Pituitary-specific Transcription Factor Pit-1 Research Articles

The study of the strength and significance of four biological parameters on the body weight of goose.

Alternative products such as those from high-value protien animals have increased the demand for the production of high-quality chicken meat in past few years. This study examines the impact of two distinct feeding types on goose body-weight, as well as the genetic variation of growth hormone (GH) and pituitary-specific transcription factor (Pit-1) genes in ten goose populations using single nucleotide polymorphism (SNP) markers and PCR-RFLP analysis. Both genes were seen as very important for productivity, especially in light of the COVID-19 and its effect on poultry industry at the time. The findings suggest that employing genetic indicators in these two genes in conjunction with a high-fat diet may be a feasible strategy for goose selection programme aiming to increase marketing body weight, as the high-fat diet outperformed the balanced diet. The study investigates the effect of gender, 2 types of diets, breeds and the genetic variation of the two genes, four SNPs were reported to be found: two at the GH gene exons C123T and C158T, and two at the Pit-1 gene exons G161A and T282G. Certain genotypes were found to have a substantial effect on the marketing body-weight of goose, which varied depending on the tested breeds. However, in terms of gender, males report higher and better performance levels than females. Diet, breeds and genotype interaction, and breeds, gender and genotype interaction were found to have a minor effect on goose body weight. However, diet, breeds, gender, SNP locus, diet and breeds interaction, and breeds and gender interaction were found to have a significant effect on goose body weight, as indicated by the effect size results.

Connection between gene markers with milk production traits of Ukrainian dairy cows

The purpose of this study was to obtain information on the genotyping of cows of Ukrainian dairy breeds for the presented loci capa-casein gene (CSN3), thyroglobulin (TG-5) gene, leptin (LEP), pituitary-specific transcription factor (Pit-1), and beta-lactoglobulin (BLG) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and to test their association with milk production. The influence of the genotype on milk yield, fat and protein content in the milk of cows of Ukrainian selection has been established. The highest levels of milk productivity traits were expected from animals with the CT genotype for the LEP gene, AA for the CSN3 gene, CC for the TG5 gene, and AA and BB for the Pit-1 gene. But at the same time, milk obtained from cows with CSN3 BB genotype were characterized by the best cheese suitability. The effect of the homo- or heterozygous state of BLG on the signs of milk production was less noticeable, and the homozygous TT genotype for the TG5 gene was not found in any of the studied breeds. The results obtained can be used in the practical work of breeding farms along with traditional methods of selection, control and preservation of the genetic diversity of specific herds at an optimal level.

Energy evaluation of cows using genetic markers

The aim of the study was to research the polymorphism of allelic variants of somatotropin GH genes and pituitary-specific transcription factor PIT-1 and its influence on bioenergetic parameters of high-yielding of Holstein breed cows in intensive milk production technology (cold method of growing heifers, early insemination of heifers. high daily operating loads on the body, etc.). Genotypes of cows were determined by PCR. Blood DNA was examined in a genetic laboratory. Isolation of genomic DNA was performed using resin “Chelex-100”. The reaction was performed in an amplifier “Tertsyk”. For the energy assessment of first-born cows used indicators of net maintenance energy (MJ per day), net milk energy (MJ per day), total net energy consumption (MJ per day), the value of the energy index (the share of energy released from milk, %), productivity index (kilogram of 4 % milk per 1 MJ), net energy consumption per 1 MJ of milk (MJ), energy released with milk per 1 kg of metabolic mass (MJ). The best energy parameters in cows by GH gene – LL genotype, by pituitary-specific transcription factor PIT-1 – AB genotype, and among paired genotypes – LL/AB and LL/BB. In these animals, bioenergetic indicators for the first and second periods of lactation were higher than their peers of LV, BB and LV/BB genotypes by 2.0–15.2 %, with a reliable result for most traits. In particular, during the first lactation period, the advantage of cows of the LL/AB genotype over the LV/BB genotype in terms of bioenergetic indicators was 3.9–15.2 %, significantly less than in the second lactation period in the range of 1.8–7.4 %. In the genome of pituitary-specific transcription factor PIT-1, better bioenergetic parameters were found in cows of genotype AB than in cows of genotype BB. The difference in bioenergetic parameters is explained by the higher lactotropic function of the L and A alleles, and is the result of a greater complementary effect on the functioning of the alleles of the associated genes in the complex genotype. This was confirmed by the analysis of variance of one-factor complexes, which revealed a stronger influence of genotypes on bioenergetic parameters during the first period of lactation than the second. However, the strength of the influence of the paired genotype on the GH and PIT-1 genes was always high regardless of the lactation period and ranged from 8.9 to 20.9 % (P < 0.01–0.001). In the future, it is of interest to study the formation of the exterior and constitution of cows of different genetic influences under the influence of polymorphisms in the GH and PIT-1 genes.

