Pituitary Transcription Factor Research Articles

Expression of the pituitary transcription factor GHF-1/PIT-1 in cell types of the adult porcine adenohypophysis.

We describe the expression of the transcription factor GHF-1/PIT-1 in adult porcine adenohypophysis by a nonradioactive in situ hybridization (ISH) method using a digoxigenin-labeled cDNA probe corresponding to the entire coding region of rat GHF-1. GHF-1 transcripts were found in 71.7% of adenohypophyseal cells. We also report the simultaneous detection of GHF-1 mRNA and pituitary hormones by combined ISH and immunocytochemistry (IC) in dispersed adenohypophyseal cells, detected with an alkaline phosphatase-NBT/BCIP technique and with an immunoperoxidase-3-amino-9-ethylcarbazole (AEC) method, respectively. The combination of the two techniques neither abolished nor diminished their sensitivity or specificity. GHF-1 is expressed in all five of the cell types in the adult porcine adenohypophysis, showing that this method is suitable for simultaneous detection of transcripts and proteins at the single-cell level.

Ontogeny of pituitary transcription factor-1 (Pit-1), growth hormone (GH) and prolactin (PRL) mRNA levels in male and female rats and the differential expression of Pit-1 in lactotrophs and somatotrophs.

Pituitary transcription factor-1 (Pit-1 or GHF-1), a transcription factor specific to the anterior pituitary, is involved in the expression and regulation of the growth hormone (GH) and prolactin (PRL) genes. Post-pubertally, the expression of both GH and PRL becomes sexually dimorphic with males having higher GH levels and females higher PRL levels; however, little is known about the postnatal regulation of their common transcription factor. Furthermore, whether the Pit-1 gene is differentially expressed in somatotrophs and lactotrophs remains to be elucidated. In this study, we used in situ hybridization histochemistry to examine Pit-1, GH and PRL mRNA levels in the anterior pituitaries of male and female rats throughout development (0, 5, 10, 20, 30, 40 and 60 days of age) to determine when GH and PRL production becomes sexually dimorphic and if this is accompanied by a dimorphism in Pit-1 gene expression. In addition, the level of Pit-1 mRNA was determined separately in both GH mRNA and PRL mRNA containing cells during the various developmental stages. We found that in both males and females the mRNA levels of Pit-1, GH and PRL remain relatively unchanged until around the time of pubertal onset (30-40 days) when there is a significant increase in all three mRNA species, which is followed by a decrease to adult levels. Also around the time of puberty, both GH and PRL mRNA levels become sexually dimorphic, with males having higher levels of GH mRNA and females higher PRL mRNA levels. In contrast, at no time during development were overall Pit-1 mRNA levels found to differ between the sexes. However, when Pit-1 mRNA content was measured separately in specific cell types, significant differences between the sexes became evident. Throughout development Pit-1 mRNA levels are higher in lactotrophs of females than in those of males, whereas in somatotrophs males have higher Pit-1 mRNA levels than females. Furthermore, within a sex there is differential expression of Pit-1 in the two cell types with females having significantly higher levels of Pit-1 in lactotrophs than in somatotrophs and males having higher levels in somatotrophs than in lactotrophs. These data support the hypothesis that a sexual dimorphism exists in the expression and pituitary specific transcription factor Pit-1; however, this dimorphism is not manifest as a difference in overall mRNA levels, but in the differential expression of this gene in lactotrophs and somatotrophs.

Dense incisors (din): a new mouse mutation on chromosome 16 affecting tooth eruption and body size.

Dense incisors (din) is a new autosomal recessive mutation in the mouse that interferes with complete eruption of the incisors. The initial eruption of incisors through the gingiva does not differ in mutants and normal littermates, but subsequent further eruption of incisors is arrested in mutants. Radiographic examinations show that, because the incisors do not erupt, continued dentin formation gradually occludes the pulp chambers of these teeth creating as dense incisor. The arrested eruption of the incisor results in an anterior open bite. The pleiotropic phenotype of din/din mutant mice also includes small body size, reduced ear pinna size, and coat color dilution. The din mutation was mapped to Chr 16 near the pituitary transcription factor gene Pit1, but din is not a mutation in Pit1.

In vivo and in vitro regulation of pituitary transcription factor-1 (Pit-1) by changes in the hormone environment.

