Pituitary-specific Transcription Factor Research Articles

A GRFa2/Prop1/Stem (GPS) Cell Niche in the Pituitary

BackgroundThe adult endocrine pituitary is known to host several hormone-producing cells regulating major physiological processes during life. Some candidates to progenitor/stem cells have been proposed. However, not much is known about pituitary cell renewal throughout life and its homeostatic regulation during specific physiological changes, such as puberty or pregnancy, or in pathological conditions such as tumor development.Principal FindingsWe have identified in rodents and humans a niche of non-endocrine cells characterized by the expression of GFRa2, a Ret co-receptor for Neurturin. These cells also express b-Catenin and E-cadherin in an oriented manner suggesting a planar polarity organization for the niche. In addition, cells in the niche uniquely express the pituitary-specific transcription factor Prop1, as well as known progenitor/stem markers such as Sox2, Sox9 and Oct4. Half of these GPS (GFRa2/Prop1/Stem) cells express S-100 whereas surrounding elongated cells in contact with GPS cells express Vimentin. GFRa2+-cells form non-endocrine spheroids in culture. These spheroids can be differentiated to hormone-producing cells or neurons outlining the neuroectoderm potential of these progenitors. In vivo, GPSs cells display slow proliferation after birth, retain BrdU label and show long telomeres in its nuclei, indicating progenitor/stem cell properties in vivo.SignificanceOur results suggest the presence in the adult pituitary of a specific niche of cells characterized by the expression of GFRa2, the pituitary-specific protein Prop1 and stem cell markers. These GPS cells are able to produce different hormone-producing and neuron-like cells and they may therefore contribute to postnatal pituitary homeostasis. Indeed, the relative abundance of GPS numbers is altered in Cdk4-deficient mice, a model of hypopituitarism induced by the lack of this cyclin-dependent kinase. Thus, GPS cells may display functional relevance in the physiological expansion of the pituitary gland throughout life as well as protection from pituitary disease.

Polymorphism of Pituitary-Specific Transcription Factor-1 (Pit-1) Gene at Locus (Pit-1-Hinf1) and its effects on dam body weight and milk production of local sheeps

Pituitary-Specific Transcription Factor-1 (Pit-1) is a transcription factor with critical role in the transcriptional regulation of multiple genes in the pituitary. The objective of this research was to identify polymorphism of Pituitary-Specific Transcription Factor-1 (Pit-1) gene at Locus (Pit-1-Hinf1) and to investigate any possible associations of Pit-1 genotypes on dam body weight, milk production and milk quality in local sheep at the Jonggol Animal Science Teaching and Research Unit (JASTRU), Fact. Anim. Sci. Bogor. Agric. Univ. A total number of 161 blood samples were collected from 3 local sheep, namely Garut from Wanaraja (55 hd), Garut from Margawati (23 hd) and lactating ewes (83 hd) from JASTRU farm in Bogor. Genomic DNAs were extracted by a standard phenol-chloroform protocol and amplified by a polymerase chain reaction (PCR) techniques, then PCR products were digested with a Hinf1 enzyme restriction. Fragments of Pit-1gene at locus Pit-1-Hinf1 was detected by a silver-staining method. A length of 637 base pairs (bps) of the Pit-1 gene of local sheep was successfully amplified. The Hinf1 restriction enzyme cut the PCR product into three different length of fragments succesively at 345, 137, and 115 bps designated as A allele; whilst B allele had four fragments at 283, 137, 115, and 62 bps respectively. The locus of Pit-1-Hinf1 was polymorphic in local sheep from Jonggol, however it was monomorfic in Garut sheep. The frequencies of A and B alleles were 0,806 and 0,194 respectively. Pit-1 genotypes had no significant effect on dam body weight and milk production. This result is indicating that the use of single locus Pit-1-Hinf1 in Pit-1 gen is less effective to be used as a candidate in selecting dam body weight and milk production in these three local sheep. Key words : Local Sheep, Pit-1 Gene, Polymorphism, Dam, Milk

