Growth Hormone Promoter Activity Research Articles

Activin/follistatin system in grass carp pituitary cells: - Regulation by local release of growth hormone and luteinizing hormone and its functional role in growth hormone synthesis and secretion.

Gonadotrophin regulation by activin/follistatin system is well-documented, but the corresponding effect on growth hormone (GH) has not been fully characterized and with little information available in lower vertebrates, especially in fish models. In grass carp, local interactions of GH and luteinizing hormone (LH) can induce GH release and gene expression at pituitary level via autocrine/paracrine mechanisms. To shed light on the role of activin/follistatin system in GH regulation by local actions of GH and LH, grass carp activin βA and βB were cloned, shown to be single-copy genes expressed in the pituitary, and confirmed to encode activin proteins capable of transactivating promoter with activin-responsive elements. In grass carp pituitary cells, activin A and B were effective in reducing GH secretion and GH cell content with concurrent drop in GH mRNA level whereas the opposite was true for follistatin, the activin-binding protein known to neutralize the effects of endogenous activin. Treatment with activin A and B not only could suppress basal but also inhibit GH mRNA expression induced by GH and human chorionic gonadotropin (hCG), a functional analogue of LH in fish model. Apparently, down-regulation of GH mRNA by activin was mediated by reducing GH transcript stability with concurrent inhibition on GH promoter activity via the SMAD pathway. In reciprocal experiments, GH treatment was found to up-regulate activin βA, activin βB and follistatin mRNA levels in carp pituitary cells but the opposite was noted by removing endogenous GH with GH antiserum. Interestingly, parallel treatment with hCG could also inhibit basal as well as GH-induced activin βA, activin βB and follistatin gene expression. These results, as a whole, indicate that the pituitary activin/follistatin system can serve as a regulatory target for local interactions of GH and LH and contribute to GH regulation by autocrine/paracrine mechanisms in the carp pituitary.

Mechanisms for luteinizing hormone induction of growth hormone gene transcription in fish model: Crosstalk of the cAMP/PKA pathway with MAPK-and PI3K-dependent cascades

In our previous studies in grass carp pituitary cells, local production of luteinizing hormone (LH) was shown to induce growth hormone (GH) production and gene expression, which constitutes a major component of the “intrapituitary feedback loop” regulating GH secretion and synthesis via autocrine/paracrine interactions between gonadotrophs and somatotrophs in the carp pituitary. To further investigate the signaling mechanisms mediating LH action at the transcriptional level, promoter studies were performed in GH3 cells co-transfected with the expression vector for carp LH receptor and luciferase-expressing reporter constructs with grass carp GH promoter. In this cell model, treatment with human chorionic gonadotropin (hCG) was effective in increasing GH promoter activity and the responsive sequence was mapped to position −616 and −572 of the grass carp GH promoter. GH promoter activation induced by hCG occurred with concurrent rise in cAMP production, CREB phosphorylation, and could be inhibited by inactivation of adenylate cyclase (AC), PKA, MEK1/2, P38 MAPK, PI3K and mTOR. AC activation, presumably via cAMP production, could mimic hCG-induced CREB phosphorylation and GH promoter activity, and these stimulatory effects were also sensitive to the blockade of PKA-, MAPK- and PI3K- dependent cascades. These results, as a whole, suggest that LH receptor activation in the carp pituitary may trigger GH gene transcription through CREB phosphorylation as a result of the functional crosstalk of the cAMP/PKA pathway with MAPK-and PI3K-dependent cascades.

Insulin-like growth factor binding protein-3 modulates osteoblast differentiation via interaction with vitamin D receptor

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is a secreted glycoprotein that reduces the bioavailability of IGFs. This glycoprotein has both IGF-dependent and -independent effects on cell growth. However, the mechanisms responsible for the IGF-independent actions of IGFBP-3 are not fully understood. In the present study, we used multiple methodologies including glutathione S-transferase pull-down assay and co-immunoprecipitation to demonstrate that IGFBP-3 can directly interact with vitamin D receptor (VDR) in vitro and in vivo. Furthermore, immunofluorescence co-localization studies showed that IGFBP-3 and VDR could co-localize in the cell nucleus. Reporter gene experiment showed that IGFBP-3 negatively regulates the growth hormone promoter activity induced by ligand-activated VDR. Moreover, real-time RT-PCR demonstrated that IGFBP-3 can inhibit the osteocalcin and CYP24a1 mRNA transcription induced by 1,25-(OH)2D3 in osteoblastic cells. Finally, alkaline phosphatase activity significantly decreased in osteoblastic cells when the cells were transfected with IGFBP-3 in the presence of 1,25-(OH)2D3. In conclusion, these studies provide evidence that overexpression of IGFBP-3 suppresses osteoblastic differentiation regulated by VDR in the presence of 1,25-(OH)2D3. These findings reveal a novel mechanism by which IGFBP-3 functions.