Pituitary transcription factor-1 (Pit-1 or GHF-1) is a transcription factor specific to the anterior pituitary and is involved in the expression and regulation of the growth hormone (GH), prolactin (PRL) and thyroid-stimulating hormone (TSH) beta-subunit genes. The expression of these three genes can be modulated by changes in the hormone environment and it is thought that some of these effects are mediated through Pit-1, but little is known about the physiological regulation of this transcription factor. Therefore, we first asked whether Pit-1 gene expression is modified as a result of changes in the in vivo gonadal steroid environment and if this could be correlated with changes in GH and/or PRL mRNA levels. Secondly, we sought to determine if sex steroids affect the mRNA levels of these three peptides by acting at the level of the pituitary and whether these effects are androgen or estrogen mediated. Finally, how sex steroids modulate the response of these three genes to the hypothalamic neuropeptides growth hormone-releasing hormone (GHRH) and somatostatin (SS) was analyzed. To this end, we compared Pit-1, GH and PRL mRNA levels in the anterior pituitary of intact, castrated, and castrated testosterone-replaced adult male rats. In addition, primary cultures of adult male pituitaries were used to study the direct effects of both androgens and estrogens on Pit-1, GH, and PRL mRNA levels. In situ hybridization histochemistry was used to compare relative levels of Pit-1, GH and PRL mRNA. Densitometric analysis of the in vivo studies showed that castration resulted in a 57, 40 and 55% decline in Pit-1, GH and PRL mRNA signal levels, respectively. Furthermore, replacement with testosterone (T) at the time of castration completely prevented the decline in all three mRNA species (ANOVA: Pit-1 mRNA, p < 0.0001; GH mRNA, p < 0.0001; PRL mRNA, p < 0.0001). In vivo, both T (10(-7) M) and estradiol (10(-9) M) were capable of stimulating Pit-1 mRNA and PRL mRNA levels, while dihydrotestosterone (DHT; 10(-7) M) had no effect. There was no effect of any of these steroid treatments on GH mRNA levels in vitro. Addition of GHRH to the cultures increased GH mRNA levels, as well as those of Pit-1 and PRL, and SS had the opposite effect on GH mRNA levels. Whereas the GH response to GHRH was not significantly modified by exposure to sex steroids, the effect of SS was. The presence of sex steroids was capable of modifying the Pit-1 and PRL responses to both GHRH and SS. These results clearly indicate that changes in circulating levels of sex steroids modulate the expression of Pit-1 in the anterior pituitary and that these changes can be correlated with commensurate modifications in GH and PRL mRNA levels. Furthermore, the effect on both Pit-1 and PRL mRNA levels occurs, at least in part, at the level of the anterior pituitary and is an estrogen-receptor-mediated event. In contrast, the effects of gonadal steroids on GH mRNA levels are less direct and are most likely mediated at the level of the hypothalamus, as well as through modulation of the response of the somatotroph to hypothalamic factors. We conclude that the transcription factor Pit-1 is actively regulated physiologically and may be involved in mediating some of the effects of sex steroids and hypothalamic factors on the synthesis of certain anterior pituitary hormones.

A novel pituitary transcription factor is produced by alternative splicing of the human GHF-1/PIT-1 gene

An alternative splice acceptor site in intron 1 of the human GHF-1/PIT-1 gene was sequenced. The use of this splice site is responsible for a 78-bp in-frame insertion upstream from exon 2 and leads to the hGHF-2/PIT-2 cDNA detected in normal human pituitary.

G(i) alpha 2- and G(o) alpha-mediated signaling in the Pit-1-dependent inhibition of the prolactin gene promoter. Control of transcription by dopamine D2 receptors.

Dopaminergic signaling in pituitary lactotrophs is dependent on coupling of D2 receptors to several inhibitory G-protein subtypes, resulting in the activation of multiple signaling pathways. In prolactin-secreting GH4 cells that express cloned D2 receptors, dopamine selectively inhibits the activity of the prolactin gene promoter, a response mediated in part by the pituitary transcription factor Pit-1. Transfected gain-of-function mutants of the G alpha subtypes, Gi alpha 2 (Q205L) and G(o) alpha (Q205L), mimic the promoter-specific and Pit-1-dependent inhibition by dopamine. Whereas the activated Gi alpha 2 subtype suppresses cAMP levels, the G(o) alpha mutant does not, demonstrating a cAMP-independent pathway in the inhibition of the prolactin gene. This alternate pathway could involve other regulators, possibly calcium. Interestingly, in Ltk- cells in which cloned D2 receptors modestly suppress cAMP, but elevate [Ca2+]i, the activity of the prolactin promoter is enhanced rather than inhibited by dopamine. The response is promoter-specific, dependent on Pit-1, and completely blocked by low concentrations of EGTA, consistent with a calcium-regulated pathway. Last, in GH4 cells, the absence of additivity between Gi alpha 2 and G(o) alpha mutants suggests a convergent mechanism in the reduction of prolactin promoter activity, in which either signaling pathway may be sufficient for maximum inhibition. This apparent redundancy in inhibitory control mechanisms may be of physiological importance for maintaining efficient tonic suppression of prolactin synthesis.

