Rat Growth Hormone Gene Research Articles

Cloning, sequencing, and polymorphisms of the Wistar-Imamichi rat growth hormone gene using PCR-SSCP

We successfully cloned the Wistar-Imamichi (WI) rat growth hormone gene (GenBank accession: GQ890681), which contained 5 exons and 4 introns. Using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique, a novel missense substitution single nucleotide polymorphism was identified and tested for Hardy-Weinberg equilibrium with the χ(2) test. This gene fragment was investigated in 50 adult rats and 50 neonatal rats by PCR-SSCP. Results showed that only intron 4 had a polymorphic locus at the 97th nucleotide position from A (in the AA genotype) to C (in the BB genotype). Further statistical analysis showed that this locus was in Hardy-Weinberg equilibrium. These results suggested that this specific pathogen-free WI rat population, which was bred in the barrier system of the Research Center for Laboratory Animal Science of Inner Mongolia University, has high hereditary stability.

Hair cycle induction by hair plucking in Mini rats

Jcl:WistarTGN(ARGHGEN)1Nts rat (Mini rat) is a transgenic rat in which the growth hormone (GH) expression is suppressed by the presence of an antisense transgene for the rat GH gene, and the hair cycle of male Mini rats enters a long-lasting telogen phase after 8 weeks old. In the present study, effects of hair plucking on the hair cycle in the dorsal skin of 11-week-old male Mini rats were examined up to 54 days after the hair plucking. As a result, hair plucking brought about an anagen phase, and hair cycle arrest in telogen phase occurred again. Male Mini rats, having follicles in anagen-inducible long-lasting telogen phase, will offer a powerful tool for investigating the molecular nature and organization of the so-called hair cycle clock.

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.

T3-activation of the rat growth hormone gene is inhibited by a zinc finger/homeodomain protein

Since the transcription factor Zfhep is expressed in somatotropes and binds the rat growth hormone (rGH) gene T3-response element (TRE), we investigated whether Zfhep regulates the response of this gene to T3. In cotransfection experiments, Zfhep did not regulate the native rGH promoter in the absence of T3. However, Zfhep repressed T3-mediated activation significantly in either GH 3 or JEG-3 cells. Up to 70% repression was mediated through the rGH TRE in a heterologous promoter (thymidine kinase), but was not observed with the idealized DR4 or chicken lysozyme F2 TREs. Zfhep apparently does not repress T3-mediated activation simply by competition for binding to DNA since the C-terminal DNA-binding domain of Zfhep (which is sufficient for DNA-binding) is not sufficient for repression and since cotransfection of excess thyroid hormone receptor (TR) did not prevent repression by Zfhep. These data indicate that the rGH TRE is a composite element that can integrate Zfhep and T3 regulation.

Gene structure of rat testicular cell adhesion molecule 1 (TCAM-1), and its physical linkage to genes coding for the growth hormone and BAF60b, a component of SWI/SNF complexes.

In the +9.7 to +21.7kb downstream region from the transcriptional start site of the rat growth hormone (GH) gene, a gene specifically expressed in the testis was found to have reverse transcriptional orientation to the GH gene. Its exon comprised 2693 bases encoded a protein having 548 amino acids (60479Da). The amino acid sequence of the testis-specific protein resembled that of the intercellular adhesion molecules (ICAM) 1 and 3. The gene was thus given the name testicular adhesion molecule (TCAM) 1 gene. The TCAM-1 gene was found to be 12041 bases with eight exons. Although exon 1 was noncoding, the remaining seven exons corresponded to the domains coding for the signal sequence, five immunoglobulin (Ig) domains, and the transmembrane plus cytoplasmic domain. The organization of TCAM-1 gene was shown to be the same as that of the ICAM-1 gene. The polyadenylation site of TCAM-1 gene was located 7.6kb downstream of that of the GH gene, whereas the 5' end of TCAM-1 gene was separated 5.9kb from that of the gene coding for BAF60b, a component of SWI/SNF complexes known as the chromatin remodeling factor. Six genes were thus mapped in the following order in the 88kb region of the rat GH locus: Na-channel (5' to 3')-B29/Ig-beta (5' to 3')-GH (5' to 3')-TCAM-1 (3' to 5')-BAF60b (5' to 3')-SUG/p45 (3' to 5').