Insulin-like growth factor binding protein-6 interacts with the thyroid hormone receptor α1 and modulates the thyroid hormone-response in osteoblastic differentiation

Insulin-like growth factor binding protein-6 (IGFBP-6) is a member of the insulin-like growth factor binding protein family, which has both Insulin-like growth factor-dependent and independent effects on cell growth. In previous studies, we have shown that recombinant IGFBP-6 could be translocated into the cell nucleus. But the effect in the nucleus of IGFBP-6 is not clear. In the present study, we use multiple methodologies including Glutathione S-transferase pull-down assay, co-immunoprecipitation, fluorescence resonance energy transfer to demonstrate that IGFBP-6 can directly interact with thyroid hormone receptor alpha 1 (TRα1) in vitro and in vivo. We also demonstrate that the DNA-binding domains and Ligand-binding domains of TRα1 and N-terminal domains and C-terminal domains of IGFBP-6 are involved in the interaction. This interaction also can block the formation of TR: retinoid X receptor heterodimers. Furthermore, immunofluorescence co-localization studies show IGFBP-6 and TRα1 could co-localize in the nucleus of the cells. Reporter gene experiment shows that IGFBP-6 negatively regulates the growth hormone promoter activity induced by ligand activated TRα1. Moreover, real-time RT-PCR demonstrates that IGFBP-6 could inhibit the osteocalcin mRNA transcription induced by Triiodothyronine (3,3',5-Triiodo-L-thyronine, T3) in osteoblastic cells. Finally, alkaline phosphatase activity was significantly decreased in osteoblastic cells when the cells were transfected with IGFBP-6 in the presence of T3. In conclusion, these studies provide evidence that overexpression of IGFBP-6 suppresses osteoblastic differentiation regulated by TR in the present of T3.

Insulin-like growth factor binding protein-3 interacts with the thyroid hormone receptor α1 and modulates transcription of thyroid hormone responsive gene.

To study the interaction between insulin-like growth factor binding protein-3 (IGFBP-3) and thyroid hormone receptor α1 (TRα1) and the modulatory effect of IGFBP-3 on transcription of the thyroid hormone responsive gene. The interaction between IGFBP-3 and TRα1 was detected with glutathione-S-transferase pull-down method, co-immunoprecipitation, fluorescence resonance energy transfer test. The cellular distribution of these two proteins was observed by confocal laser scanning microscopy. The effect of IGFBP-3 on the growth hormone promoter activity stimulated by triiodothyronine (T3) was determined by dual-luciferase reporter assay. IGFBP-3 interacted with TRα1 both in vivo and in vitro. IGFBP-3 and TRα1 were shown to co-localize in the nucleus of HEK-293 cells. The overexpressed IGFBP-3 inhibited the growth hormone promoter activity stimulated by T3 (P<0.01). IGFBP-3 interacts with TRα1 and inhibits T3 responsive gene transcription. This finding further confirms the insulin-like growth factor-independent role of IGFBP-3 in the nucleus.

Over-expression of protein disulfide isomerase reduces the release of growth hormone induced by bisphenol A and/or T 3

We previously isolated a bisphenol A (BPA)-binding protein from rat brain and showed that it was identical to the protein disulfide isomerase (PDI), which also serves as a 3,3′,5-triiodo- l-thyronine (T 3)-binding protein. In this study, we investigated the effects of BPA on the production of growth hormone (GH) in GH3 cells and examined the possible involvement of PDI in this process. When administered singly, BPA and T 3 each induced GH release in GH3 cells. When cells were treated with the combination of BPA and T 3, the release of GH was much greater than that by T 3 alone, but GH mRNA and promoter activity were not increased. These results suggested that the synergistic effect of T 3 plus BPA on GH release was due to posttranslational regulation. Over-expression of PDI suppressed GH mRNA expression and GH release, suggesting that PDI modulates the T 3-induced gene expression. We conclude that BPA can disrupt the thyroid hormone function in GH3 cells, and GH release induced by T 3 is influenced by expression of PDI.

