Human Growth Hormone Promoter Research Articles

Stereotactic injection of adenoviral vectors that target gene expression to specific pituitary cell types: implications for gene therapy.

Gene therapy is a potentially useful strategy for the treatment of pituitary adenomas or hormone deficiency disorders. We investigated the feasibility of targeting gene expression to specific pituitary cell types in vivo, using a combination of stereotactic injection and adenoviral vectors that carry pituitary-specific promoters. Recombinant adenoviruses containing the human growth hormone promoter (AdGHGal) or the human glycoprotein hormone alpha-subunit promoter (AdalphaGal) were used to drive expression of the beta-galactosidase gene. The expression of beta-galactosidase activity in the pituitary was analyzed after the administration of recombinant adenoviruses via the peripheral vein or the carotid artery, or by stereotactic injection into the rat pituitary. Double-label histology was used to evaluate cell-type expression in the pituitary. Intravascular injection of AdGHGal or AdalphaGal failed to deliver the marker gene to the pituitary. However, direct stereotactic injection of recombinant adenoviral vectors into the pituitary achieved a high level of transgene expression. In addition, immunohistochemical staining revealed selective expression of the AdGHGal or AdalphaGal transgenes in pituitary cells that normally produce the respective hormones. These findings indicate that adenoviral vectors carrying pituitary gland-specific promoters may be useful for targeted gene therapy of pituitary diseases. However, because of low transduction after peripheral administration, stereotactic injection or local administration of viruses at the time of pituitary surgery is probably required for efficient gene expression.

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.

High-Affinity Binding Sites to the Vitamin D Receptor DNA Binding Domain in the Human Growth Hormone Promoter

The regulation of the human growth hormone (hGH) gene by 1,25(OH) 2D 3is a mechanism which is poorly understood. The objective of this study was to investigate whether the hGH gene has DNA recognition elements for the DNA binding domain of the vitamin D receptor. Using gel retardation assays and footprinting techniques, two high-affinity binding sites, denominated F1 and F2, were identified in the 5′-flanking sequence of hGH. The distal site, F1, located at −59 bp is made up of an imperfect direct repeat separated by 3 bp and showed a high degree of similarity with other known vitamin D response elements (VDREs). The proximal site, F2, located at −36 bp showed a single 7-bp sequence, which is different from other known VDREs. The location of both sites (F1 near the GHF-1/Pit-1 response element, F2 contacting the TATA box) suggests that the vitamin D receptor by itself or through interference with other transcriptional factors may modulate hGH expression.

Transcriptional activation of the human growth hormone gene by ras oncogene

ras Oncogenes play an important role in causing cellular transformation and proliferation. They have been implicated in the formation of many human tumors but only rarely been identified in pituitary adenomas. We studied the effect of ras activation on growth hormone (GH) production. Transcriptional regulation of human GH was investigated by transient transfections in a pituitary cell line GH 4 using different promoter fragments cloned 5′ of the luciferase reporter gene (−344 to −83). Co-transfection of the constitutively active valine 12 mutant ras oncogene (V-12 ras) resulted in a selective and dose-dependent stimulation of −344-GH/Luc activity. This effect is pituitary-cell specific as activation of the human GH promoter by ras was absent in a human chorion carcinoma cell line JEG3. Co-transfection of protein kinase inhibitor did not influence ras mediated stimulation of the human GH promoter. Investigations of several deletion constructs of the human GH promoter revealed that elements between −145 and −83 are sufficient to transduce ras signaling. This region contains two Pit-1 bindings sites as well as a Zn-15 binding site. These studies demonstrate transduction of ras signaling to the human GH promoter through a protein kinase A (PKA) independent signaling pathway. This separate transduction mechanism may convey regulation by yet unknown factors.

