Rat Growth Hormone Gene Expression Research Articles

The transcription factor GHF-1, but not the splice variant GHF-2, cooperates with thyroid hormone and retinoic acid receptors to stimulate rat growth hormone gene expression

The rat growth hormone (GH) promoter was significantly activated in non-pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand-dependent activation was found in the presence of the pituitary-specific transcription factor GHF-1. When compared with GHF-1, the splice variant GHF-2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF-2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over-expression of GHF-2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF-1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors on GH gene expression.

9-cis retinoic acid regulation of rat growth hormone gene expression: potential roles of multiple nuclear hormone receptors.

Rat GH (rGH) gene expression is increased by both thyroid hormone (T3) and all-trans retinoic acid (RA) via a composite hormone response element (HRE) containing three putative half-sites (rGH-HRE). However, it is not known whether 9-cis RA (9cRA) also can regulate rGH gene expression. In this study, we performed a Northern blot analysis that demonstrated that 9cRA, as well as T3 and RA, increased rGH messenger RNA expression in rat pituitary GH3 cells. Transient transfection studies in GH3 cells, using reporter plasmids containing the rGH-HRE and mutated half-sites, revealed that 9cRA-stimulation of rGH transcription was mediated by the rGH-HRE and that all three half-sites are necessary. Additionally, we performed cotransfection studies to elucidate the particular receptor complexes involved in 9cRA regulation of rGH gene expression using CV-1 cells, which contain little or no endogenous RA receptors, and thyroid hormone receptors. Interestingly, in the presence of either retinoid X receptor alone, RA receptors alone, or both receptors, 9cRA caused similar induction of transcriptional activity. However, cotransfection of thyroid hormone receptors with these receptors repressed basal and blocked 9cRA-induced transcriptional activity in the absence of T3. Our data suggest that 9cRA-stimulation of rGH transcription is likely mediated by 9cRA-bound retinoid X receptor- and/or RA receptor-containing complexes but not by thyroid hormone receptor-containing complexes. Our studies provide evidence that several different members of the nuclear hormone receptor family can interact on this composite DNA element, with transcription stimulated or blocked depending on the presence or absence of cognate ligands.

9-cis retinoic acid regulation of rat growth hormone gene expression: potential roles of multiple nuclear hormone receptors

9-cis retinoic acid regulation of rat growth hormone gene expression: potential roles of multiple nuclear hormone receptors

Synergistic interactions between Pit-1 and other elements are required for effective somatotroph rat growth hormone gene expression in transgenic mice.

The role of the pituitary-specific POU-domain protein, Pit-1, in GH gene activation has been established by in vitro analyses and by the observation that mutations affecting the Pit-1 genomic locus result in genetically transmitted dwarfism. To define the quantitative contribution of the two Pit-1 response elements and the potential role of other factors in GH gene activation, we systematically assessed the ability of a series of GH promoter regions to activate transgenes in the mouse anterior pituitary gland. These studies revealed that the two GH Pit-1 binding sites are necessary, but not sufficient, for efficient transcriptional activation. Transgenes containing information including only these cis-active regions are expressed at extremely low levels in the pituitary glands of transgenic mice. The addition of 35 base pairs of 5'-flanking information, contributing other elements including a thyroid hormone/retinoic acid response element, results in much higher levels of transgene expression. Sequences located upstream of this segment contribute a further 5- to 10-fold activation. Thus, while Pit-1 is required for GH gene activation, it alone can only direct minimal expression in transgenic animals. Rather, synergistic interactions between other promoter elements and Pit-1 appear to be required for expression of the transgenes at approximately the 100-fold higher levels that are characteristic of somatotrophs, and are therefore likely to be critical components of somatotroph-specific expression of the GH gene.

Synergistic interactions between Pit-1 and other elements are required for effective somatotroph rat growth hormone gene expression in transgenic mice

Synergistic interactions between Pit-1 and other elements are required for effective somatotroph rat growth hormone gene expression in transgenic mice

Ligand-dependent, Pit-1/growth hormone factor-1 (GHF-1)-independent transcriptional stimulation of rat growth hormone gene expression by thyroid hormone receptors in vitro.

