Pituitary-specific Transcription Factor Pit-1 Research Articles

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.

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.

Dual regulation of somatostatin receptor subtype 1 gene expression by pit-1 in anterior pituitary GH3 cells.

Somatostatin represents a major release inhibiting factor for hypophyseal hormones and mediates its action via five receptor subtypes, sst1-sst5, that are all present in the anterior pituitary. The pituitary specific transcription factor Pit-1 is essential for the pituitary development and pituitary-specific gene expression. Here the transcriptional regulation of the sst1 gene, which contains putative Pit-1-binding sites, was studied in anterior pituitary GH3 cells. We found that a fragment of 2 kb suffices to drive the expression of a reporter gene specifically in this cell line. Positive and negative cis-regulatory elements contributed to the promoter activity. Among these elements two functional binding sites for Pit-1 were identified. While the proximal site mediated transcriptional activation, the distal site attenuated transcription of reporter gene constructs. Mutations of the proximal Pit-1 site prevented expression of the reporter gene. Targeting Pit-1 mRNA by antisense oligonucleotides caused inhibition of transcription of reporter gene constructs containing the proximal Pit-1-binding site. Moreover, the expression of the endogenous sst1 gene in GH3 anterior pituitary cells was blocked. This resulted in reduced sst1 levels at the plasma membrane. Reduced sst1 levels were associated with a diminished antisecretory response to the sst1-specific agonist CH-275 and somatostatin. These results demonstrate the importance of Pit-1 for the expression of the sst1 gene, which hence is placed under common genetic control with the genes for hypophysiotropic hormones and the gene for the receptor of GH-releasing hormone.

CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression.

Hypothalamic growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene expression in anterior pituitary somatotrophs by binding to the GHRH receptor, a G-protein-coupled transmembrane receptor, and by mediating a cAMP-mediated protein kinase A (PKA) signal-transduction pathway. Two nonclassical cAMP-response element motifs (CGTCA) are located at nucleotides -187/-183 (distal cAMP-response element; dCRE) and -99/-95 (proximal cAMP-response element; pCRE) of the human GH promoter and are required for cAMP responsiveness, along with the pituitary-specific transcription factor Pit-1 (official nomenclature, POU1F1). Although a role for cAMP-response element binding protein (CREB) in GH stimulation by PKA has been suggested, it is unclear how the effect may be mediated. CREB binding protein (CBP) is a nuclear cofactor named for its ability to bind CREB. However, CBP also binds other nuclear proteins. We determined that CBP interacts with Pit-1 and is a cofactor for Pit-1-dependent activation of the human GH promoter. This pathway appears to be independent of CREB, with CPB being the likely target of phosphorylation by PKA.

The rat growth hormone-releasing hormone receptor gene: structure, regulation, and generation of receptor isoforms with different signaling properties.

The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 5' flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMP-mediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism.

The Rat Growth Hormone-Releasing Hormone Receptor Gene: Structure, Regulation, and Generation of Receptor Isoforms with Different Signaling Properties

The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 5′ flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMP-mediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism.

Structural and functional organization of the gene encoding the human thyrotropin-releasing hormone receptor.

The thyrotropin-releasing hormone (TRH) receptor (TRHR) is widely distributed throughout the central and peripheral nervous systems. In addition to its role in controlling the synthesis and secretion of thyroid-stimulating hormone and prolactin from the anterior pituitary, TRH is believed to act as a neurotransmitter as well as a neuromodulator. We have isolated genomic lambda and P1-derived artificial chromosome clones encoding the human TRHR. The gene was found to be 35 kb with three exons and two introns. A 541-bp intron 1 (-629 to -89 relative to the translation start site) is conserved between human and mouse. A large intron 2 of 31 kb disrupts the open reading frame (starting in position +790) in the sequence encoding the supposed junction between the third intracellular loop and the putative sixth transmembrane domain. A similar intron was found in chimpanzee and sheep but not in rat and mouse. Promoter analysis of upstream regions demonstrated cell type-specific reporter activation, and sequencing of 2.5 kb of the promoter revealed putative cis-acting regulatory elements for several transcription factors that may contribute to the regulation of the TRHR gene expression. Functional analysis of potential response elements for the anterior pituitary-specific transcription factor Pit-1 revealed cell type-specific binding that was competed out with a Pit-1 response element from the GH gene promoter.