A Novel Dominant Negative Mutation of OTX2 Associated with Combined Pituitary Hormone Deficiency

Combined pituitary hormone deficiency (CPHD) is characterized by deficiencies in more than one anterior pituitary hormone. Mutations in developmental factors responsible for pituitary cell specification and gene expression have been found in CPHD patients. OTX2, a bicoid class homeodomain protein, is necessary for both forebrain development and transactivation of the HESX1 promoter, but as of yet, has not been associated with CPHD. The goal of this study was to identify and characterize novel mutations in pituitary specific transcription factors from CPHD patients. Genomic DNA was isolated from patients with hypopituitarism to amplify and sequence eight pituitary specific transcription factors (HESX1, LHX3, LHX4, OTX2, PITX2, POU1F1, PROP1, and SIX6). Characterization of novel mutations is based on structural and functional studies. We describe two unrelated children with CPHD who presented with neonatal hypoglycemia, and deficiencies of GH, TSH, LH, FSH, and ACTH. Magnetic resonance imaging revealed anterior pituitary hypoplasia with an ectopic posterior pituitary. A novel heterozygous OTX2 mutation (N233S) was identified. Wild-type and mutant OTX2 proteins bind equivalently to bicoid binding sites, whereas mutant OTX2 revealed decreased transactivation. A novel mutation in OTX2 binds normally to target genes and acts as a dominant negative inhibitor of HESX1 gene expression. This suggests that the expression of HESX1, required for spaciotemporal development of anterior pituitary cell types, when disrupted, results in an absent or underdeveloped anterior pituitary with diminished hormonal expression. These results demonstrate a novel mechanism for CPHD and extend our knowledge of the spectrum of gene mutations causing CPHD.

Basic science and clinical research advances in the pituitary transcription factors: Pit-1 and Prop-1

The pituitary-specific transcription factors, Pit-1 (also called Pou1f1) and prophet of Pit-1 (Prop-1), are critical for normal pituitary development and function. The aim of the present review is to highlight the recent basic science and clinical research advances in these transcription factors, as they relate to signaling pathway interactions, gene regulation, and mutations in human diseases. A number of important basic research discoveries have been made in the area of the pituitary-specific transcription factors, Pit-1 and Prop-1. Among these findings include: the effects of the Pit-1 coactivators, GATA-2 and TRAP-220, on the transcriptional regulation of the TSHbeta gene and thyrotropin expression, characterization of a novel pituitary regulator of Pit-1 expression, Atbf1, elucidation of the roles of Wnt and Notch signaling on Prop-1-mediated specification of the Pit-1 cell lineage and gonadotropes, and the identification of regulatory regions of the Prop-1 gene. Advances in clinical research include: the identification of novel mutations in the human POU1F1 and PROP-1 genes, and screening guidelines for patients with combined pituitary hormone deficiencies and possible mutations in these transcription factors. Research into the complex interplay of signaling pathways and transcription factors that regulate the pituitary gland are important areas of developmental biology and normal physiology. Clinically, such research has important implications for human diseases by identifying inheritable transcription factor mutations that may cause pituitary hormonal deficiencies.

Genetic effects of <I>MC4R</I> and <I>POU1F1 </I>gene on the growth performance in Jinghai yellow chicken

Melanocortin-4 receptor (MC4R) gene and pituitary specific transcription factor 1 (POU1F1) gene were studied as candidate genes on the Jinghai yellow chicken in this study. Polymorphisms of MC4R and POU1F1 gene had been analyzed by PCR-SSCP and DNA sequencing method. The relationship between two candidate genes and the growth performance of Jinghai yellow chicken had also been analyzed by GLM model. The frequency of A/B alleles for MC4R gene in the population was 0.929 and 0.071, and that of C/D alleles for POU1F1 gene was 0.500 and 0.500. The results showed that AA genotype of MC4R gene had higher bodyweight at 4, 8, 12 weeks compared to AB and BB genotype (P<0.05); CD genotype of POU1F1 gene had significantly higher bodyweight at all weeks compared to CC and DD genotype (P<0.01). So, it could be preliminarily deduced that MC4R and POU1F1 gene were probably the major gene on growth performance or a QTL linked gene which associated with growth performance in chicken.