Comprehensive circular RNA profiling reveals that hsa_circ_0001368 is involved in growth hormone-secreting pituitary adenoma development

Growth hormone-secreting pituitary adenoma (GHPA) represents about 20% of all histological subtypes of pituitary adenoma (PA), which may result in serious complications and shortened lifespan via growth-hormone (GH) hypersecretion. To date, no biomarkers of early diagnosis or therapeutic targets for GHPA treatment have yet been found. Recently, growing evidence has indicated that circular RNAs (circRNAs) are critical for the development and progression of numerous diseases, including cancers; however, their role in the pathogenesis of GHPA has not been reported. Here, we revealed the expression profile of circRNAs in GHPA using a circRNA microarray, and found 1938 circRNAs were upregulated and 1601 circRNAs were downregulated in GHPA versus normal control. Then the ten most up-regulated circRNAs were selected for the mapping of a circRNA–miRNA–target gene interaction network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses further indicate that target genes were mostly enriched in the mTOR and the Wnt signaling pathway. Among these differentially expressed circRNAs, hsa_circ_0001368 was verified significant up-regulated by qRT-PCR, which was specific up-regulated in GHPA and correlated with the invasiveness and serum GH level of GHPA; functional studies indicated that knockdown of hsa_circ_0001368 significantly inhibited the proliferation, invasion and GH secreting level of GHPA primary culture cells. Moreover, hsa_circ_0001368 had a significant positive correlation with the pituitary-specific transcription factor Pit-1. In conclusion, our study identified a wealth of candidate circRNAs involved in GHPA and proposed that hsa_circ_0001368 may represent a novel potential biomarker and therapeutic target of GHPA.

A novel thymoma-associated autoimmune disease: Anti-PIT-1 antibody syndrome

Anti-PIT-1 antibody syndrome has recently been reported and characterized by acquired growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiencies associated with autoimmunity to a pituitary specific transcription factor PIT-1, which plays an essential role in GH-, PRL-, and TSH-producing cells. Although circulating anti-PIT-1 antibody and PIT-1-reactive cytotoxic T cells (CTLs) were detected in the patients, the pathophysiology and precise mechanisms for the autoimmunity remain unclarified. During the follow up, thymoma was diagnosed in all 3 cases with anti-PIT-1 antibody syndrome. Immunohistochemical analysis revealed that PIT-1 was strongly expressed in neoplastic cortical thymic epithelial cells. Importantly, after thymectomy, the titer of anti-PIT-1 antibody decreased and reactivity of CTLs toward PIT-1 diminished. These data strongly suggest that the aberrant expression of PIT-1 in the thymoma plays a causal role in the development of this syndrome. Thus, we define that this syndrome is a novel thymoma-associated autoimmune disease.

Polymorphisms in the 5'-flanking regions of the GH, PRL, and Pit-1 genes with Muscovy duck egg production.

Somatotropic axis-related genes contribute to reproduction of ducks. Five SNP in the 5'-flanking regions of the growth hormone (GH), prolactin (PRL), and pituitary-specific transcription factor (Pit-1) genes were identified and genotyped in a female population of Muscovy ducks. Association analysis of these SNP with Muscovy duck egg production traits was performed. Results showed that SNP C-515G of GH was significantly associated with egg number in ducks at age 59 wk (E59W; P = 0.0009) and egg number in ducks at age 300 d (E300D; P = 0.0022). Single nucleotide polymorphism C-441T of GH was significantly associated with E59W (P = 0.0014). Significant associations of SNP T-884C and T-335C of PRL with the age at first egg (A1D), E59W, and E300D were detected in this population (P < 0.0001). It was concluded that these 4 SNP might be useful markers to use with the aim of increasing Muscovy duck E59W. On the basis of genetic parameter estimation, the heritability of A1D, E300D, E59W, and molting time were 0.43 ± 0.04, 0.45 ± 0.04, 0.36 ± 0.04, and 0.04 ± 0.03, respectively. Strong positive genetic correlation was noted between E59W and E300D (correlation coefficient = 0.80), whereas a negative association was noted between E59W and A1D (correlation coefficient = -0.80). Therefore, the selection for improved A1D should also increase E59W.