The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.

Ligand-dependent, Pit-1/growth hormone factor-1 (GHF-1)-independent transcriptional stimulation of rat growth hormone gene expression by thyroid hormone receptors in vitro.

The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.

Posttranscriptional Regulation of Rat Growth Hormone Gene Expression: Increased Message Stability and Nuclear Polyadenylation Accompany Thyroid Hormone Depletion

In thyroid hormone-depleted rats, the rate of transcription of the growth hormone (GH) gene in the anterior pituitary gland is lower than the rate in euthyroid controls, and there is a corresponding reduction in the abundance of the GH mRNA. Concomitantly, the poly(A) tail of the GH mRNA increases in length. Examination of nuclear RNA from anterior pituitary glands of control and thyroid hormone-depleted rats revealed no difference in the length of pre-mRNAs containing the first and last introns of the GH gene. However, mature nuclear GH RNA is differentially polyadenylated in euthyroid and hypothyroid animals. We suggest that the extent of polyadenylation of the GH transcript is regulated in the cell nucleus concomitant with or subsequent to the splicing of the pre-mRNA. Experiments with anterior pituitary gland explant cultures demonstrated that the GH mRNA from thyroid hormone-depleted rats is more stable than its euthyroid counterpart and that the poly(A) tail may contribute to the differential stability of free GH ribonucleoproteins.

Posttranscriptional regulation of rat growth hormone gene expression: increased message stability and nuclear polyadenylation accompany thyroid hormone depletion.

In thyroid hormone-depleted rats, the rate of transcription of the growth hormone (GH) gene in the anterior pituitary gland is lower than the rate in euthyroid controls, and there is a corresponding reduction in the abundance of the GH mRNA. Concomitantly, the poly(A) tail of the GH mRNA increases in length. Examination of nuclear RNA from anterior pituitary glands of control and thyroid hormone-depleted rats revealed no difference in the length of pre-mRNAs containing the first and last introns of the GH gene. However, mature nuclear GH RNA is differentially polyadenylated in euthyroid and hypothyroid animals. We suggest that the extent of polyadenylation of the GH transcript is regulated in the cell nucleus concomitant with or subsequent to the splicing of the pre-mRNA. Experiments with anterior pituitary gland explant cultures demonstrated that the GH mRNA from thyroid hormone-depleted rats is more stable than its euthyroid counterpart and that the poly(A) tail may contribute to the differential stability of free GH ribonucleoproteins.

A major thyroid hormone response element in the third intron of the rat growth hormone gene.

The rat growth hormone (RGH) gene constitutes a well-documented model system for the direct regulation of transcription by thyroid hormones. In order to analyse its interaction with sequences in the RGH gene, we have overproduced the thyroid hormone receptor-alpha (c-erbA) protein using a vaccinia virus expression system. The expressed protein bound T3 and DNA-cellulose with expected affinities, and the major binding site for the receptor protein was found to be located in the third intron of the RGH gene. This site displayed significantly higher affinity for the receptor protein than a previously described thyroid hormone response element (TRE) in the promoter of this gene, and also conferred stronger hormone responsiveness in vivo to a heterologous promoter. The data suggest that this novel TRE plays a major role in the regulation of rat growth hormone gene expression by thyroid hormones.

Expression of the rat growth hormone gene in transfected CV-1 cells

Expression of the cloned rat growth hormone gene was studied in transfected CV-1 cells. Cell lines expressing rat growth hormone synthesized and secreted the 22 kilodalton form and the 20 kilodalton variant. The results confirm that the 20 kilodalton variant arises from an alternatively spliced mRNA rather than expression from a variant gene in rat. The 5' end of the mRNA from expressing cell lines was located upstream of the normal initiation site in rat. Transcription initiated within a 5' flanking AT rich sequence. Results presented indicate that transcription from normally silent promoter or promoter-like sequences dictated rat growth hormone gene expression in transfected cells. Finally, no hormone was expressed by CV-1 cells transfected with a plasmid containing the rat growth hormone gene in the same transcriptional reading frame as the neomycin resistance gene of pSV2neo indicating an effect of cloning orientation on expression in CV-1 cells.