Reduction in hypophyseal growth hormone and prolactin expression due to deficiency in ghrelin receptor signaling is associated with Pit-1 suppression: Relevance to the immune system

In mice and in rats, reduced levels of the growth hormone secretagogue receptor (GHS-R1a) results in reduced body weight and lower levels of serum insulin-like growth factor I (IGF-I). However, the mechanism leading to these impairments has not been elucidated. Studies in primary cultures of pituitary cells from very young mice have shown that GHS-R1a agonists, including ghrelin, increase expression of the pituitary-specific transcription factor (Pit-1) that is critical for differentiation of pituitary cells into somatotrophs, lactotrophs, and thyrotrophs. Hence, we hypothesized that ablation of Ghsr would reduce Pit-1 expression and as a consequence reduce growth hormone (GH) production explaining the lower body weight of Ghsr−/− mice. Here, we now show that Pit-1 mRNA levels are significantly lower in the pituitary gland of Ghsr−/− mice compared to wild-type littermates and also with advancing age. This Pit-1 loss is associated with reduced GH mRNA and fewer GH producing cells. To determine whether reduced GH is caused by reduced expression of Pit-1 in Ghsr−/− mice, we also measured prolactin (PRL) expression in the pituitary gland and in the circulation. PRL mRNA was significantly reduced in Ghsr−/− mice compared to wild-type littermates and fewer cells expressed PRL. The reduction in expression of both GH and PRL is consistent with a Pit-1 regulated pathway and demonstrates that the GHS-R has an important role in the pituitary gland as a modulator of Pit-1 expression and provides a possible mechanism to explain the lower plasma IGF-1 and modestly reduced body weight exhibited by Ghsr−/− mice. We also believe that lower systemic and lymphoid hormone expression may also account, in part, for the enhanced thymic involution and reduced thymic output in Ghsr−/− mice.

Genetic polymorphisms of osteopontin (OPN), prolactin (PRL) and pituitary-specific transcript factor-1 (PIT-1) in South Anatolian and East Anatolian Red cattle

The objective of the present study was to determine the genotype and allele frequencies for osteopontin (OPN), prolactin (PRL) and pituitary-specific transcription factor-1 (PIT-1) that are claimed to be associated with milk production traits in cattle in South Anatolian Red (SAR) and East Anatolian Red (EAR) cattle. In this study, 40 cattle for each of SAR and EAR were used. OPN in intron 4, PRL in exon 3 and PIT-1 in exon 6 were amplified by polymerase chain reaction (PCR) and the resulting PCR products were subjected to digestion using BseNI, RsaI and HinfI enzymes, respectively, to determine the single nucleotide polymorphisms (SNPs). Results showed that frequencies of the OPN allele C, PRL allele B, and PIT-1 allele A which are associated with milk production tarits were found low in SAR and EAR. As a result, SAR and EAR cattle breeds have the lower allel frequencies that influence the milk production traits for three genes than high-yielding European dairy cattle breeds.