Role of GHF-1 in the Regulation of the Rat Growth Hormone Gene Promoter by Thyroid Hormone and Retinoic Acid Receptors

In non-pituitary HeLa cells the unliganded thyroid hormone or retinoic acid receptors cause a strong activation of the rat growth hormone promoter that is repressed by their ligands. In contrast, after expression of the pituitary-specific transcription factor GHF-1, thyroid hormone and retinoic acid produce a stimulation similar to that found in pituitary cells. Therefore, GHF-1 changes a ligand-dependent inhibition into a ligand-dependent activation. The essential role of GHF-1 on the rat growth hormone promoter was also demonstrated with AF-2-defective T3 receptor mutants that show a normal activation of this promoter in the presence of GHF-1. Furthermore, a truncated T3 receptor, which lacks the N-terminus and the DNA binding domain, was able to stimulate this promoter in the presence of GHF-1 and exogenous RXR receptors, suggesting the importance of protein to protein interactions in this regulation. This study shows that the final transcriptional effect depends not only on the type of regulatory promoter response elements but also on the presence of other transcriptional activators, in the case of the growth hormone promoter, the tissue-specific transcription factor GHF-1, which plays a coactivator-like role in this promoter.

A direct protein-protein interaction is involved in the cooperation between thyroid hormone and retinoic acid receptors and the transcription factor GHF-1.

The nuclear receptors for thyroid hormone (TRs) and retinoic acid (RARs and RXRs) cooperate with the pituitary-specific transcription factor GHF-1 to activate the rat growth hormone (GH) gene. The GH promoter contains a hormone response element (HRE), which binds TR/RXR and RAR/RXR heterodimers, located close to two binding sites for GHF-1. GHF-1 inhibits binding of TR/RXR and RAR/RXR heterodimers to an isolated HRE. Similarly, the receptors inhibit binding of GHF-1 to its cognate site. These results suggest the existence of direct protein to protein interactions between the receptors and the pituitary transcription factor. This was confirmed by in vitro binding studies with GST fusion proteins, which demonstrated a strong association of GHF-1 with RXR and a weaker interaction with RAR and TR. GHF-1 and the receptor heterodimers form a ternary complex with a fragment of the rat GH promoter, which contains binding sites for both, and GHF-1 increases receptor binding to the promoter when present in limiting conditions. These results suggest that the synergistic activation of the rat GH gene involves protein-DNA interactions as well as a physical association between the nuclear receptors and the pituitary-specific transcription factor GHF-1.

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.

Comparison of Age-Related Bone Changes between Mini Rats and Wistar Rats.

It has been reported that in Mini rats, rats of transgenic Jcl: Wistar-TgN (ARGHGEN) 1Nts strain, the expression of rat growth hormone (GH) gene was suppressed by the presence of an antisense transgene, resulting in lowered plasma GH levels compared with nontransgenic Wistar rats. GH is thought to have an important role in bone growth, but bone changes of Mini rats have not been reported. In the present study, we examined the femurs of both male Mini rats and male Wistar rats at different ages, i.e. 8 and 22 weeks of age, and compared them between both rat strains. As a result, Mini rats showed smaller sizes of the femur with low mineral densities histologically characterized by the metaphyseal and diaphyseal bone be a possible animal model for osteopenia.

Gene structure of rat BAF60b, a component of mammalian SWI/SNF complexes, and its physical linkage to the growth hormone gene and transcription factor SUG/proteasome p45 gene

In the +27.6 to +36.7 kb downstream region from the transcriptional start site of the rat growth hormone (GH) gene, a gene encoding BAF60b, a component of mammalian SWI/SNF complexes, was found to have the same transcriptional orientation as the GH gene. The 5′ end of the BAF60b gene was heterogeneous and the longest gene was 9060 bp long with 13 exons. The largest of all exons was estimated to be 2774 bases. Deduced rat BAF60b protein was made of 531 amino acids and its amino acid sequence was 97% identical with the human counterpart. No TATA box was found up to the −100 bp region but five GC boxes corresponding to the Sp1 binding site were observed up to 640 bp upstream from the transcriptional start site. Sixty-three bases downstream from the BAF60b gene, the polyadenylation site of the gene encoding transcription factor SUG/proteasome p45, whose expression is constant in many tissues, was identified. The BAF60b gene was expressed as 3.0 kb poly(A)-rich RNA in seven tissues and one cell line from rat but its expression varied considerably according to the tissue.

