Pituitary-specific Transcription Factor Research Articles

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.

Novel Function of the Transactivation Domain of a Pituitary-specific Transcription Factor, Pit-1

Pit-1 stimulates the expression of growth hormone, prolactin, and thyrotropin beta subunit genes. Consequently, abnormality of the Pit-1 gene results in combined pituitary hormone deficiency (CPHD). In this study, we analyzed the function of Pit-1 with a mutation (proline to leucine at codon 24) in the transactivation domain, P24L, which has a normal POU domain important for binding to DNA, because this mutation had been reported in a patient with CPHD. We found that codon 24 proline in the transactivation domain as well as the POU domain of Pit-1 was crucial to recruit coactivator CREB-binding protein (CBP) in the cultured cells. P24L completely lost the responsiveness to cAMP to stimulate the expression of the Pit-1-targeted genes. Furthermore, CBP and Pit-1, but not P24L, markedly enhanced the expression of the Pit-1-targeted gene to cAMP, and adenovirus E1a that binds to CBP and abrogates its function blocked the induction by cAMP of Pit-1-stimulated gene transcription in the pituitary-derived GH3 cells. These results suggest that CBP and proline at codon 24 in the transactivation domain of Pit-1 are important for the cAMP-induced activation of Pit-1-targeted genes. However, P24L maintained basal transcriptional activity, suggesting that CBP is unlikely to be an essential coactivator for Pit-1.

Molecular basis of combined pituitary hormone deficiencies.

Pituitary gland commitment from oral ectoderm occurs in response to inductive signals from the neuroepithelium of the ventral diencephalon. Invagination of the oral ectoderm leads to the creation of Rathke's pouch. Intensified cell proliferation within Rathke's pouch results in formation of the anterior pituitary lobe. Subsequently, highly differentiated cell types arise sequentially due to overlapping, but distinct, spatial and temporal patterns of signaling molecules and transcription factors. Mutations in some of the pituitary-specific transcription factors have been identified in patients with hypopituitarism, confirming the role of these factors in pituitary development.

Characterization of transactivation domain and developmental expression of pituitary specific transcription factor, Pit-1 of ayu ( Plecoglossus altivelis)

Pit-1 is a pituitary-specific transcription factor, which regulates the expression of growth hormone, prolactin, and thyroid stimulating hormone-β genes. We previously reported the expression of a Pit-1 gene from ayu ( Plecoglossus altivelis), which is an important cultivated food fish in Taiwan and Japan. Comparison of ayu Pit-1 with that of salmon, turkey, and rodent, revealed that the Pit-1 structure is highly conserved through vertebrates, especially in POU-specific and POU-homeo domains. The variation among fish, bird, and mammal are mainly found in transactivation domain by alternative splicing and initiation. Three insertions were found. The γ-insert in fish Pit-1 is homologous to the exon 2a of avian Pit-1, which is not found in mammals. The β-insert of fish Pit-1 is homologous to the 28 amino acids (a.a.) and 26 a.a. insert of avian Pit- 1 β ∗ and mammalian Pit-1β, respectively. An additional similarity was noticed between fish and bird, as both of them contain 7 a.a. insert that is not present in mammalian Pit-1. By site directed mutagenesis, we demonstrated that the β, γ, and the 7 a.a. inserts of ayu Pit-1 are critical for activation of zebrafish growth hormone promoter. The ayu Pit-1 protein was found to be expressed specifically in pituitary gland, and its mRNA was first detected at embryonic day 4, significantly increased at embryonic day 5, then sustained to time of hatching at day 8.

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.

Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon ( Salmo salar)

Alkylphenols such as 4-nonylphenol (NP) are one of the wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) mRNA and protein synthesis in the liver are widely used biomarkers for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the pituitary. To evaluate pituitary effects and develop new potential biomarkers for xenoestrogens, the influences of NP and 17β-estradiol (E2) on the mRNA levels of pituitary gonadotropic hormone (GTH) beta subunits [leutinizing hormone beta (LHβ or GTH IIβ) and follicle stimulating hormone beta (FSHβ or GTH Iβ)], prolactin (PRL), growth hormone (GH) and the pituitary specific transcription factor (Pit-1) were investigated in individual male and female juvenile Atlantic salmon ( Salmo salar), 3 days after a single intraperitoneal (i.p.) injection. In one experiment, fish were injected with NP (125 mg/kg body weight (BW)) or E2 (5 mg/kg BW) and a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze LHβ and FSHβ mRNA levels. In the second experiment, fish were injected with three doses of NP (10, 50, 125 mg/kg BW) or a single dose of E2 (5 mg/kg BW) and Northern blot analysis was used to quantify LHβ, FSHβ, PRL, GH and Pit-1 mRNAs. Both NP (50 and 125 mg/kg BW) and E2 significantly induced LHβ mRNA levels ( P<0.01), but only in females. The highest dose of NP (125 mg/kg BW) significantly induced Pit-1 mRNA in males ( P<0.01). NP did not have significant effects on any of the other pituitary transcripts. NP induced LHβ mRNA synthesis in females by up to 6-fold and the changes appeared to correlate with the increases in hepatic Vtg and Zrp mRNA levels. The results show that LHβ mRNA assay in female juvenile salmonids may be used as a marker for pituitary effects of xenoestrogens. The data also suggest that NP may have the potential to perturb the regulation of LHβ gene expression by mimicking E2.

Transcriptional regulation of the human PRL-releasing peptide (PrRP) receptor gene by a dopamine 2 Receptor agonist: cloning and characterization of the human PrRP receptor gene and its promoter region.

PRL-releasing peptide receptor (PrRPR) mRNA was expressed in pituitary adenomas but was not detected in patients treated with bromocriptine, a specific agonist of dopamine 2 (D2) receptor. Although medical treatment with bromocriptine is effective for patients with pituitary adenomas, little is known about the molecular mechanisms of gene regulation mediated by D2 receptors. The cloned human PrRPR gene spanned approximately 2.0 kb and contained two exons and one intron. Two functional polyadenylation signals located at 510 and 714 bp downstream from the stop codon. A primer extension analysis demonstrated two major transcriptional start sites at 139 and 140 bp upstream from the translational start site and an additional minor site at -161. The promoter region contained several putative binding sites for transcriptional factors including pituitary-specific transcription factor (Pit 1), activator protein 1 (AP-1), and specificity protein (Sp1), but no typical TATA or CAAT box. This promoter showed the strong activity in the pituitary-derived GH4C1 cells, and the region between -697 and -596 bp was responsible for the stimulation both by forskolin and overexpression of cAMP response element binding protein (CREB). These stimulations were significantly suppressed by incubation with bromocriptine in a dose- and time-dependent manner, and the mutant CREB (S133A) completely abolished the inhibitory events of bromocriptine. However, EMSA studies demonstrated that CREB did not bind to this region, to which an approximately 60-kDa protein was strongly bound, and that antibodies against CREB, c-Fos, and Sp1 did not supershift this complex. Furthermore, the amount of this unknown protein was apparently reduced by treatment with bromocriptine. A series of mutation analyses demonstrated that the specific sequence, 5'-cccacatcat-3', was required for both the binding to the 60-kDa protein and the repression by bromocriptine. Therefore, the transcriptional repression of the PrRPR gene by bromocriptine required CREB but was independent of direct binding of CREB to the gene and that the sequence -663 -- -672, 5'-cccacatcat-3', bound to the 60-kDa protein appeared to be critical for this event.

Expression of ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: its purification and immunohistochemical detection using monoclonal antibody.

