2 bp) were made in an inverted CCAAT box between nt -50 and -46, a cyclicAMP response element (CRE; between nt -75 and -68), a fibroblast growth factor-2 response element (FRE; -92 to -85), and a pituitary-specific transcription factor-1 motif (Pit-1; nt -111 to -105) within the pLUC3 construct. Transcriptional stimulation by SMF was almost completely abrogated in constructs that included 2-bp mutations in the FRE and Pit-1. Binding of nuclear proteins to a radiolabeled FRE was increased and that to a Pit-1 was decreased in nuclear extracts prepared from SMF-stimulated UMR 106 cells. Further, the stimulatory and inhibitory effects of SMF on FRE and Pit-1 DNA-protein complexes were completely abolished by HA treatment. These studies, therefore, show that SMF increases BSP transcription through a tyrosine kinase-dependent pathway and that the SMF effects are mediated through juxtaposed FRE and Pit-1elements in the proximal promoter of the BSP gene.

Glucocorticoid induction of lactotrophs and prolactin gene expression in chicken embryonic pituitary cells: a delayed response relative to stimulated growth hormone production.

We reported that corticosterone (CORT) induces GH cell differentiation in chicken embryonic pituitary cells in culture and in living embryos. The present study tested whether CORT could also induce prolactin (PRL) cell differentiation in vitro. CORT increased the percentage of GH cells in cultures of embryonic d (e) 13 pituitary cells after 1 d of treatment and PRL cells after 3 d of incubation. Dual immunofluorescence showed that the PRL cells induced by CORT did not contain GH, indicating they were separate cell populations. Similar PRL cell responses were also observed in cultures of e11, e15, and e17 pituitary cells. ZK98299, a glucocorticoid receptor (GR) antagonist, suppressed CORT effects, verifying involvement of GR. Northern blot analysis indicated that CORT increased GH mRNA levels after 1 d of treatment. In contrast, increases in PRL mRNA levels were delayed and observed after 3 d of treatment. Induction of a luciferase reporter driven by the PRL promoter was also delayed until 3 d of CORT treatment. Dual-labeling immunofluorescence indicated that the majority of PRL cells induced by CORT were not labeled with bromodeoxyuridine, suggesting that lactotrophs induced by CORT do not result from cell proliferation. Proportions of pituitary-specific transcription factor (Pit-1)-containing cells and the total amount of Pit-1 protein spontaneously increased with increasing culture time. However, no effect of CORT on Pit-1 levels or the number of Pit-1-containing cells were observed. We conclude that CORT can induce lactotroph differentiation in culture and that longer CORT exposure is needed for lactotroph induction compared with somatotroph induction. The effects of CORT on PRL cell induction are GR dependent and involve an increase in PRL gene expression. PRL cell induction by CORT is not associated with an increase in Pit-1-containing cells.

Functional modification of pituitary somatotropes in the aromatase knockout mouse and the effect of estrogen replacement.

Available data on the influence of estradiol (E(2)) on GH levels remains controversial. A factor contributing to this uncertainty is a lack of knowledge of both E(2) action on somatotropes as well as the molecular mechanisms involved. In this study we investigated gene expression implicated in GH secretion in somatotropes derived from female aromatase knockout (ArKO) mice. In these mice E(2) production is blocked due to disruption of the Cyp19 gene encoding aromatase, the enzyme responsible for estrogen biosynthesis. The effect of E(2) replacement was also studied by in vivo treatment of mice with E(2) for 3 wk. It was demonstrated that somatotropes from ArKO mice had a low expression of GH, GH secretagogue receptor, GHRH receptor (GHRH-R), and pituitary-specific transcription factor (Pit-1). On the other hand, the somatotropes exhibited elevated expression of somatostatin receptors (sst1-5). Overall, these effects resulted in a reduction in GH secretion. E(2) replacement increased GHRH-R, Pit-1, and GH mRNA levels to 185%, 193%, and 157% and reduced the levels of sst1, sst2, sst4, and sst5 mRNA expression in ArKO mice, respectively. E(2) replacement did not affect the levels of pituitary estrogen (alpha and beta) and androgen receptor mRNA expression. It is concluded that the expression of important genes involved in GH synthesis in somatotropes of the female ArKO mouse are functionally down-regulated, and such a down-regulation is reversed to normal levels by E(2) replacement. The levels of GH secretagogue receptor, GHRH-R, and Pit-1 mRNA expression were also reduced, and sst1 and sst3 mRNA expression enhanced in aging ArKO and wild-type mice, resulting in a decrease in GH mRNA expression. It is suggested that aging is another important impact factor for the pituitary expression and regulation of GH mRNA in female mice.