Quantitative Analysis of Growth Hormone (GH) Pre-mRNA Expression in Cultured Rat Anterior Pituitary Cells by an Intron-Specific and Competitive PCR Method

We have developed a novel method of quantifying growth hormone(GH) pre-mRNA expression in anterior pituitary cells. DNA-free total RNA extracted from cultured rat anterior pituitary cells was reverse transcribed(RT) to cDNA, and RT products were subsequently quantitated by competitive PCR using intron-specific primers of rat GH gene. After 6-h of incubation in treated cells, dexamethasone(Dex) and triiodo-L-thyronine(T3) significantly increased GH pre-mRNA levels(3.2- and 2.2-fold compared to non-treated cells, respectively). However, Northern blot analysis did not detect significant changes in GH mRNA levels. After 24-h incubation with Dex and T3, significant increases in GH mRNA levels were detected on Northern blots, but GH pre-mRNA levels did not differ between treated and non-treated cells. These findings suggest that both Dex and T3 treatments rapidly increase GH pre-mRNA levels in normal somatotropes. This method has high sensitivity and widespread application to the analysis of pre-mRNAs of target genes.

Gene coding for the transcription factor, SUG/proteasome, p45 is located nearly 40 kb downstream from the rat growth hormone gene

About 40 kilobases (kb) downstream of the rat growth hormone gene, a gene was found to be expressed in the liver and placenta as 1.5 kb poly(A)-rich RNA. Using the genomic DNA fragment as a probe, the corresponding cDNA clone containing a 1.3 kb insert was isolated from the rat liver cDNA library. The deduced amino acid sequence having 406 residues was identical with that of the mouse transcription factor, SUG and human proteasome subunit, p45. The gene was thus identified as the rat SUG/p45 ( rSUG/p45) gene. The 5′ end of the gene was determined by the primer-extension analysis and the exon was noted to comprise 1409 bases. The rSUG/p45 gene, 6.0 kb in length and possessing 12 exons, started at 42.8 and ended at 36.8 kb downstream from the transcription start site of the GH gene. From exon 2 to 11, the size of each rSUG/p45 exon was identical with the corresponding exon of the 4.4 kb pig gene. Rat SUG/p45 mRNA was similarly expressed in seven different tissues and one cell line.

DNA sequence variation in the porcine growth hormone promoter region from Danish and exotic pigs

An extension of the porcine growth hormone (GH) gene sequence from ‐178 to ‐331 was performed. Comparison of the porcine GH promoter between ‐331 and +169 with the published sequences from rat, human and bovine GH genes revealed conservation of several transcription factor binding sites. The sequence of the GH promoter between position ‐294 and +145 was determined in a total of ten animals from four Danish breeds (Duroc, Hampshire, Landrace and Yorkshire). The animals were homozygous for five different haplotypes defined by five polymorphic restriction sites. The sequences revealed nine additional polymorphic base pairs of which the most notable are located within the TATA‐box, within the recognition sequence for transcription factor Spl, and within a recognition sequence for the thyroid hormone receptor. Sequence was determined in the same region of the porcine GH promoter from one wild boar and three Meishan animals. At any single nucleotide position, the sequence was shared with at least one Danish animal. However, including data from restriction enzyme digests, the haplotypes of the Meishan animals were different from all haplotypes detected in Danish pigs.