The pituitary-specific transcription factor Pit-1 belongs to the family of POU-domain proteins and is known to play an important role in the differentiation of pituitary cells. Here we report the complete nucleotide sequence of cDNA encoding Pit-1 from the brackish water fish, ayu (Plecoglossus altivelis). Nucleotide sequence analysis of 1910 bp of ayu Pit-1 cDNA revealed an open reading frame of 1074 bp that encodes a protein of 358 amino acids containing a POU-specific domain, POU homeodomain, and an STA (Ser/Thr-rich activation) transactivation domain. We inserted the coding region of Pit-1 cDNA, obtained by PCR, into a pET-20b(+) plasmid to produce recombinant Pit-1 in Escherichia coli BL21 (DE3) pLysS cells. Upon induction with isopropyl beta-D-thiogalactopyranoside, Pit-1 was expressed and accumulated as inclusion bodies in E. coli. The protein was then purified in one step by affinity chromatography on a nickel-nitrilotriacetic acid agarose column under denaturing conditions. This method yielded 0.7 mg of highly pure and stable protein per 200 ml of bacterial culture. A band of 40 kDa, resolved as recombinant ayu Pit-1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, agrees well with the molecular mass calculated from the translated cDNA sequence. The purified recombinant Pit-1 was confirmed in vitro through Western blot analysis, using its monoclonal antibody. This monoclonal antibody detected Pit-1 in the nuclei of ayu developing pituitary by immunohistochemical reaction. It serves as a good reagent for the detection of ayu Pit-1 in situ.

Trout GH promoter analysis reveals a modular pattern of regulation consistent with the diversification of GH gene control and function in vertebrates

In vertebrates, growth hormone (GH) gene expression requires the pituitary-specific transcription factor Pit-1/GHF1 but is differently regulated by a variety of factors in different vertebrate species. Here, we have studied the transcriptional activity of the trout GH (tGH) promoter, which is synergistically stimulated by cAMP and glucocorticoid. Gel shift assays indicated that Pit-1 binds as a dimer to three high affinity sites in the −226/+24 tGH region, and that recombinant cAMP response element (CRE)-binding protein (CREB) binds to a CRE situated between the two distal Pit-1 sites. Deletional and mutational transfection experiments, performed in pituitary Pit-1-expressing GC cells, showed that the different Pit-1 sites play distinct roles and are obligatory elements in the mechanisms mediating cAMP and glucocorticoid responses. Remarkably, the results suggest a hierarchical modular model of regulation of the tGH promoter, according to which a critical module, triggered by Pit-1 bound to the proximal Pit-1 site, is necessary and sufficient to turn on and drive basal levels of transcription. The latter may be stimulated synergistically by two Pit-1-dependent reciprocally non-cooperative auxiliary modules, activated by cAMP and glucocorticoid, respectively. Such modularity explains, in evolutionary terms, the crucial role played by Pit-1 in transcriptional activation and the emergence of the wide variety of mechanisms regulating transcriptional levels of GH, prolactin and other Pit-1-target genes in vertebrates.

Extrapituitary TSH in early chick embryos: Pit-1 dependence?

The expression of the thyrotropin (TSH) gene in the pituitary gland is thought to be dependent upon the pituitary-specific transcription factor, Pit-1. TSH immunoreactivity is, however, widespread in extrapituitary tissues, and the possibility that this may reflect a widespread distribution of Pit-1 was therefore investigated in embryonic chicks, prior to the ontogenic differentiation of the pituitary gland. TSH immunoreactivity in chick embryos at the end of the first trimester of the 21d incubation period was present in discrete cells in the developing brain (particularly in ependymal cells lining the diocoele and mesocoele and in cells lining the otic vesicle), spinal cord (ependymal cells), liver (hepatocytes), lungs (in the linings of the bronchi), gut (in the linings of the proventriculus) and limb bud (in skin, muscle, bone and nerve fibers). In some of these tissues (particularly in brain and spinal cord ependymal cells, cells in the otic vesicle and in liver hepatocytes), the distribution of TSH immunoreactivity was overlapped by the distribution of immunoreactive Pit-1, suggesting Pit-1 involvement in TSH expression in these sites. However, in other tissues (e.g., the trigeminal nerve in the head and the marginal mantle layer of the spinal cord), Pit-1 immunoreactivity was intense but TSH immunoreactivity was marginal. Conversely, other tissues (e.g., cells in the skin, blood vessels, limb bud, bronchus, proventriculus, and cardiopleural cavities) had intense TSH staining but little, if any, Pit-1 immunoreactivity. The expression of the TSH gene in these tissues would thus appear to be Pit-1 independent. These results demonstrate the presence of Pit-1 in pituitary and extrapituitary tissues of the domes tic fowl and suggest the involvement of Pit-1 in the extrapituitary expression of TSH in chick embryos may be tissue-specific.