Analysis of caprine pituitary specific transcription factor-1 gene polymorphism in indigenous Chinese goats

Since mutations on POU1F1 gene possibly resulted in deficiency of GH, PRL, TSH and POU1F1, this study revealed the polymorphism of goat POU1F1-AluI locus and analyzed the distribution of alleles on 13 indigenous Chinese goat breeds. The PCR-RFLP analysis showed the predominance of TT genotype and the frequencies of allele T varied from 0.757 to 0.976 in the analyzed populations (SBWC, Bo, XH and HM). Further study, distributions of genotypic and allelic frequencies at this locus were found to be significantly different among populations based on a chi(2)-test (P < 0.001), suggesting that the breed factor significantly affected the molecular genetic character of POU1F1 gene. The genetic diversity analysis revealed that Chinese indigenous populations had a wide spectrum of genetic diversity in goat POU1F1-AluI locus. However, the ANOVA analysis revealed no significant differences for gene homozygosty, gene heterozygosty, effective allele numbers and PIC (polymorphism information content) among meat, dairy and cashmere utility types (P > 0.05), suggesting that goat utility types had no significant effect on the spectrum of genetic diversity.

Functional Sequestration of Transcription Factor Activity by Repetitive DNA

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) to the mouse major alpha-satellite repetitive DNA sequesters C/EBPalpha in the transcriptionally inert pericentromeric heterochromatin. We find that this sequestration reduces the transcriptional capacity of C/EBPalpha. Functional sequestration of C/EBPalpha was demonstrated by experimentally reducing C/EBPalpha binding to the major alpha-satellite DNA, which elevated the concentration of C/EBPalpha in the non-heterochromatic subcompartment of the cell nucleus. The reduction in C/EBPalpha binding to alpha-satellite DNA was induced by the co-expression of the transcription factor Pit-1, which removes C/EBPalpha from the heterochromatic compartment, and by the introduction of an altered-specificity mutation into C/EBPalpha that reduces binding to alpha-satellite DNA but permits normal binding to sites in some gene promoters. In both cases the loss of alpha-satellite DNA binding coincided with an elevation in the binding of C/EBPalpha to a promoter and an increased transcriptional output from that promoter. Thus, the binding of C/EBPalpha to this highly repetitive DNA reduced the amount of C/EBPalpha available for binding to and regulation of this promoter. The functional sequestration of some transcription factors through binding to repetitive DNAs may represent an underappreciated mechanism controlling transcription output.

Pituitary-Specific Expression and Pit-1 Regulation of the Rat Growth Hormone-Releasing Hormone Receptor Gene

The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.

Analysis of bovine pituitary specific transcription factor- HinfI gene polymorphism in Indian zebuine cattle

The pituitary specific transcription factor ( Pit-1) controls the expression of prolactin, thyrotropin, and growth hormone genes and is considered as an important candidate gene for several quantitative traits. The study was aimed to reveal the distribution pattern of known variants at Pit-1/ HinfI locus in 16 distinct Indian native cattle ( Bos indicus) breeds. PCR-RFLP analysis of 1350 bp fragment revealed the predominance of BB genotype and B allele with a mean frequency of 0.881 and 0.937, respectively, irrespective of utility type (dairy, dual, draft), geographical region (northern, central and southern) and coat colour (red/brown and grey/white). AB genotype was the next most frequent with a mean frequency of 0.119. Interestingly, only a single individual out of 723 screened was observed to have AA genotype. The significantly low frequency of A allele observed in present study differs from Bos taurine breeds. Chi square test showed non-significant ( P > 0.05) difference in pattern of variant distribution, regardless of the geographical location, agroclimatic regions and coat colour. However, significant differences ( P < 0.05) were observed between utility type categories (dual vs draft and dairy vs draft). This study further validates the utility of PCR-RFLP as efficient means of exploring the distribution pattern of known variants at important candidate genes in Indian zebu cattle for future breeding plans.