The Pituitary Transcription Factor Ghf-1 /Pit-1: an Evolutionary Overview

Growth hormone (GH) transcription factor 1 (GHF-1) or pituitary transcription factor 1 (Pit-1) is a pituitary gland-specific homeodomain protein of the POU subclass. POU proteins exert critical developmental and transcriptional functions. Two functional regions, the DNA-binding or POU domain and the transcriptional serine-threonine activation domain (STA) are highly conserved between fishes and primates. However, a variability in the length of the STA domain (between 71 and 128 amino acid residues) is found in different species as well as isoforms generated by alternative splicing.

Molecular basis of familial growth hormone deficiency.

A significant proportion of cases of GH deficiency (5-30%) may be due to genetic causes. At least four Mendelian types of isolated GH deficiency (IGHD) have been delineated based on the mode of inheritance and the degree of GH deficiency: IGHD type IA, autosomal recessive with absent endogenous GH; type IB, autosomal recessive with diminished GH; type II, autosomal dominant with diminished GH; and type III, X-linked with diminished GH. Most patients with IGHD type IA have heterogeneous deletions, ranging in size from 6.7 kb to 45 kb, that encompass the entire gene encoding for pituitary GH, GH-1. Nonsense, frameshift and splice GH-1 mutations that predict a complete lack of bioactive GH synthesis in homozygotes have also been reported in association with IGHD IA. Additionally, some cases of IGHD type II have dominant negative mutations in one allele of the GH-1 gene. Panhypopituitary Dwarfism (PD), a condition characterized by deficiency of at least other pituitary trophic hormone in addition to GH deficiency, can have autosomal and X-linked modes of inheritance. Interestingly, both recessive and dominant mutations at the gene encoding for the pituitary transcription factor Pit-1 have been found in a specific subtype of PD that combines GH, prolactin and TSH deficiencies. In contrast, the loci and mutations responsible for the other Mendelian forms of IGHD and PD remain unknown. Linkage studies using genetic markers have excluded the GH locus on chromosome 20 in all the studied families (types IB and II) in whom the mutation cannot be traced to defects in these genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired growth hormone-releasing hormone signal transduction in the dwarf (dw) rat is independent of a generalized defect in the stimulatory G-protein, Gs alpha.

The homozygous dwarf (dw) rat pituitary is characterized by a 95% reduction in GH content as well as a 75-80% reduction in the number of somatotrophs. The nature of the mutation responsible for this phenotype is unknown. Previous investigations from our laboratory indicate that dw somatotrophs exhibit decreased sensitivity and a reduced GH secretory response to GH-releasing hormone in vitro, accompanied by a decreased generation of cAMP. We hypothesized that dw rats have a defect in the pathway linking the GH-releasing hormone receptor to adenylyl cyclase and focused on the expression and function of the stimulatory G-protein, Gs alpha. When corrected for differences in pituitary size and cell number, GH mRNA content was reduced by 78% compared to that in controls. However, there was no difference in Gs alpha mRNA content or size in dw pituitaries. Similarly, there was no difference in the content or size of mRNA for the pituitary transcription factor pit-1 in dw pituitaries. Immunoblot analysis of pituitary membrane proteins using a Gs alpha-specific antibody revealed no differences in size, quantity, or relative distribution of Gs alpha peptides between control and dw pituitaries. In addition, cholera toxin effectively ribosylated dw Gs alpha, and there were no differences in the size, quantity, or relative distribution of ribosylated membrane proteins between dw and control pituitaries. Finally, to examine for mutations in other regions of the Gs alpha-coding sequence, we cloned the full-length Gs alpha cDNA from dw rat pituitaries by polymerase chain reaction. The sequence of this clone was identical to that of normal rat Gs alpha cDNA. These results indicate that 1) 52-kilodalton Gs alpha appears to predominate in both normal and dw pituitary; 2) the content, function, and sequence of Gs alpha in adult dw rats are normal; and 3) a generalized Gs alpha regulatory or structural mutation as the cause of the observed phenotype can be excluded. The results also demonstrate that there is no decrease in pit-1 expression in the adult dw pituitary.

Absence of somatotrophs, lactotrophs, and thyrotrophs in the pituitary of two dwarfs with hypothyroidism: Deficiency of pituitary transcription factor-1?