Characterization of the human somatostatin receptor type 4 promoter

Somatostatin (SRIF) exerts inhibitory effects on virtually all endocrine and exocrine secretions. Five distinct SRIF receptor subtypes (sst 1–5) have been identified. In contrast to the other subtypes, very little is known about specific functions of sst4. We investigated structure and regulation of the human sst4 gene. A genomic clone containing the 5′ region of the sst4 gene was isolated. 1.5 kb of the promoter was sequenced and putative transcription factor binding sites were identified. The transcription start site was located 88 nucleotides upstream of the translation start site. A −984 sst4 promoter directed significant levels of luciferase expression in GH4 rat pituitary cells, Skut-1B endometrium cells, and BEAS-2B human bronchial epithelial cells, whereas only low activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal −209 promoter allowed cell specific expression, its activity in COS-7 cells is not enhanced by co-transfection of the pituitary-specific transcription factor Pit-1. An enhancer element was localized between nt −459 and −984. We did not find any regulation of the sst4 promoter region analyzed by SRIF, forskolin, TPA, IGF-1, EGF, T3, glucocorticoids or 17β-estradiol. These studies identify the 5′ region of the sst4 gene. Furthermore, specific activity of the promoter in various cell lines is demonstrated.

Regulation of Pit-1 expression by ghrelin and GHRP-6 through the GH secretagogue receptor.

GH secretagogues are an expanding class of synthetic peptide and nonpeptide molecules that stimulate the pituitary gland to secrete GH through their own specific receptor, the GH-secretagogue receptor. The cloning of the receptor for these nonclassical GH releasing molecules, together with the more recent characterization of an endogenous ligand, named ghrelin, have unambiguously demonstrated the existence of a physiological system that regulates GH secretion. Somatotroph cell-specific expression of the GH gene is dependent on a pituitary-specific transcription factor (Pit-1). This factor is transcribed in a highly restricted manner in the anterior pituitary gland. The present experiments sought to determine whether the synthetic hexapeptide GHRP-6, a reference GH secretagogue compound, as well as an endogenous ligand, ghrelin, regulate pit-1 expression. By a combination of Northern and Western blot analysis we found that GHRP-6 elicits a time- and dose-dependent activation of pit-1 expression in monolayer cultures of infant rat anterior pituitary cells. This effect was blocked by pretreatment with actinomycin D, but not by cycloheximide, suggesting that this action was due to direct transcriptional activation of pit-1. Using an established cell line (HEK293-GHS-R) that overexpresses the GH secretagogue receptor, we showed a marked stimulatory effect of GHRP-6 on the pit-1 -2,500 bp 5'-region driving luciferase expression. We truncated the responsive region to -231 bp, a sequence that contains two CREs, and found that both CREs are needed for GHRP-6-induced transcriptional activation in both HEK293-GHS-R cells and infant rat anterior pituitary primary cultures. The effect was dependent on PKC, MAPK kinase, and PKA activation. Increasing Pit-1 by coexpression of pCMV-pit-1 potentiated the GHRP-6 effect on the pit-1 promoter. Similarly, we showed that the endogenous GH secretagogue receptor ligand ghrelin exerts a similar effect on the pit-1 promoter. These data provide the first evidence that ghrelin, in addition to its previously reported GH-releasing activities, is also capable of regulating pit-1 transcription through the GH secretagogue receptor in the pituitary, thus giving new insights into the physiological role of the GH secretagogue receptor on somatotroph cell differentiation and function.