Regulatory regions in the promoter and first intron of Sparus aurata growth hormone gene: Repression of gene activity by a polymorphic minisatellite

Transcriptional activity of the gilthead sea bream ( Sparus aurata) growth hormone (saGH) gene promoter has been investigated using transient transfection assays in GH3 cells. Analysis of two fragments (1.7 kb and a 5′-deleted 0.9 kb) of saGH gene promoter directed luciferase reporter gene activity, indicating transcriptional activity of both fragments in vitro. The shorter fragment, containing five potential binding sites for the pituitary-specific transcription factor GHF-1/Pit-1, two cAMP-response elements and two glucocorticoid-response elements conferred higher reporter gene activity than the longer fragment. This result suggests presence of an inhibitory region upstream of the saGH promoter. Transient transfection assays were also used to investigate the effect of the polymorphic minisatellite saGHFIM found in the first intron of saGH gene on gene expression in four cell lines: fish pituitary (RTP-2) and non-pituitary (RTH-149) and mammalian pituitary (GH3) and non-pituitary (HEK293T). Luciferase activity was repressed by saGHFIM containing a high number of tandem repeats compared to a Control construct and to a construct with a smaller number of tandem repeats. Moreover, this repression was dependent on the orientation of the repeats relative to the viral promoter. These in vitro results imply that long GH introns might influence GH gene expression in vivo.

The chicken pituitary-specific transcription factor pit-1 is involved in the hypothalamic regulation of pituitary hormones

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. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.

Identification and Functional Analysis of the Novel S179R POU1F1 Mutation Associated with Combined Pituitary Hormone Deficiency

The pituitary-specific transcription factor 1 plays a key role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Several mutations of the human gene (called POU1F1) have been shown to be responsible for a phenotype of combined pituitary hormone deficiency involving GH, prolactin (PRL), and TSH. We have identified a novel homozygous C to G mutation in exon 4 of the POU1F1 gene (S179R) in a patient with this rare phenotype. We analyzed the functional consequences of this S179R mutation associated with a single-amino acid change in the POU-specific domain. Consequences of this mutation on transcriptional activities by transfection studies in alphaT3 cells, DNA binding ability by EMSA, structural properties, and nuclear accumulation of POU1F1 were investigated. The transactivation capacity of this mutant was markedly decreased on the GH1, PRL, TSHbeta, and POU1F1 genes. Interestingly, this mutation abolished the functional interaction of POU1F1 on the PRL promoter with the coactivator cAMP response element-binding protein-binding protein but not with the transcription factor LIM homeodomain transcription factor 3. The S179R mutant displayed normal nuclear accumulation but a markedly decreased binding to a DNA response element in keeping with crystallographic data, suggesting that the S179R mutation might interfere with DNA binding. Together with previous data, our study indicates that both DNA binding and interaction with cofactors like cAMP response element-binding protein-binding protein are critical for POU1F1 function and that functional and structural properties of abnormal POU1F1 proteins are variously influenced by the type of mutations.

Involvement of the Pituitary-Specific Transcription Factor Pit-1 in Somatolactotrope Cell Growth and Death: An Approach Using Dominant-Negative Pit-1 Mutants

The anterior pituitary-specific transcription factor Pit-1 was initially identified and cloned as a transactivator of the prolactin (PRL) and GH genes and later as a regulator of the TSHb gene. It was found to be a major developmental regulator, because natural Pit-1 gene mutations cause a dwarf phenotype in mice and cause combined pituitary hormone deficiency associated with pituitary hypoplasia in humans. To further investigate the growth-promoting effects of Pit-1, we used a strategy based on the use of dominant-negative Pit-1 mutants as an alternative means of inactivating endogenous Pit-1 functions. R271W, a Pit-1 mutant identified in one allele in patients with severe combined pituitary hormone deficiency, and Pit-1Delta1-123, a deletion mutant in which only the DNA binding domain of Pit-1 is conserved, were generated, and their ability to abolish the effects of the endogenous native Pit-1 in the differentiated proliferating somatolactotrope GH4C1 cell line was investigated. Enforced expression of the dominant-negative mutants in GH4C1 cells using recombinant lentiviral vectors decreased the levels of expression of known Pit-1 target genes such as PRL and GH, abolished the hormone release, and reduced cell viability by decreasing the growth rate and inducing apoptosis via a caspase-independent pathway. These results show for the first time that the growth-promoting effects of Pit-1 are at least partly due to the fact that this transcription factor prevents apoptotic cell death.