Hormonal and tissue-specific control of rat prolactin and rat growth hormone gene expression.

Hormonal and tissue-specific control of rat prolactin and rat growth hormone gene expression.

Rat growth hormone gene expression. Both cell-specific and thyroid hormone response elements are required for thyroid hormone regulation.

The elements involved in mediating cell-specific and thyroid hormone stimulation of rat growth hormone gene expression have been defined by transfection studies and by nuclease footprinting. 5'-Flanking DNA extending to -104 can mediate cell-specific expression, and this is enhanced 3- to 4-fold with DNA extending to -145. Cell-specific factors, found only in rat growth hormone producing cells, bind within the -137/-107 and -95/-65 regions, and competition studies suggest that the same factor binds to both sites. The sequence A (A or T) TAAAT is found at the center of both footprints at -80 and -122, suggesting that it is a core component of the recognition sequence of the cell-specific factor. Disruption of the spatial and/or distance relationships between the two regions eliminates the enhanced level of cell-specific expression, suggesting a cooperative interaction of the proteins which bind to these elements. Sequences located between -208 and -178 can confer thyroid hormone-regulated expression when linked in either orientation in close proximity to one or both cell-specific elements. The thyroid hormone and cell-specific elements function as an enhancer-like unit and are both required to confer regulated expression to heterologous promoters. We propose that thyroid hormone acts via its receptor to enhance the function of the cell-specific element by forming a more "active" transcription complex which stimulates the level of gene expression.

Glucocorticoid control of rat growth hormone gene expression: effect on cytoplasmic messenger ribonucleic acid production and degradation.

The effect of the glucocorticoid dexamethasone on the production and degradation of rat GH (rGH) cytoplasmic mRNA was studied in cultured rat pituitary tumor (GC) cells. The incorporation of [3H]uridine into both rGH cytoplasmic mRNA and the pyrimidine nucleotide precursor pool was determined in hormone-treated and control cells. From these measurements glucocorticoid effects on absolute production rates of rGH cytoplasmic mRNA were determined and compared to effects on rGH mRNA accumulation. Rat GH mRNA half-life was then calculated based on a first-order decay model. Rat GH mRNA half-life was also directly assayed by: 1) pulse-chase studies and 2) measuring the kinetics of decay of rGH mRNA in cells after transfer from serum-containing to hormone-deficient media. From these independent analyses rGH mRNA half-life estimates ranged from 28-55 h in different experiments. Within individual experiments there was little variability of rGH mRNA decay rates; glucocorticoids were found not to alter the stability of rGH cytoplasmic mRNA. Glucocorticoid induction of rGH cytoplasmic mRNA accumulation was accounted for solely on the basis of increased mRNA production.

Glucocorticoid regulation of rat growth hormone gene expression in GH3 cells

Glucocorticoid regulation of rat growth hormone gene expression in GH3 cells

Rat growth hormone gene expression is correlated with an unmethylated CGCG sequence near the transcription initiation site.

The methylation status of the rat growth hormone (GH) gene was compared in DNA obtained from GH-producing and GH-nonproducing sources by digestion with three methylation-sensitive restriction enzymes. GH gene expression was correlated with an unmethylated ThaI site (CGCG) 144-bp upstream of the GH RNA transcription initiation site. This ThaI site was unmethylated in nine GH-producing subclones of the rat pituitary tissue culture cell line GH3 and in greater than 50% of the DNA isolated from rat anterior pituitary, a gland containing GH-producing somatotroph cells as well as GH-nonproducing cells. In DNA prepared from GH-nonproducing tissues, e.g., rat spleen, kidney, liver, and brain, this ThaI site was entirely methylated. Furthermore, this site was entirely methylated in hybrid cells formed by the fusion of GH3 cells with mouse fibroblasts in which GH production has been extinguished. DNA methylation at 10 other restriction sites located throughout the rat GH gene region failed to correlate with GH expression in GH-producing subclones of GH3 cells as well as in GH-nonproducing GH3 X LB82 hybrid cells. We suggest that the conserved absence of methylation 144 bp 5' of the RNA transcription initiation site of transcribed GH genes identifies a potential GH gene control region.