Involvement of a Pit-1 binding site in the regulation of the rat somatostatin receptor 1 gene expression.

It was shown that at least four regions in the 2.2 kb upstream DNA of the sst1 gene are important for the cell type-specific promoter activity in GH3 and RIN cells. Moreover, the 48 bp region located between -165 to -117 harbors positive regulatory elements that are active in RIN and GH3 cells. This region is recognized by the pituitary-specific transcription factor Pit-1. It is therefore concluded that Pit-1 represents a major regulator of GH secretion at the genetic level by regulating transcriptional activity not only of the GH gene itself but also of the genes for the receptors that mediate stimulation and inhibition of GH secretion.

Evidence for the presence of the tissue-specific transcription factor Pit-1 in lancelet larvae.

Recent molecular studies have noted the affinity among cephalochordates and vertebrates. In particular, a cluster of vertebrate-like homeobox genes regulates the development of the lancelet Branchiostoma lanceolatum. A previous study has outlined the expression pattern of the pituitary-specific transcription factor Pit-1 in adult lancelets. Pit-1 belongs to the POU family of transcription factors, which, like homeotic proteins, are members of the helix-turn-helix superfamily of proteins. POU is an acronym for Pit-1, Oct-1 and Oct-2, and Unc-86. In the present work, we investigated the head region of premetamorphic larvae of B. lanceolatum, by means of scanning electron microscopy, wholemount and tissue sections immunocytochemistry, and Western blotting assay, to verify the presence and distribution of Pit-1. Immunoreactive Pit-1 protein was detected in the rostral nerves and in a cluster of photoreceptor cells of the frontal eye. At the same time, an electrophoretic band of 33 kDa was shown from extracts of premetamorphic larvae and recognized by a monoclonal antibody to rat Pit-1. On the basis of the immunocytochemical and electrophoretic results, we can assume that Pit-1 may play a neuromodulatory role in the larval central nervous system. Moreover, the spatial and temporal distribution of Pit-1 protein in larva and adult lancelets agrees only in part with that described in embryonic and adult mice, suggesting different molecular controls of regional identity in the nervous system of cephalochordates and vertebrates.

Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy.

The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 A. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.

Glucocorticoids Repress Transcription from a Negative Glucocorticoid Response Element Recognized by Two Homeodomain-containing Proteins, Pbx and Oct-1

Several studies have established that the prolactin (PRL) gene is expressed not only in lactotrophs and somatotrophs of the anterior pituitary but, albeit to a lesser extent, in non-pituitary cells like human thymocytes, decidualized endometrium, mammary glands during lactation, and some human non-pituitary cell lines. Despite the requirement in the pituitary for the pituitary-specific transcription factor Pit-1/GHF-1 for PRL expression, the expression in non-pituitary cells occurs in the absence of Pit-1/GHF-1 and can be repressed by glucocorticoids. This prompted us to investigate the transcription factors in non-pituitary cells which are involved in controlling expression and glucocorticoid repression of a previously characterized negative glucocorticoid response element from the bovine prolactin gene (PRL3 nGRE). Here we have demonstrated that non-pituitary cells (COS-7 and mouse hepatoma Hepa1c1c7 cells) conferred increased expression via the PRL3 nGRE mainly because of the binding of the ubiquitously expressed POU-homeodomain-containing octamer transcription factor-1 (Oct-1) to an AT-rich sequence present in the PRL3 sequence. However, full transcriptional activity required the binding of a second ubiquitously expressed homeodomain-containing protein, Pbx, previously shown to bind cooperatively with several homeotic selector proteins. The Pbx binding site in the PRL3 nGRE, located just upstream of the Oct-1 binding site, showed a strong sequence similarity with known Pbx binding sites and bound Pbx with an affinity similar to that of other established Pbx target sequences. Interestingly, both Oct-1 and Pbx binding to the PRL3 nGRE were found to be required for glucocorticoid repression. Addition of in vitro translated glucocorticoid receptor DNA binding domain to the nuclear extract prevented Oct-1 and Pbx from binding to the PRL element. The involvement of the homeobox protein Pbx in glucocorticoid repression via an nGRE identifies a new role for this protein.