Examination of the direct effects of metabolic factors on somatotrope function in a non-human primate model, Papio anubis

In humans, circulating GH levels are increased in catabolic states and suppressed in obesity. In both extremes, normalization of the metabolic environment normalizes GH release, leading to the conclusion that changes in metabolic hormones and/or metabolites promote changes in GH synthesis and release. Metabolic regulation of GH secretion can be mediated centrally by modulation of hypothalamic GHRH and somatostatin input to the pituitary and/or by direct regulation of pituitary somatotrope function. Although data are available showing glucocorticoids, free fatty acids (FFA), IGF-I, and insulin have direct effects on rat somatotrope function, little information is available regarding the direct pituitary effects of these metabolic factors in primates. Therefore, this study examined the effects of glucocorticoids (dexamethasone (0.1-100 nM) and hydrocortisone (10 nM)), FFA (oleic and linoleic acid, 100 and 400 microM each), IGF-I (0.5-50 nM), and insulin (0.5-50 nM) on GH release and GH, GHRH-receptor (GHRH-R) and ghrelin-receptor (GHS-R) mRNA levels, in primary pituitary cell cultures of baboons (Papio anubis) after 24 h treatment. A commercial ELISA kit was used to determine the amount of GH released into the media, while quantitative real-time reverse transcription-PCR was used to determine mRNA levels. To design species-specific primers for baboon GH, GHRH-R, GHS-R, insulin receptor (INSR), IGF-I receptor (IGF-IR), pituitary-specific transcription factor-1 (Pit-1), and cyclophilin A (used as a housekeeping gene) cDNA, sequence data for each baboon transcript were obtained and this data were submitted to Genbank. Glucocorticoids, FFA, insulin and IGF-I treatment did not significantly alter the expression of Pit-1, a transcription factor essential for normal somatotrope development and function. However, as previously reported in the rat, glucocorticoids increased, while FFA, IGF-I and insulin decreased GH release in baboon pituitary cell cultures, where changes in GH release were reflected by comparable changes in GH mRNA levels. In addition, glucocorticoids increased, while FFA, IGF-I and insulin decreased the expression of the GH stimulatory receptors, GHRH-R and GHS-R, without significantly altering cyclophilin A mRNA levels. A role of insulin/INSR pathway, independent of IGF-I, in regulating pituitary function is supported by the fact that (1) IGF-I and insulin significantly suppressed somatotrope function at doses (0.5 and 5 nM respectively) not anticipated to activate their respective receptors, and (2) the baboon pituitary expresses INSR mRNA at levels comparable to or greater than that of tissues commonly considered as insulin sensitive (i.e. liver, skeletal muscle, and fat). Taken together, these results demonstrate that metabolic factors can directly modulate primate somatotrope function through regulating GH synthesis and release, as well as mediating the expression of receptors important in central (GHRH) and systemic (ghrelin) regulation of GH secretion.