Most patients with dwarfism due to growth hormone (GH) deficiency have normal pituitary somatotroph morphology and GH response to GH-releasing hormone (GRH), consistent with decreased GRH synthesis, release, or delivery. Primary pituitary hyposecretion of GH may result from adenohypophysial tissue destruction caused by tumors, such as craniopharyngioma. We report a hitherto undescribed form of primary pituitary dwarfism associated with absence of adenohypophysial GH, prolactin (PRL), and thyrotropin (TSH). Two sisters had dwarfism, hypothyroidism, and hypoglycemia. The first child had craniofacial abnormalities and died at age 11/2 months of fluid overload. The second sibling died at age 4 years of pulmonary congestion. At autopsy, both pituitaries were small and acidophils were conspicuously absent. They contained a normal number of corticotrophs with intense adrenocorticotropin (ACTH) immunoreactivity, and gonadotrophs with normal content of α-subunit and β-subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). No GH, PRL, or β-TSH immunoreactivity was identified. The thyroid glandswere atrophic. Both children had marked pancreatic nesidioblastosis, with an increased number of insulin-containing cells. This clinicopathologic entity appears to represent a familial disorder in which there is defective development of three adenohypophysial cell types: somatotrophs, lactotrophs, and thyrotrophs. A common pituitary transcription factor, Pit-1, has been implicated in the differentiation of these three cell types. The pituitary changes in these two children resemble those described in Snell, Jackson, and Ames dwarf mice, which have recently been shown to be due to abnormalities of the Pit-1 gene. We suggest that this novel human disease is due to deficient or abnormal pituitary transcription factor Pit-1.

Thyrotropin (TSH)-releasing hormone-responsive elements in the rat TSH beta gene have distinct biological and nuclear protein-binding properties.

Two TRH-responsive elements have been identified in the rat TSH beta gene by deletion/mutation analysis of the 5'-flanking region of the gene and transfection of TSH beta-luciferase constructs into the GH3 pituitary cell line. Biological responsiveness was confirmed by inserting synthetic oligonucleotides next to the heterologous viral thymidine kinase (tk) promoter in tk luciferase (tkLUC) constructs. Both DNA regions, termed TSH A (at -274 to -258 bp) and TSH C (-402 to -385 bp), have a high level of sequence similarity to binding sites for the POU domain pituitary transcription factor Pit-1/GHF-1. In transfection assays, the TSH A region had no basal enhancer activity, but did confer 3- to 6-fold TRH- and PMA-stimulated transcriptional responses to the tk promoter. The TSH C region conferred basal enhancer activity (3- to 10-fold above control tkLUC) as well as a 2- to 3-fold TRH or PMA response. Combinations of TSH A and TSH C elements conferred both enhancer activity and a TRH- or PMA-stimulated response, but more than two copies of the regions resulted in no further stimulatory effect. Both TSH beta gene regions bound to nuclear proteins from GH3 cells, as determined by gel retardation analysis. The TSH A region DNA formed three prominent DNA-protein complexes, ranging from slowly to rapidly migrating bands and with calculated affinities of 32, 0.5, and 208 nM, respectively. The TSH C region formed two major complexes, which corresponded on the basis of mobility to the most slowly and rapidly migrating complexes formed by TSH A, but with calculated affinities of 3.1 and 33 nM. TSH C also formed a rapidly migrating minor complex unique for this gene region. The more rapidly migrating complexes appeared to be specific to nuclear proteins from GH3 cells. Treatment of cells with TRH did not significantly alter the affinity of protein binding. Mutation of TSH A and TSH C DNA by T to G substitutions abolished the ability of the DNA to confer a TRH response and severely inhibited the ability of the DNA to bind to GH3 nuclear proteins. Thus, transcriptional regulation of the rat TSH beta gene by TRH is correlated with the ability of the two TRH-sensitive elements to bind nuclear proteins. The differences noted in basal enhancer activity or the degree of TRH responsiveness may be related to some unique proteins bound to each DNA or to the differences in affinity of binding of the proteins common to both elements.

Regulation of cell‐type‐specific transcription and differentiation of the pituitary

The transcription of rat prolactin and growth hormone genes in vitro requires a pituitary transcription factor, specific to certain cell types in the pituitary, which currently appears to be the PUF-I/Pit-1/GHF-1 protein. This factor binds to cis-regulatory elements in the 5' region of both genes and exerts a positive influence on transcription initiation presumably by interacting with general transcription factors. The PUF-I/Pit-1/GHF-1 transcriptional regulatory protein probably has an important role in not only the differentiation of the pituitary lactotroph/somatotroph cell lineage; it is also expressed in the early development of the nervous system but its function there is less well documented. It appears to be one member of a family of trans-activator proteins involved in differential gene expression in several cell types.