Fret Imaging of Pit-1 Protein Interactions in Living Cells

The combined use of fluorescence resonance energy transfer (FRET) microscopy and expression of genetic vectors encoding protein fusions with green fluorescent protein (GFP) and blue fluorescent protein (BFP) provides an exceptionally sensitive method for detecting the interaction of protein partners in living cells. The acquisition of FRET signals from GFP- and BFP-fusion proteins expressed in living cells was demonstrated using an optimized imaging system and high sensitivity charge coupled device camera. This imaging system was used to detect energy transfer signals from a fusion protein containing GFP physically linked to BFP expressed in living HeLa cells. In contrast, the co-localization of noninteracting GFP- and BFP-fusion proteins was not sufficient for energy transfer. The FRET imaging system was then used to demonstrate dimerization of the pituitary-specific transcription factor Pit-1 within the living cell nucleus. © 1998 Society of Photo-Optical Instrumentation Engineers.

Structure and regulation of the human growth hormone-releasing hormone receptor gene.

The GHRH receptor (GHRH-R) acts as a critical molecule for proliferation and differentiation of somatotrophic pituitary cells. A role in the pathogenesis of GH hypersecretion and GH deficiency has been implicated. We investigated structure and regulation of the human GHRH-R gene. A genomic clone including approximately 12 kb of 5'-flanking region was isolated. The gene is of complex structure consisting of more than 10 exons. Two kilobase pairs of the promoter were sequenced, and putative transcription factor binding sites were identified. The transcription start site was defined by ribonuclease protection assay. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 108-1456 bp. GHRH-R promoter (1456 bp) directed high levels of luciferase expression in GH4 rat pituitary cells whereas no activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal 202-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells is enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHRH-R promoter by forskolin, phorbol-myristate-acetate, or T3. Glucocorticoids lead to a significant stimulation, and estrogen leads to a significant inhibition. Further mapping suggests a glucocorticoid-responsive element between -1456 and -1181 and an estrogen-responsive element between -202 and -108. These studies demonstrate the complex nature of the human GHRH-R gene and identify its 5'-flanking region. Furthermore, specific activity of the promoter and regulation by various hormones are demonstrated.

Pit-1 Gene Polymorphism, Milk Yield, and Conformation Traits for Italian Holstein-Friesian Bulls

The growth hormone factor-1/pituitary-specific transcription factor Pit-1 is responsible for the expression of growth hormone in mammals. Mutations in Pit-1 have been found in growth hormone disorders of mice and humans. We studied the eventual association between Pit-1 polymorphism using the HinfI enzyme and the milk yield and conformation traits of 89 Italian Holstein-Friesian bulls. A strategy employing polymerase chain reaction was used to amplify a 451-bp fragment from semen DNA. Digestion of polymerase chain reaction products with HinfI revealed two alleles: allele A was not digested (451-bp fragment), and allele B was cut at one restriction site, generating two fragments of 244 and 207 bp. Three patterns were observed; frequencies were 2.2, 31.5, and 66.3% for AA, AB, and BB, respectively. Fixed and mixed linear models were fitted on daughter yield deviations for milk yields and on deregressed proofs for conformation traits. Predictions were weighted using the inverse of the estimated variance of records. The models used contained mean and gene substitution effects for Pit-1 A allele as fixed effects and random sire effect for the mixed model. The A allele was found to be superior for milk and protein yields, inferior for fat percentage, and superior for body depth, angularity, and rear leg set, which is difficult to explain. A canonical transformation revealed that Pit-1 had three actions, one linked to milk yield traits and angularity, a second linked to body depth and rear leg set, and a third linked to lower fat yields and to higher angularity.