Behavioural rhythmicity in transgenic growth hormone mice: trade-offs, energetics, and sleep–wake cycles

Transgenic mice with extra rat growth hormone (GH) genes (TRrGH mice) are behaviourally lethargic and sleep 3.4 h/d longer than normal on standard diets. We tested the hypothesis that the doubling of the growth rate of TRrGH mice reduced the energy available for behaviour. Provision of sucrose supplements ad libitum normalized the durations of activity and sleep. Our results support a new allocative theory suggesting that sleep serves as an umbrella function for a suite of synergistic anabolic functions (e.g., growth, immunity, repair). Relegating these to the period of sleep in a secure nest allows full dedication of waking resources to niche interfacing (resource acquisition, risk avoidance and environmental stress resistance). Energy stress in TRrGH mice may arise via specific diversion of energy from waking functions via GH-induced insulin resistance. GH is normally secreted during sleep, but any causal relationship remains unresolved. We examined the circadian and ultradian behaviour of TRrGH mice to determine how a chronically elevated GH level impacts sleep. Remarkably, even the major hormonal distortion in TRrGH mice had little impact on the timing of ultradian or circadian rhythms. Increased sleeping of TRrGH mice on normal diets was due to an increased likelihood and duration of sleep at permitted times. GH did, however, appear to increase the depth of sleep.

Histone acetylation influences thyroid hormone and retinoic acid-mediated gene expression.

Thyroid hormone (T3) and retinoic acid (RA) receptors regulate transcription of the rat growth hormone (GH) gene through binding to a common hormone response element (HRE) in the promoter. We have investigated the effect of histone acetylation on hormone-dependent expression of the rat GH gene. We examined the effect of butyrate, which induces histone hyperacetylation, and trichostatin A (TSA), a highly specific inhibitor of histone deacetylases. GH-mRNA levels were significantly increased in pituitary GH4C1 cells incubated with T3 and RA, and this response was further stimulated in the presence of 1 mM butyrate. The effect of butyrate was mimicked by TSA. Butyrate and TSA also enhanced the activity of recombinant constructs containing the GH promoter directing chloramphenicol acetyl transferase (CAT) reporter gene expression. CAT activity increased by 4- to 8-fold after incubation with 1 nM T3 and 1 microM RA, and this response was stimulated 2- to 4-fold further in the presence of 0.25 mM butyrate. This concentration of butyrate did not influence basal expression of CAT. TSA produced a dose-dependent increase of CAT activity in the absence of ligands, and between 5 and 200 nM potentiated the effect of T3 and RA. These compounds also increased the hormonal response of constructs in which the HRE was linked to heterologous [mouse mammary tumor virus (MMTV) and thymidine kinase (TK)] promoters. With butyrate >1 mM, basal activity of the GH promoter increased by more than 10-fold and the effect of T3 and RA was no longer observed. Overexpression of T3 receptors was able to counteract the stimulation of basal CAT levels caused by butyrate. Thus, in the absence of ligand, the T3 receptor acts as a constitutive repressor of gene expression. Upon binding of the hormone, the T3 receptor is converted into an activator. Our findings suggest that histone acetylation, which alters chromatin structure, may play an important role in hormone-mediated transcriptional regulation.

Selective constraints on the activation domain of transcription factor Pit-1.

The POU transcription factor Pit-1 activates members of the prolactin/growth hormone gene family in specific endocrine cell types of the pituitary gland. Although Pit-1 is structurally conserved among vertebrate species, evolutionary changes in the pattern of Pit-1 RNA splicing have led to a notable "contraction" of the transactivation domain in the mammalian lineage, relative to Pit-1 in salmonid fish. By site-directed mutagenesis we demonstrate that two splice insertions in salmon Pit-1, called beta (29 aa) and gamma (33 aa), are critical for cooperative activation of the salmon prolactin gene. Paradoxically, Pit-1-dependent activation of the prolactin gene in rat is enhanced in the absence of the homologous beta-insert sequence. This apparent divergence in the mechanism of activation of prolactin genes by Pit-1 is target gene specific, as activation of rat and salmon growth hormone genes by Pit-1 splice variants is entirely conserved. Our data suggest that efficient activation of the prolactin gene in the vertebrate pituitary has significantly constrained the pattern of splicing within the Pit-1 transactivation domain. Rapid evolutionary divergence of prolactin gene function may have demanded changes in Pit-1/protein interactions to accommodate new patterns of transcriptional control by developmental or physiological factors.