The R271W mutant form of Pit-1 does not act as a dominant inhibitor of Pit-1 action to activate the promoters of GH and prolactin genes.

Genetic abnormalities of the pituitary specific transcription factor, Pit-1, have been reported in several patients with GH, prolactin (PRL) and TSH deficiencies. The most common is a mutation altering an arginine to a tryptophan in codon 271 (R271W) in one allele of the Pit-1 gene. According to the previous in vitro expression study, R271W acted as a dominant negative inhibitor of the wild type to activate the GH promoter. However, healthy carriers with this mutation, who should be affected by the dominant negative effect of R271W, have also been reported. The aim of this study was to clarify in more detail the function of this mutant form of Pit-1. Transcriptional activity of R271W for the expression of Pit-1-associated genes was investigated in COS7 cells with the aid of transient transfection assays. The 1.8 kb rat GH, 0.6 kb rat PRL or 1.9 kb rat PRL 5'-flanking regions were inserted upstream of the luciferase reporter gene and were used for functional analysis of R271W. Another reporter gene containing seven Pit-1 responsive elements was also used. The same experiments were also performed using JEG3 and CHO cells. We could not confirm the dominant negative effect of R271W on wild type Pit-1. Furthermore, our expression study revealed that R271W could activate the promoters of GH and PRL genes to levels similar to the wild type. Taken together with the evidence that phenotypically normal cases have been reported with this mutation, our results deny the relationship between R271W and combined pituitary hormone deficiency.

Expression of growth hormone and its transcription factor, Pit-1, in early bovine development.

During bovine embryogenesis, bovine growth hormone (bGH) contributes to proliferation, differentiation, and modulation of embryo metabolism. Pituitary-specific transcription factor-1 (Pit-1) is a transcription factor that binds to promoters of GH, prolactin (PRL), and thyroid-stimulating hormone-beta (TSHbeta) encoding genes. A polymorphism in the fifth exon of the bGH gene resulting in a leucine (Leu) to valine (Val) substitution provides an Alu I restriction site when the Leu allele is present. To determine the onset of embryonic expression of the bGH gene, oocytes derived from ovaries homozygous for Leu alleles were fertilized in vitro with spermatozoa obtained from a Val homozygote. For each developmental stage examined, three separate pools of embryos composed of approximately 100 cell samples underwent RNA isolation, reverse transcription to cDNA, and amplification by nested PCR (nPCR). Bovine GH gene transcripts were identified at 2- to 4-cell (n = 162), 8- to 16-cell (n = 73), morulae (n = 51), and blastocyst (n = 15) stages. Likewise, transcripts for Pit-1 were detected at 2-cell (n = 125), 4-cell (n = 114), 8-cell (n = 56), 12-to-32-cell (n = 32), morulae (n = 68), and blastocyst (n = 14) stages. After digestion with Alu1, bGH cDNA was genotyped by restriction fragment length polymorphism (RFLP) analysis. Bovine GH mRNA was present in all pools of stages examined. Both Leu and Val alleles (maternal and paternal) were only detected in pools of embryos that had reached 8- to 16-cell stage. Results suggest that transcription of the bGH gene begins at the 8- to 16-cell stage in bovine embryos, possibly under control of the transcription factor, Pit-1, and that RFLP analysis of the bGH gene can be used to determine parental origin of transcripts in early embryonic development.

