PROP1与联合垂体激素缺乏症

垂体特异性转录因子祖先蛋白(PROP l),是成对同源转录因子,在垂体腺中呈特异性表达,参与早期胚胎垂体的发育,因此,PROP1基因对于垂体前叶的发育是必需的。PROP1启动胚胎期垂体特异性转录因子(PIT-1)的起始表达并维持个体出生后的持续表达,且可直接促使PIT-1细胞系的前体分化为促性腺细胞系。其基因突变可使人、鼠患有联合垂体激素缺乏症(CPHD),表现为生长激素(GH)、促乳素(PRL)、促甲状腺素(TSH)以及促黄体激素(LH)、促卵泡激素(FSH)或促肾上腺皮质激素(ACTH)缺乏,垂体核磁共振成像显示垂体萎缩。在其它哺乳动物中PROP1突变也会引起垂体和性腺激素异常。就PROP1基因的结构与功能,以及与CPHD间的关系作一综述。     

Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.     

Tissue-specific activity of the pro-opiomelanocortin gene promoter.

The pro-opiomelanocortin (POMC) gene is specifically expressed in corticotroph cells of the anterior pituitary. To define the POMC promoter sequences responsible for tissue-specific expression, we assessed POMC promoter activity by gene transfer into POMC-expressing pituitary tumor cells (AtT-20) and fibroblast L cells. The rat POMC promoter was only efficiently utilized and correctly transcribed in AtT-20 cells. 5'-End deletion analysis revealed two promoter regions required for activity in AtT-20 cells. When tested by fusion to a heterologous promoter, DNA fragments corresponding to both regions exhibited tissue-specific activity, suggesting the presence of at least two tissue-specific DNA sequence elements within the promoter. In summary, POMC promoter sequences from -480 to -34 base pairs appear sufficient to mimic the specificity of anterior pituitary expression.     

Truncated K+ channel DNA sequences specifically suppress lymphocyte K+ channel gene expression.

We have constructed a series of deletion mutants of Kv1.3, a Shaker-like, voltage-gated K+ channel, and examined the ability of these truncated mutants to form channels and to specifically suppress full-length Kv1.3 currents. These constructs were expressed heterologously in both Xenopus oocytes and a mouse cytotoxic T cell line. Our results show that a truncated mutant Kv1.3 must contain both the amino terminus and the first transmembrane-spanning segment, S1, to suppress full-length Kv1.3 currents. Amino-terminal-truncated DNA sequences from one subfamily suppress K+ channel expression of members of only the same subfamily. The first 141 amino acids of the amino-terminal of Kv1.3 are not necessary for channel formation. Deletion of these amino acids yields a current identical to that of full-length Kv1.3, except that it cannot be suppressed by a truncated Kv1.3 containing the amino terminus and S1. To test the ability of truncated Kv1.3 to suppress endogenous K+ currents, we constructed a plasmid that contained both truncated Kv1.3 and a selection marker gene (mouse CD4). Although constitutively expressed K+ currents in Jurkat (a human T cell leukemia line) and GH3 (an anterior pituitary cell line) cells cannot be suppressed by this double-gene plasmid, stimulated (up-regulated) Shaker-like K+ currents in GH3 cells can be suppressed.     

The chimpanzee GH locus: composition, organization, and evolution

In most mammals the growth hormone (GH) locus comprises a single gene expressed primarily in the anterior pituitary gland. However, in higher primates multiple duplications of the GH gene gave rise to a complex locus containing several genes. In man this locus comprises five genes, including GH-N (expressed in pituitary) and four genes expressed in the placenta, but in other species the number and organization of these genes vary. The situation in chimpanzee has been unclear, with suggestions of up to seven GH-like genes. We have re-examined the GH locus in chimpanzee and have deduced the complete sequence. The locus includes five genes apparently organized in a fashion similar to that in human, with two of these genes encoding GH-like proteins, and three encoding chorionic somatomammotropins/placental lactogens (CSHs/PLs). There are notable differences between the human and chimpanzee loci with regard to the expressed proteins, gene regulation, and gene conversion events. In particular, one human gene (hCSH-L) has changed substantially since the chimpanzee/human split, potentially becoming a pseudogene, while the corresponding chimpanzee gene (CSH-A1) has been conserved, giving a product almost identical to the adjacent CSH-A2. Chimpanzee appears to produce two CSHs, with potentially differing biological properties, whereas human produces a single CSH. The pattern of gene conversion in human has been quite different from that in chimpanzee. The region around the GH-N gene in chimpanzee is remarkably polymorphic, unlike the corresponding region in human. The results shed new light on the complex evolution of the GH locus in higher primates.     

Cell-type-specific function of the C-type natriuretic peptide gene promoter in rat anterior pituitary-derived cultured cell lines.

The promoter function of the human C-type natriuretic peptide (CNP) gene in various cultured cells was examined by transient transfection assays. The CNP promoter functioned very effectively in GH3 cells, which originated from the growth hormone-producing tumor of the rat anterior pituitary and somatomammotroph phenotype, but functioned much less effectively in GH1 cells, another type of rat pituitary-derived cell with a somatotroph phenotype, and rat primary cardiocytes. The CNP promoter did not function at all in other cells, including AtT20 cells of murine pituitary corticotroph origin. Functional analyses of the deleted promoters with various 5' deletion breakpoints revealed the existence of at least two negative and one positive regulatory regions. Within the positive regulatory region (positions -54 to -19), which conferred 90% of the promoter activity in GH3 cells, two equipotent GC-rich cis elements (positions -49 to -45 and -40 to -35) were identified. Both sites shared half of the promoter activity and binding properties to the nuclear protein in GH3 cells. Rat anterior pituitary tissue contained the binding protein of the identified cis element, which was identical or similar to that of GH3 cells. With Southwestern (DNA-protein) analysis, a 70-kDa specific binding protein distinct from known factors such as SP-1, AP-2, and Pit-1 was identified in the nuclear extract of GH3 cells.     

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.     

The homeodomain protein, Pit-1/GHF-1, is capable of binding to and activating cell-specific elements of both the growth hormone and prolactin gene promoters

Studies were conducted to determine whether the trans-acting protein Pit-1/GHF-1 can bind to and activate promoter elements in both the GH and PRL genes that are necessary for cell-specific expression. Four pituitary cell lines that differentially express the endogenous GH and PRL genes were examined for their ability to activate GH and PRL promoter constructs containing sequences necessary for cell-specific expression (CSEs). Plasmids containing one CSE, -96 PRL and -104 GH, were similarly expressed in each of the four cell lines. Of the plasmids containing two CSEs, -173 PRL was always activated to a greater extent than -145 GH, with this relative activation being stronger in GC and GH1 cells than in 235-1 and GH4C1 cells. Protein-DNA binding assays were used to show that the GH and PRL CSEs specifically bound two highly abundant nuclear proteins (31 and 33 kDa). The two proteins were present at similar levels in all four pituitary cell lines and were recognized by a Pit-1/GHF-1 antibody. In contrast, HeLa and Rat2 cells did not activate transfected GH or PRL plasmids and did not contain nuclear proteins that specifically bound to the GH and PRL CSEs. However, cotransfection of these cells with the expression vector RSV-Pit-1/GHF-1 resulted in the activation of -173 PRL and -145 GH (PRL greater than GH). HeLa cells transfected with RSV-Pit-1/GHF-1 also contained 31- and 33-kDa nuclear proteins that bound to the GH and PRL CSEs. These results show that Pit-1/GHF-1 is present at levels in pituitary cell lines that are sufficient to activate the minimal elements in both the GH and PRL promoters necessary for cell-specific expression of these genes.