Identification of a Negative Regulatory Site in the Upstream Region of Bovine Growth Hormone Gene

The 5′ upstream region of bovine growth hormone (bGH) gene was analyzed. When the region between nucleotides −336 and −240 was deleted, the expression of the reporter chloramphenicol acetyltransferase (CAT) gene increased about 5 fold in HeLa cells and 2.2 fold in rat GH3cells which produce growth hormone. This region was named negative regulatory site (NRS). When NRS was inserted in front of promoter for rat growth hormone (rGH) gene or human thymidine kinase (TK) gene, the CAT activity decreased by 75–80% in HeLa cells and 5–30% in GH3cells. It also repressed the expression of CAT gene from several promoter–enhancer combinations tested. By fine deletion analysis negative elements in the NRS were mapped and found to contain sequences similar to the binding elements of YY1.

DNase I-hypersensitive sites in the chromatin of rat growth hormone gene locus and enhancer activity of regions with these sites.

In this study, a determination was made of the chromatin structure of the rat growth hormone (GH) gene locus by DNase I sensitivity analysis using GC [GH+, prolactin (PRL)-], 235 (GH-, PRL+), GH3 (GH+, PRL+) and liver (GH-, PRL-) cells. From 7 kb upstream from the transcription start site to 19 kb downstream from the polyadenylation site, two major DNase I-hypersensitive sites (M-DHS; UIA, UIIA) and three M-DHS (DIA, DII, DIII) were found within 2 kb upstream and 7 kb downstream regions, respectively. Two minor DHS (m-DHS; UIB, UIIB) in the upstream region and one m-DHS (DIB) downstream were shown to be associated with M-DHS. Thus, a total of five M-DHS and three m-DHS were mapped on the rat GH gene locus. Among these, five (UIIB, UIA, UIB, DIB, DIA) including two (UIA, DIA) M-DHS were specific for GH-producing cells. UIIA and DIII were M-DHS only in PRL-producing 235 cells while the major hypersensitivity of DII was detected in GH-producing cells and liver cells. Assessment of the enhancing activity of the DHS regions indicated novel enhancers in one upstream and two downstream regions that function well with the GH promoter in GC cells. These enhancers, each appearing different, coincided with m-DHS but not M-DHS in GC cells, and were not activated by Pit-1. Based on these observations, the following functions of five M-DHS and three m-DHS regions were defined: enhancer; locus control region (LCR); switch region serving for conversion from GH/PRL-producing cells to PRL-producing cells; and a region having a structural function in chromatin.

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T3 injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about approximately 35-40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly approximately 25-32 and approximately 29-41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by approximately 72-81 and approximately 51-70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels.

Evidence for a trade-off between growth and behavioural activity in giant "Supermice" genetically engineered with extra growth hormone genes

The "Supermouse," transgenic strain (Tg(MT-1,rGH),Bri2), contains multiple copies of rat growth hormone genes. Supermice exhibit doubled growth rates and body sizes but no increase in mass-specific feeding rates or assimilation efficiencies. Extra growth is achieved entirely by means of increased production efficiency by diverting energy from other processes. Reduction of behavioural activity is one potential mechanism for diverting energy to growth. To test this hypothesis we compared the behavioural time budgets (resting, sleeping, locomotion, wheel running, feeding, drinking, and grooming) of transgenic and normal male mice. The activities of individual Supermice and normal mice in artificial enclosures were videotaped and compared over 24-h periods. The duration of each activity was recorded and compared. Transgenic mice slept 126% as much as their normal counterparts (an increase of 3.4 h/d), and locomotion and wheel running combined were only 53.8% that of normal mice (a decrease of 2.95 h/d). Supermice spent only 77% as much time drinking and 69% as much time grooming as normal mice. No differences in the duration of feeding were found. The evidence suggests a direct trade-off between growth rate and behavioural activity consistent with the "principle of allocation" paradigm for life-history evolution.