Selective repression of rat prolactin gene by stable expression of dominant-negative Ets in GH4 pituitary cells.

Members of the Ets family of transcription factors are key regulators controlling prolactin (PRL) gene expression. Utilizing a transient transfection approach and the GH4 rat pituitary cell line, we have shown that Ets- 1 acts synergistically with the pituitary-specific POU homeodomain transcription factor, Pit-1, to mediate basal and Ras-induced regulation of the proximal (-425) rat PRL (rPRL) promoter. Although the transient transfection approach has provided important information regarding rPRL proximal promoter regulation, the role of Ets factors in the regulation of the intact, endogenous PRL promoter has not been explored. To address this area of question, we created several clonal GH4 cell lines that stably express either dominant-negative Ets (dn-EtsZ) or dominant-active Ets (VP16 Ets) constructs and used these cell lines as a model system to analyze the role of Ets factors on endogenous PRL gene expression. Northern blot analysis of these cells showed that PRL mRNA levels were dramatically reduced, by an average of 80%, in the cell lines expressing dn-Ets compared to vector-only controls. Conversely, stable expression of the dominant-active VP16 Ets led to an average threefold increase in PRL mRNA. GH4 cells expressing dn-EtsZ displayed significantly lower levels of intracellular PRL protein content and greatly diminished secretion of PRL into the cell culture medium, compared to vector-only controls. Consistent with our previous observations, the mRNA levels for growth hormone were unaffected by either dn-EtsZ or VP16 Ets expression. Expression of dn-EtsZ reduced Pit-1 mRNA levels by about 30%; however, the intracellular levels of Pit-1 protein were unchanged. Taken together, these results verify and strengthen the view that Ets factors play a critical role in the regulation of endogenous PRL gene expression and PRL protein production.

Regulation of the rat follicle-stimulating hormone beta-subunit promoter by activin.

FSH is controlled by a variety of positive and negative stimuli, and the unique FSHbeta-subunit is a major target for this regulation. Activin is a key modulator of FSHbeta transcription and hormone secretion. The signal transduction pathway leading to FSH expression was previously unknown. Here, we show that the transcription factors Smad3 and Smad4 mediate activin-stimulated activity of the rat FSHbeta promoter in a pituitary-derived cell line, LbetaT2. Cells were transiently transfected with the rat FSHbeta promoter fused to a luciferase reporter gene (-338rFSHbeta-Luc), and a minimal activin-responsive region was identified. Transfection of Smad3, but not the highly related Smad2, led to a ligand-independent stimulation of the FSHbeta promoter activity. As expected, activin caused an additional increase of luciferase expression, which was blocked by cotreatment with follistatin. Although Smad4 alone had no effect on FSHbeta transcription, it significantly augmented Smad3 and activin-mediated stimulation of the promoter. A palindromic consensus Smad-binding element in the proximal promoter was found to bind Smad4, and elimination of the region resulted in a loss of activin-mediated FSHbeta transcription. The activin signaling pathway is conserved in a number of cells, but FSHbeta expression is restricted to gonadotropes. A pituitary-specific transcription factor necessary for activin-dependent induction of the FSHbeta promoter has been identified that permits FSHbeta expression in nongonadotrope cells. Pitx2 is a member of Pitx subfamily of bicoid-related homeodomain factors that is required for pituitary development and is present in the adult pituitary. This factor was transfected into LbetaT2 cells, where it caused up-regulation of basal and activin-mediated FSHbeta promoter activity. Furthermore, cotransfection of Pitx2c with Smad3 in kidney-derived TSA cells resulted in activin-regulated FSHbeta response, suggesting its important role in tissue-restricted regulation of FSHbeta by activin. A Pitx2c binding site was identified within the proximal promoter, and elimination of this region also resulted in a loss of activin-regulated FSHbeta promoter activity. Taken together, these studies suggest that the regulation of FSHbeta is dependent on activin-mediated signaling factors in concert with pituitary-derived nuclear regulatory proteins.