Signaling mechanisms for α2-adrenergic inhibition of PACAP-induced growth hormone secretion and gene expression grass carp pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent growth hormone (GH)-releasing factor in lower vertebrates. However, its functional interactions with other GH regulators have not been fully characterized. In fish models, norepinephrine (NE) inhibits GH release at the pituitary cell level, but its effects on GH synthesis have yet to be determined. We examined adrenergic inhibition of PACAP-induced GH secretion and GH gene expression using grass carp pituitary cells as a cell model. Through activation of pituitary alpha2-adrenoreceptors, NE or the alpha2-agonist clonidine reduced both basal and PACAP-induced GH release and GH mRNA expression. In carp pituitary cells, clonidine also suppressed cAMP production and intracellular Ca2+ levels and blocked PACAP induction of these two second messenger signals. In GH3 cells transfected with a reporter carrying the grass carp GH promoter, PACAP stimulation increased GH promoter activity, and this stimulatory effect could be abolished by NE treatment. In parallel experiments, clonidine reduced GH primary transcript and GH promoter activity without affecting GH mRNA stability, and these inhibitory actions were mimicked by inhibiting adenylate cyclase (AC), blocking protein kinase A (PKA), removing extracellular Ca2+ in the culture medium, or inactivating L-type voltage-sensitive Ca2+ channels (VSCC). Since our recent studies have shown that PACAP can induce GH secretion in carp pituitary cells through cAMP/PKA- and Ca2+/calmodulin-dependent mechanisms, these results, taken together, suggest that alpha2-adrenergic stimulation in the carp pituitary may inhibit PACAP-induced GH release and GH gene transcription by blocking the AC/cAMP/PKA pathway and Ca2+ entry through L-type VSCC.

Identification and characterization of four splicing variants of ovine POU1F1 gene

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone ( GH), prolactin ( PRL), thyroid-stimulating hormone ( TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-β, with a 78 bp insert in the trans-activation domain; POU1F1-γ that lacks exon 3 and POU1F1-δ that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the β variant presented only 12% of the Wild-type trans-activation capacity. Variants γ and δ showed no capacity to trans-activate PRL promoter. Both γ and δ variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone ( p < 0.05). Concerning the GH promoter, the β variant presented a trans-activation capacity 10% higher than Wt. Wt and β variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants γ and δ showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly ( p < 0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp. I. Functional Coupling of Cyclic Adenosine 3′,5′-Monophosphate and Ca2+/Calmodulin-Dependent Signaling Pathways in PACAP-Induced GH Secretion and GH Gene Expression in Grass Carp Pituitary Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, has been recently proposed to be the ancestral GH-releasing factor. Using grass carp as a model for bony fish, we examined the mechanisms for PACAP regulation of GH synthesis and secretion at the pituitary level. Nerve fibers with PACAP immunoreactivity were identified in the grass carp pituitary overlapping with the distribution of somatotrophs. At the somatotroph level, PACAP was shown to induce cAMP synthesis and Ca(2+) entry through voltage-sensitive Ca(2+) channels (VSCC). In carp pituitary cells, PACAP but not vasoactive intestinal polypeptide increased GH release, GH content, total GH production, and steady-state GH mRNA levels. PACAP also enhanced GH mRNA stability, GH promoter activity, and nuclear expression of GH primary transcripts. Increasing cAMP levels, induction of Ca(2+) entry, and activation of VSCC were all effective in elevating GH secretion and GH mRNA levels. PACAP-induced GH secretion and GH mRNA expression, however, were abolished by inhibiting adenylate cyclase and protein kinase A, removing extracellular Ca(2+) or VSCC blockade, or inactivating calmodulin (CaM)-dependent protein kinase II (CaM kinase II). Similar sensitivity to VSCC and CaM kinase II blockade was also observed by activating cAMP production as a trigger for GH release and GH gene expression. These results suggest that PACAP stimulates GH synthesis and secretion in grass carp pituitary cells through PAC(1) receptors. These stimulatory actions probably are mediated by the adenylate cyclase/cAMP/protein kinase A pathway coupled to Ca(2+) entry via VSCC and subsequent activation of CaM/CaM kinase II cascades.

Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters

Despite important advances in human therapeutics, no specific treatment for both non-functioning gonadotroph and resistant somatotroph adenomas is available. Gene transfer by viral vectors can be considered as a promising way to achieve a specific and efficient treatment. Here we show the possibility of efficient gene transfer in human pituitary adenoma cells in vitro using a human immunodeficiency virus (HIV)-type 1-derived vector. Using enhanced green fluorescent protein (eGFP) gene as a marker placed under the phosphoglycerate kinase (PGK) promoter, gonadotroph and somatotroph adenomas were transduced even with moderate viral loads. The expression started at day 2, reached a peak at day 5, and it was still present at day 90. For targeting somatotroph and gonadotroph adenomas, human growth hormone (GH) promoter (GH -481, +54 bp) and two fragments of the human glycoprotein hormone alpha-subunit promoter (alpha-subunit 1 -520, +33 bp, and alpha-subunit 2 -907, +33 bp) were tested. In gonadotroph adenomas, the percentage of identified fluorescent cells and the fluorescence intensity analyzed by fluorescence-activated cell sorting indicated that the strength of the alpha-subunit 1 and alpha-subunit 2 promoters were comparable to that of the PGK promoter. Primary cultures of rat pituitary cells showed that alpha-subunit 1 is more selective to thyreotroph and gonadotroph phenotypes than alpha-subunit 2. GH promoter activity appeared weak in somatotroph adenomas. The human GH enhancer did not increase the GH promoter activity at all but the human prolactin promoter (-250 bp) allowed 4-fold more fluorescent cells to be obtained than the GH promoter. Several cell lines appeared too permissive to test cell-specificity of pituitary promoters. However, on human non-pituitary cell cultures, the tested pituitary promoters seemed clearly selective to target endocrine pituitary phenotypes. This study gives a starting point for a gene-therapy program using lentiviral vectors to transfer therapeutic genes in human pituitary adenomas.

Effects of Hormones Targeting Nuclear Receptors on Transcriptional Regulation of the Growth Hormone Gene in the MtT/S Rat Somatotrope Cell Line

We have examined the effects of nuclear receptor hormones such as glucocorticoid, gonadal steroid hormones, thyroid hormone and retinoids on the transcriptional regulation of the 5′-promoter activity of growth hormone (GH) gene using the MtT/S rat pure somatotrope cell line or MtT/SGL, a subclone of MtT/S in which the rat GH gene 5′-promoter (1.7 Kb)-luciferase fusion gene was stably incorporated. RT-PCR analyses revealed that receptors for all the hormones except androgen receptor were expressed in the cell line. Triiodothyronine (T₃, 10 nM) transiently but significantly stimulated the promoter activity of GH gene, whereas retinoic acids (9-cis and all-trans, both 1 µM) showed sustained stimulation. There were no additive effects among the T₃, all-trans, and9-cis retinoic acids. Synthetic glucocorticoid hormone dexamethasone (100 nM) showed an inhibitory effect but, interestingly, significantly enhanced T₃-stimulated GH promoter activity during long-term incubation. Among the gonadal steroid hormones tested, estradiol and estriol had significant stimulatory effects, and deletion analysis showed that the estrogen effect was maintained with the shortest construct examined (–150 to +6, +1 denotes the transcription start site). These results suggest that thyroid hormone and retinoids stimulate the transcription of GH gene, probably through a common response element, whereas glucocorticoid has both negative and positive effects on GH expression, depending on the combination with other hormones and the time of exposure. Estrogens also have direct stimulatory effects through the proximal promoter region of GH gene.

Continuous Measurement of Targeted Promoter Activity by a Secreted Bioluminescence Reporter, Vargula hilgendorfii Luciferase

The promoter activity of growth hormone (GH) was continuously monitored in rat pituitary adenoma cells (GH3) by a secreted bioluminescence reporter, Vargula hilgendorfii (Vh) luciferase. The sensitivity of the reporter was approximately 60-times higher than that of the firefly luciferase. GH3 cells were transfected with a plasmid containing a DNA sequence of the GH promoter (1.8 kb) and a full length of the Vh luciferase cDNA. Using the stable transformants, the Vh luciferase activity was monitored in the plate culture through the bioluminescence of Vh luciferase secreted into the culture medium. The reporter activity was well correlated with GH mRNA as well as GH when the GH promoter activity was activated by thyroid hormone. To develop a continuous monitoring system of the promoter, the reporter activity was sequentially measured in the perfusion system. When the promoter activity of the stable transformants was suppressed by a transcription inhibitor, the reporter activity and GH in the perfusate were simultaneously decreased. The Vh luciferase reporter is a sensitive and convenient tool for continuous and prolonged measurement of promoter activity in living cell culture systems.