Genomic structure and transcriptional regulation of the human growth hormone secretagogue receptor.

Synthetic GH secretagogues stimulate GH release through binding to a recently cloned specific GH secretagogue receptor (GHS-R). The endogenous ligand of this receptor may be part of a new endocrine pathway controlling GH secretion. Two different receptor variants, type 1a and 1b, have been described that differ in their 3'-terminal amino acids. We investigated the genomic structure and transcriptional regulation of the human GHS-R. An 18-kb genomic clone including sequences encoding for the two GHS-R variants was isolated. Sequencing revealed that the two variants originate from specific RNA processing of a single gene that spans approximately 4.1 kb. The transcription start site was defined by 5'-inverse PCR analysis at position -227. RT-PCR analysis points to differential transcriptional initiation and processing. Type 1a is encoded by two exons; 2152 bp of intronic sequence are removed by splicing at position 796/797 relative to the translation start site. Type 1b is encoded by a single exon. A putative polyadenylation signal consensus motif was identified at position +4118; 2.7 kb of the 5'-flanking region were sequenced, and putative transcription factor binding sites were identified. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 168-1745 bp; 1745 bp of the GHS-R promoter directed significant levels of luciferase expression in GH(4) rat pituitary cells, whereas no activity was detected in monkey kidney COS-7 cells, human endometrium Skut-1B cells, mouse hypothalamic LHRH neuronal GT1-7 cells, or mouse corticotroph pituitary AtT20 cells. A minimal 309-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells was enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHS-R promoter by forskolin, somatostatin, insulin-like growth factor I, or 12-O-tetraphorbol 12-myristate 13-acetate. Thyroid hormone and estrogen lead to a significant stimulation; glucocorticoids lead to a significant inhibition. Further mapping suggests a thyroid hormone-responsive element, an estrogen-responsive element, and a glucocorticoid-responsive element located between -309 and the translation start codon. These studies demonstrate the nature of the human GHS-R gene and identify its 5'-flanking region. Furthermore, pituitary-specific activity of the promoter and regulation by various hormones are demonstrated.

Genomic Structure and Transcriptional Regulation of the Human Growth Hormone Secretagogue Receptor

Synthetic GH secretagogues stimulate GH release through binding to a recently cloned specific GH secretagogue receptor (GHS-R). The endogenous ligand of this receptor may be part of a new endocrine pathway controlling GH secretion. Two different receptor variants, type 1a and 1b, have been described that differ in their 3′-terminal amino acids. We investigated the genomic structure and transcriptional regulation of the human GHS-R. An 18-kb genomic clone including sequences encoding for the two GHS-R variants was isolated. Sequencing revealed that the two variants originate from specific RNA processing of a single gene that spans approximately 4.1 kb. The transcription start site was defined by 5′-inverse PCR analysis at position −227. RT-PCR analysis points to differential transcriptional initiation and processing. Type 1a is encoded by two exons; 2152 bp of intronic sequence are removed by splicing at position 796/797 relative to the translation start site. Type 1b is encoded by a single exon. A putative polyadenylation signal consensus motif was identified at position+ 4118; 2.7 kb of the 5′-flanking region were sequenced, and putative transcription factor binding sites were identified. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 168-1745 bp; 1745 bp of the GHS-R promoter directed significant levels of luciferase expression in GH4 rat pituitary cells, whereas no activity was detected in monkey kidney COS-7 cells, human endometrium Skut-1B cells, mouse hypothalamic LHRH neuronal GT1–7 cells, or mouse corticotroph pituitary AtT20 cells. A minimal 309-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells was enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHS-R promoter by forskolin, somatostatin, insulin-like growth factor I, or 12-O-tetraphorbol 12-myristate 13-acetate. Thyroid hormone and estrogen lead to a significant stimulation; glucocorticoids lead to a significant inhibition. Further mapping suggests a thyroid hormone-responsive element, an estrogen-responsive element, and a glucocorticoid-responsive element located between −309 and the translation start codon. These studies demonstrate the nature of the human GHS-R gene and identify its 5′-flanking region. Furthermore, pituitary-specific activity of the promoter and regulation by various hormones are demonstrated.