Pituitary Transcription Factors: From Congenital Deficiencies to Gene Therapy

Despite the existence of interspecies phenotypic variability, animal models have yielded valuable insights into human pituitary diseases. Studies on Snell and Jackson mice known to have growth hormone, prolactin and thyroid-stimulating hormone deficiencies involving the hypoplastic pituitary gland have led to identifying alterations of the pituitary specific POU homeodomain Pit-1 transcription factor gene. The human phenotype associated with rare mutations in this gene was found to be similar to that of these mice mutants. Terminal differentiation of lactotroph cells and direct regulation of the prolactin gene both require interactions between Pit-1 and cell type specific partners, including panpituitary transcriptional regulators such as Pitx1 and Pitx2. Synergistic activation of the prolactin promoter by Pitx factors and Pit-1 is involved not only in basal condition, but also in responsiveness to forskolin, thyrotrophin-releasing-hormone and epidermal growth factor. In corticotroph cells, Pitx1 interacts with Tpit. Tpit mutations have turned out to be the main molecular cause of neonatal isolated adrenocorticotrophin deficiency. This finding supports the idea that Tpit plays an essential role in the differentiation of the pro-opiomelanocortin pituitary lineage. The effects of Pit-1 are not restricted to hormone gene regulation because this factor also contributes to cell division and protects the cell from programmed cell death. Lentiviral vectors expressing a Pit-1 dominant negative mutant induced time- and dose-dependent cell death in somatotroph and lactotroph adenomas in vitro. Gene transfer by lentiviral vectors should provide a promising step towards developing an efficient specific therapeutic approach by which a gene therapy programme for treating human pituitary adenomas could be based.

Modular changes of cis‐regulatory elements from two functional Pit1 genes in the duplicated genome of Cyprinus carpio

The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth hormone (GH). In the ectotherm teleost Cyprinus carpio, Pit1 plays an important role in regulation of the adaptive response to seasonal environmental changes. Two Pit1 genes exist in carp, a tetraploid vertebrate and transcripts of both genes were detected by RT-PCR analysis. Powerful comparative analyses of the 5'-flanking regions revealed copy specific changes comprising modular functional units in the naturally evolved promoters. These include the precise replacement of four nucleotides around the transcription start site embedded in completely conserved regions extending upstream of the TATA-box, an additional transcription factor binding site in the 5'-UTR of gene-I and, instead, duplication of a 9 bp element in gene-II. Binding of nuclear factors was assessed by electro mobility shift assays using extracts from rat pituitary cells and carp pituitary. Binding was confirmed at one conserved Pit1, one conserved CREB and one consensus MTF1. Interestingly, two functional Pit1 sites and one putative MTF1 binding site are unique to the Pit1 gene-I. In situ hybridization experiments revealed that the expression of gene-I in winter carp was significantly stronger than that of gene-II. Our data suggest that the specific control elements identified in the proximal regulatory region are physiologically relevant for the function of the duplicated Pit1 genes in carp and highlight modular changes in the architecture of two Pit1 genes that evolved for at least 12 MYA in the same organism.

Positive association between POU1F1 and mental retardation in young females in the Chinese Han population

Genetic defects attributable to the genes involved in the hypothalamus-pituitary-thyroid (HPT) gland axis can cause abnormal thyroid hormone function and mental retardation (MR). Pit-1, encoded by the POU1F1 gene on human chromosome 3p11, is a pituitary-specific transcription factor responsible for the expression of several pituitary hormones. Thyrotropin is one of these hormones and is an important regulator in the HPT axis. One of the symptoms of patients with POU1F1 mutations is hypothyroidism and abnormalities of the nervous system early in the period after birth. We performed a case-control association study and a quantitative analysis of IQ to investigate the possible genetic contribution of POU1F1 in the Chinese Han population. Pairwise linkage disequilibrium (LD) analysis showed that rs300996, snp-7057 and rs300977 were in strong LD. There were significant differences of allele, genotype and haplotype frequencies of these three single nucleotide polymorphisms (SNPs) between cases and controls. When we conducted a breakdown comparison between cases and controls within different gender groups, no positive results in males were found. In females, however, we found significant differences between cases and controls in allele frequency distribution of rs300996 (P=0.0003), snp-7057 (P=0.0001) and rs300977 (P=0.0005) and in the distributions of common haplotypes combined by these SNPs (global P=0.0050). The P-value was 0.0301 for rs300996 and 0.0397 for the haplotype combination of rs300996-snp-7057-rs300977 in the analysis of the quantitative effects of the alleles and haplotypes on IQ in females. Our data suggest that POU1F1 may affect MR through a gender-specific mechanism.

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.