Characterization of the Promoter-Directing Expression of Growth Hormone in a Monocyte Cell Line

Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis-elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (–299/–193 bp) and negative (–193/–107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between –299 and –107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line.

Tissue-specific differential repression of gene expression by a dominant negative mutant of thyroid hormone beta1 receptor.

Resistance to thyroid hormone (RTH) is a genetic disease caused by the mutations of the thyroid hormone beta receptor (TRbeta) gene, producing receptors with a dominant negative action. The present study addressed the question as to whether tissue-specific factors modulate the dominant negative function in different tissues. We prepared stably transfected pituitary GH3 (GH3-PV) and liver SK-Hep-1 (SK-Hep-1-PV) cell lines with a potent dominant negative mutant, PV. The growth hormone (GH) and the malic enzyme genes (ME) in GH3 and SK-Hep-1, respectively, are directly regulated by the thyroid hormone, 3,3,'5-triiodo-L-thyronine (T3). The ratio of the expressed PV/endogenous TRbeta1 proteins was approximately 20 and 5 for GH3-PV and SK-Hep-1-PV cells, respectively. However, the T3-activated expression of the GH gene in GH3-PV and ME gene in SK-Hep-1-PV was repressed by approximately 30% and 90%, respectively, indicating the lack of correlation of PV/TRpbeta1 protein ratio with the dominant negative potency of mutant PV. Furthermore, the synergistic effect of the pituitary-specific factor 1 on the TR-mediated GH promoter activity was not repressed by mutant PV. Taken together, these results suggest that the dominant negative effect of mutant TR is variable in the tissues studied.

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

Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'- monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A

Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.

Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'-monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A.

Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.

A Pit-1 phosphorylation mutant can mediate both basal and induced prolactin and growth hormone promoter activity

A Pit-1 phosphorylation mutant can mediate both basal and induced prolactin and growth hormone promoter activity

Dexamethasone control of growth hormone mRNA levels in GH 3 pituitary cells is cycloheximide-sensitive and primarily posttranscriptional

To clarify the mechanism of growth hormone (GH) gene activation by glucocorticoids in GH 3 pituitary cells, GH mRNA accumulation in nuclear and cytoplasmic compartments was measured in the presence and absence of cycloheximide. In dexamethasone-treated cells, levels of GH mRNA were increased in the nucleus by 6 h and in the cytoplasm by 12 h. Dexamethasone treatment caused a 5- to 24-fold rise in total GH mRNA levels by 48–72 h. The differential elevation of nuclear levels of GH mRNA relative to the amount of cytoplasmic GH mRNA persisted for 48 h. A transient accumulation of GH mRNA in the nucleus was followed by a brief rise in cytoplasmic GH mRNA levels in GH 3 cells treated simultaneously with dexamethasone and cycloheximide. In GH 3 cells pretreated for 2 h with cycloheximide, the rise in nuclear and cytoplasmic GH mRNA levels mediated by dexamethasone was blocked completely. Levels of glucocorticoid receptor were unaffected by cycloheximide. These data suggest that the stimulation of GH mRNA levels by glucocorticoids is initiated within the nucleus and that cycloheximide-sensitive events are essential for this stimulation to occur. To assess the importance of GH gene transcriptional activation by glucocorticoids, nuclear transcription run-on reactions and assays of GH promoter activity in an aminoglycoside 3'-phosphotransferase (Neo) fusion gene within stably transformed GH 3 cells were performed. Evidence for a weak, transient transcriptional activation of the GH gene by dexamethasone in nuclear run-on assays was obtained. Consistent with this idea, a 30–72 h exposure to dexamethasone raised levels of Neo mRNA in GH-Neo GH 3 cell transformants by ⩽ 2-fold. We conclude that glucocorticoid stimulation of GH mRNA in GH 3 cells requires ongoing protein synthesis and can occur largely independently of GH gene transcriptional activation.