Combined pituitary hormone deficiency due to the F135C human Pit-1 (pituitary-specific factor 1) gene mutation: functional and structural correlates.

The pituitary-specific transcription factor Pit-1 (pituitary-specific factor 1) is known to play a key role in the differentiation of PRL-, GH-, and TSH-secreting cells, and in the regulation of expression of the corresponding genes. In recent years, 12 distinct mutations of the Pit-1 gene have been shown to be responsible for a phenotype of multiple congenital pituitary hormone deficiency involving PRL, GH, and TSH. We had previously identified, in four siblings with GH, PRL, and TSH deficiencies, a mutation (F135C) resulting in a single amino acid change within the POU-specific binding domain of the Pit-1 molecule. In the present report, we have explored the functional effect of the F135C mutation. In vitro activity tests performed by transfection in human HeLa cells showed decreased transactivation capacity on the PRL, GH, and Pit-1 genes. The DNA binding experiments performed by gel shift showed that the F135C mutation generated a protein capable of binding to DNA response elements. To analyze how the F135C mutation might affect functionality of the transcription factor despite a normal DNA binding, we used a structure modelization approach and also analyzed two other Pit-1 mutant proteins (F135A and F135Y). The loss of functionality in these two mutants was similar to that of F135C. This finding was in keeping with our molecular modeling studies. According to structural data derived from the crystallographic analysis of the DNA/Pit-1 POU domain complex, the conformation of the first helix of the F135C-mutated POU-specific domain could be perturbed to such an extent that any interaction with other transcription cofactors might be definitively prevented.

Pituitary and extrapituitary growth hormone: Pit-1 dependence?

Growth hormone (GH) is primarily produced in pituitary somatotrophs. The synthesis of this hormone is thought to be dependent upon a pituitary-specific transcription factor (Pit-1). However, many extrapituitary tissues are now known to express GH genes. The extrapituitary production of GH may therefore indicate an extrapituitary distribution of the Pit-1 gene. The extrapituitary production of GH may, alternatively, indicate that GH expression occurs independently of Pit-1 in extrapituitary tissues. These possibilities are considered in this brief review.

Complementary DNA Cloning and Ontogenic Expression of Pituitary-Specific Transcription Factor of Chickens (Gallus domesticus) from the Pituitary Gland

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and transactivates promoters of growth hormone (GH)-, prolactin (PRL)-, and thyroid-stimulating hormone-beta (TSHβ)-encoding genes. Pit-1 has been identified in several mammalian species. In birds, it has been identified only in the turkey and chicken pituitary. Several (gg) Pit-1 cDNA sequences have been identified in the anterior pituitary of the chicken (Gallus domesticus). As in turkey, three different transcripts (ggPit-1*, ggPit-1β*, and ggPit-1W*) of the Pit-1 gene were identified. This suggests that the chicken pituitary expresses more variants than reported earlier. Previous studies have concentrated on expressions during posthatch life of the turkey and chicken. The present study has determined the ontogeny of Pit-1 during the embryonic life of the chicken (day 1–day 21 of incubation). The mRNA for Pit-1 was first detected in the pituitary on day 5 of embryonic life. The expression of the mRNA was maintained until hatch. The presence of Pit-1 at this stage of embryonic development suggests that Pit-1 may be physiologically important during embryonic development of birds, as it precedes the gene expression and secretion of GH, PRL, and TSHβ. A similar sequence for the expression of Pit-1 relative to GH and PRL during embryonic/fetal development has been reported for rodents.

