Presence Of Pit-1 Research Articles

Cloning of a protein binding to the most proximal Pit-1 binding element of prolactin gene from human pituitary cDNA library

A human pituitary cDNA library was screened using a yeast one-hybrid system to find a factor binding Pit-1 binding elements in the PRL gene other than Pit-1. Beside colonies containing Pit-1 or Oct-1 cDNA, three colonies contained mPOU cDNA, a member of the POU protein family. Immunohistochemical analysis showed mPOU-like immunoreactivity was present in human PRL-producing pituitary tumors but not in non-functioning pituitary tumors. Mobility shift analysis revealed that mPOU bound to Pit-1 binding elements of the PRL gene, 1P and 3P. mPOU activated the expression of 0.6 k PRL and 7×1P reporter genes in the presence of Pit-1 and cAMP, although it did not enhance Pit-1-induced expression of 7×3P reporter gene. These findings suggest that mPOU is involved in the activation of the PRL gene by cAMP through 1P in the presence of Pit-1.

Pit-1/GHF-1 and GH expression in the MCF-7 human breast adenocarcinoma cell line.

GH expression in mammary tumors has been related to the increase and spreading of cell proliferation. Using the MCF-7 human breast adenocarcinoma cell line, it has been demonstrated that autocrine GH-stimulated mammary carcinoma cell proliferation decreased the apoptosis rate and enhanced cell spreading. Surprisingly, no data are available about the presence of Pit-1 (the main pituitary regulator of GH) or GH expression in this cell line. Using RT-PCR, Western blot and immunohistochemistry, we have demonstrated the presence of both mRNA coding Pit-1 and GH as well as Pit-1 and GH protein in the MCF-7 cell line. These data could imply that Pit-1 may be an adequate target to inhibit breast cell proliferation.

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.

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.

The glucocorticoid receptor inhibits the human prolactin gene expression by interference with Pit-1 activity

Glucocorticoids have been shown to inhibit the activity of the human prolactin (hPRL) promoter. Using transient expression experiments in rat pituitary cells, we located the sequence conferring glucocorticoid inhibition to a region which contains Pit-1 binding sites, responsible for pituitary-specific expression, but does not seem to contain a glucocorticoid receptor (GR) binding site. Co-transfection experiments in non-pituitary cell lines, using expression vectors for Pit-1 and different mutants of the human GR show that inhibition of the hPRL gene is seen only in the presence of Pit-1 and GR, and that the DNA binding function of the receptor is not required. Immunoprecipitation studies show that either anti-GR or anti-Pit-1 antibodies are able to co-precipitate GR and Pit-1, suggesting an interaction between these factors. We conclude that the activated GR functionally interferes with the pituitary specific factor Pit-1, thereby leading to the observed transcriptional repression.

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1.

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1