Characterization and association of the Pit-1 gene in the Indian buffalo

Pituitary-specific transcription factor (Pit-1) is responsible for pituitary development and hormone expression in mammals. In present study, a comparison of buffalo Pit-1 gene sequences with Bos taurus revealed no nucleotide changes however, 7 single nucleotide polymorphisms (SNPs), one of which was non-synonymous were found within Indian buffalo breeds (Bubalus bubalis). SNP c.160 T&gt;A alters the encoded amino acid from methionine to lysine. Association of all SNPs with milk production traits was assessed in Mehsana buffalo populations and the results demonstrated that SNP c.160 T&gt;A was significantly associated with the milk fat percentage (P=0.0358). Sequence analysis showed that the buffalo Pit-1 protein shares high homology with cattle (Bos taurus) (100%), goat (99%), sheep (99%), human (96%) and pig (98%). Polymorphisms detected in buffalo Pit-1 gene are expected to have functional significance and might be useful as genetic markers in association studies for milk fat and milk yield in buffalo.

DNase I Hypersensitive Site II of the Human Growth Hormone Locus Control Region Mediates an Essential and Distinct Long-range Enhancer Function

Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5' to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this "neutral" DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.

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.

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.

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.

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.

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.

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.

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.

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.

Identification of an Upstream Pituitary-Active Promoter of Human Somatostatin Receptor Subtype 5

Somatostatin receptor subtype 5 (sst5) has been linked to inhibition of PRL and insulin secretion. We characterized the genomic structure of the human sst5. The transcription start site was located 94 nucleotides upstream of the initiator ATG codon. Sequence analysis of 5′-inverse PCR products revealed the presence of a 6.1-kb intron in the 5′-untranslated region. RT-PCR analysis indicated tissue-specific activation of the newly identified upstream promoter in pituitary, but not in small intestine, lung, or placenta. A −1741 promoter directed significant levels of luciferase expression in GH4 rat pituitary cells, Skut-1B endometrium cells, and JEG3 chorion carcinoma cells, which was absent in COS-7 monkey kidney cells. A minimal −101 promoter was sufficient to allow tissue-specific expression. Its activity in COS-7 cells was not enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. Analysis of deletion constructs revealed a GC-rich region immediately upstream of the transcription start site, which is necessary for promoter activity. Somatostatin led to a significant inhibition, and forskolin and thyroid hormone to a significant stimulation of pituitary-specific promoter activity. Further mapping suggested a cAMP-responsive element located between −101 and the transcription start site, and thyroid hormone-responsive elements between −1741 and −1269 and between −317 and −101. These studies identified an upstream promoter of the sst5 gene with tissue-specific activity.

Identification of an upstream pituitary-active promoter of human somatostatin receptor subtype 5.

Somatostatin receptor subtype 5 (sst5) has been linked to inhibition of PRL and insulin secretion. We characterized the genomic structure of the human sst5. The transcription start site was located 94 nucleotides upstream of the initiator ATG codon. Sequence analysis of 5'-inverse PCR products revealed the presence of a 6.1-kb intron in the 5'-untranslated region. RT-PCR analysis indicated tissue-specific activation of the newly identified upstream promoter in pituitary, but not in small intestine, lung, or placenta. A -1741 promoter directed significant levels of luciferase expression in GH(4) rat pituitary cells, Skut-1B endometrium cells, and JEG3 chorion carcinoma cells, which was absent in COS-7 monkey kidney cells. A minimal -101 promoter was sufficient to allow tissue-specific expression. Its activity in COS-7 cells was not enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. Analysis of deletion constructs revealed a GC-rich region immediately upstream of the transcription start site, which is necessary for promoter activity. Somatostatin led to a significant inhibition, and forskolin and thyroid hormone to a significant stimulation of pituitary-specific promoter activity. Further mapping suggested a cAMP-responsive element located between -101 and the transcription start site, and thyroid hormone-responsive elements between -1741 and -1269 and between -317 and -101. These studies identified an upstream promoter of the sst5 gene with tissue-specific activity.