Parathyroid hormone regulation of bone sialoprotein (BSP) gene transcription is mediated through a pituitary-specific transcription factor-1 (Pit-1) motif in the rat BSP gene promoter.

Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. Parathyroid hormone (PTH), which regulates serum calcium through its actions on bone cells, increases the expression of BSP in the rat osteosarcoma cell line (ROS 17/2.8). At 10(-8) M PTH (human 1-34 PTH), stimulation of BSP mRNA was first evident at 3 h ( approximately 3.8-fold), reached maximal levels at 6 h ( approximately 4.7-fold), and declined slowly thereafter. The effects of PTH, which were abrogated by cycloheximide (28 microg/ml), did not alter the stability of the BSP mRNA. The increased transcription was mimicked by both forskolin (10(-6) M) and isoproterenol (10(-7) M), and was also increased by 3-isobutyl-1-methylxanthine (IBMX; 10(-5) M), while the transcriptional activity induced by PTH was inhibited by the protein kinase A inhibitor, H89 (5x10(-6) M). From transient transfection assays using various BSP promoter-luciferase constructs, a pituitary-specific transcription factor-1 (Pit-1) regulatory element (nts -111 to -105) was identified as the target of transcriptional activation by PTH. Thus, transcriptional activity of constructs including the Pit-1 was enhanced approximately 4.7-fold by 10(-8) M PTH while 5'-ligation of the Pit-1 element conferred PTH regulation in an SV40 promoter construct. Binding of a nuclear protein, recognized by anti-Pit-1 antibodies, to a radiolabelled Pit-1-BSP probe was decreased in nuclear extracts prepared from PTH, forskolin and isoproterenol-stimulated ROS 17/2.8 cells. Moreover, co-transfection of ROS cells with a double-stranded Pit-1 oligonucleotide also increased luciferase activity. Collectively, these results indicate that PTH acts through a protein kinase A pathway involving cAMP to stimulate BSP transcription by blocking the action of a Pit-1-related nuclear protein that suppresses BSP transcription by binding a cognate element in the BSP promoter. Thus, we have identified a novel Pit-1 suppressor element in the rat BSP gene promoter that is the target of PTH-stimulated transcription of the BSP gene.

Combined Pituitary Hormone Deficiency Caused by a Novel Mutation of a Highly Conserved Residue (F88S) in the Homeodomain of PROP-11

Mutations in the pituitary-specific paired-like homeodomain transcription factor, PROP-1, result in combined pituitary hormone deficiency. We studied a Brazilian girl, offspring of first cousins, who presented with short stature and deficiencies of GH, TSH, PRL, LH, and FSH. Her cortisol response to hypoglycemia was determined at age 4.9, 10.7, and 14.1 yr and remained normal. Magnetic resonance imaging at the age of 9 yr revealed an anterior pituitary lobe of diminished height (3 mm; normal, 4.5 +/- 0.6), but radiography revealed a sella turcica volume above the normal mean. Direct sequencing of the PROP-1 gene revealed homozygosity for a novel 263T>C transition that results in the replacement of a highly conserved phenylalanine by serine at codon 88 (F88S). F88 constitutes the hydrophobic core of the first helix of the homeodomain of PROP-1, and the substitution by the polar residue serine is expected to alter the secondary structure and impair binding of the mutated PROP-1 to DNA target sequences. The F88S mutation (which corresponds to murine F85S) was introduced into the murine Prop-1 complementary DNA and its consequences on DNA binding and trans-activation were assessed in vitro. In contrast to wild-type Prop-1, the F88S mutant showed no significant DNA binding to a PRDQ9 Prop-1 response element in gel shift assays. Transcriptional activation of a luciferase reporter gene containing a PRDQ9 site upstream of a simian virus 40 promoter was reduced to approximately 34% compared with that of wild-type Prop-1 in transiently transfected TSA-201 human embryonic kidney cells. The F88S mutation further expands the repertoire of mutations in PROP-1.