Molecular cloning of gilthead seabream ( Sparus aurata) pituitary transcription factor GHF-1/Pit-1

We report here the complete nucleotide sequence of a cDNA clone encoding Sparus aurata GHF-1/Pit-1 isolated from an expression library prepared from gilthead seabream pituitary gland poly(A) +RNA. The cDNA sequence (saGHF-1/Pit-1) encodes a protein of 371 amino acids (aa) containing a POU domain (aa 194–343) and a transactivation, STA domain (aa 1–128). Northern blot hybridization of pituitary RNA detected a single 3.0 kb band and a rat GHF-1/Pit-1 antiserum was found to immunoreact with pituitary protein species of 42 kDa by Western blot analysis. When compared with mammalian GHF-1/Pit-1 aa sequence, the POU and STA domains of saGHF-1/Pit-1 protein show 83% and 48% aa identity, respectively. In spite of the low homology of the transactivation domain, saGHF-1/Pit-1 is able to activate the transcription of the human growth hormone promoter.

Triiodothyronine inhibits transcription from the human growth hormone promoter

Three DNA constructs, the natural human growth hormone gene (hGH-hGH) its 500 bp promoter linked to the chloramphenicol acetyl transferase reporter gene (hGH-CAT), and its structural part linked to the herpes virus thymidine kinase promoter (TK-hGH) were introduced into rat pituitary GC cells by DEAE-dextran transfection. Transient expression was followed as a function of triiodothyronine (T 3) concentration. The hGH-CAT expression was specifically inhibited by T 3 following a typical dose-response curve while hGH-GH gene expression was not significantly modified. The transient expression of TK-hGH increased as a function of T 3 concentration. These results indicate that T 3 exerts two opposite effects on hGH gene expression. First, it down-regulates expression by acting on the promoter; second, it up-regulates expression by acting on the structural part of the gene. These action could be due to regulation of transcription and mRNA stabilization, respectively.

Induction of Human Growth Hormone Promoter Activity by the Adenosine 3′,5′-Monophosphate Pathway Involves a Novel Responsive Element

Regulation of GH gene expression by GRF involves cAMP as a second messenger. We have demonstrated that a 500-basepair fragment of the human GH (hGH) gene 5' flanking region can confer cAMP inducibility upon the chloramphenicol acetyltransferase transcription unit in transient transfections of rat pituitary tumor cells treated with forskolin, an activator of adenyl cyclase. The same hGH construct is not induced by forskolin in nonpituitary-derived cells. Experiments with hGH deletion constructs reveal that binding sites for transcription factor AP-2 and the pituitary-specific factor GHF-1 are not required for forskolin stimulation, but that GHF-1 may potentiate the effect. RNA analyses reveal that forskolin also stimulates accumulation of transcripts initiated at the hGH promoter. Other agents that elevate cAMP levels also stimulate hGH expression. Since the hGH 5' flanking region contains no sequences homologous to the cAMP-responsive element of the somatostatin gene, and the AP-2 sites do not appear to be required for the forskolin response, these results suggest that a novel cAMP-responsive element exists within 82 basepairs upstream from the transcriptional start of the hGH gene and that hGH regulation by GRF may involve interaction between a tissue-specific element and a cAMP-inducible element.

Insulin regulates expression of the human growth hormone gene in transfected cells.

Insulin has been shown to inhibit rat growth hormone (GH) gene transcription. The effects of insulin were therefore tested on the expression of a transfected human GH gene. A 2.6-kilobase EcoRI fragment of the human GH gene was propagated in pUC18 and transfected by calcium-phosphate shock into HeLa and GC cells, respectively. Transfected cells grown in serum-free medium for 72 h expressed human GH measured by specific radioimmunoassay, incorporation of [35S] methionine into newly synthesized GH, and the presence of the appropriately sized protected transcripts seen after RNase protection assay. Immunoprecipitation analysis showed that insulin (0.7-7 nM) suppressed both the basal as well as the hydrocortisone (100 nM)-stimulated expression of newly synthesized 22-kDa GH in a dose-dependent fashion. Insulin (7 nM) also suppressed the basal and hydrocortisone-stimulated GH mRNA transcripts in these cells. Control nontransfected cells did not express human GH. Cells transfected with the truncated pOGH gene and pTKGH gene failed to respond to insulin treatment, whereas the human GH promoter was able to confer insulin responsiveness to the chloramphenicol acetyltransferase reporter gene. cis-Acting regulatory sequences residing on the 497-base pair 5'-flanking region of the human GH gene therefore appear to be a requirement for human GH gene response to the insulin signal.