Growth Hormone Gene Transcription Research Articles

Estradiol and corticosterone stimulate the proliferation of a GH cell line, MtT/S: Proliferation of growth hormone cells

ObjectivesEstrogens are known as a potent growth-stimulator of the anterior pituitary cells such as prolactin cells and somatomammotroph cell lines, while glucocorticoids often inhibit cellular proliferation in the pituitary gland as well as in the extra-pituitary tissues. In this study, the involvement of these steroid hormones in the regulation of proliferation was examined in the MtT/S cells, secreting growth hormone (GH). DesignEffects of estrogens and glucocorticoids were examined in MtT/S cells grown in the medium containing dextran-coated charcoal treated serum. The relative cell density after culture was estimated by the Cell Titer-Glo Luminescent Cell Viability Assay System, and the proliferation rate was determined by the BrdU incorporation method. The mRNA levels were determined by real-time PCR. ResultsEstradiol and the specific agonist for both estrogen receptor (ER) α and ERβ stimulated MtT/S growth at a dose dependent manner. The membrane impermeable estrogen, 17β-estradiol-bovine serum albumin conjugate also stimulated the MtT/S proliferation. The effects of all estrogens were inhibited by an estrogen receptor antagonist, ICI182780. Corticosterone stimulated the proliferation of MtT/S cells at doses lower than 10nM without stimulating GH gene transcription, whereas it did not change the proliferation rate at 1μM. The effects of corticosterone were inhibited by glucocorticoid receptor inhibitor, RU486, but not by the mineralocorticoid receptor antagonist, spironolactone. Both estrogens and glucocorticoids were found to stimulate the proliferation of MtT/S, increasing the mRNA expression of cyclins D1, D3, and E. ConclusionsThe results suggest that estrogens and glucocorticoids may be involved in the mechanisms responsible for the proliferation of GH cells in the course of pituitary development, to maintain the population of GH cells in the adult pituitary gland, and also in the promotion of GH cell tumors.

Leptin activates chicken growth hormone promoter without chicken STAT3 in vitro.

Leptin is an adipocyte-derived hormone that not only regulates food intake and energy homeostasis but also induces growth hormone (GH) mRNA expression and release, thereby controlling growth and metabolism in mammals. The molecular mechanism of leptin-induced regulation of GH gene transcription is unclear. The current study investigated the effects of leptin on the chicken GH (cGH) promoter and the molecular mechanism underlying leptin-induced cGH gene expression in vitro. Leptin activated the cGH promoter in the presence of chPit-1α in CHO cells stably expressing the chicken leptin receptor. Promoter activation did not require STAT-binding elements in the cGH promoter or STAT3 activity. However, JAK2 activation was required for leptin-dependent activity. JAK2-dependent pathways include p42/44 MAPK and PI3K, and inhibition of these pathways partially blocked leptin-induced cGH gene transcription. Although CK2 directly activates JAK2, a CK2 inhibitor blocked leptin-dependent activation of the cGH gene without affecting JAK2 phosphorylation. The CK2 inhibitor suppressed Erk1/2 and Akt phosphorylation. Additional data implicate Src family kinases in leptin-dependent cGH gene activation. These results suggest that leptin activates the cGH gene in the presence of chPit-1α via several leptin-activated kinases. Although further study is required, we suggest that the leptin-induced JAK2/p42/44 MAPK and JAK2/PI3K cascades are activated by Src-meditated CK2, leading to CBP phosphorylation and interaction with chPit-1α, resulting in transactivation of the cGH promoter.

β-Hydroxybutyric Sodium Salt Inhibition of Growth Hormone and Prolactin Secretion via the cAMP/PKA/CREB and AMPK Signaling Pathways in Dairy Cow Anterior Pituitary Cells

β-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs). The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA) activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB) phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX). In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1) could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows.

Rapid induction of the Growth Hormone Gene Transcription by Glucocorticoids In Vitro: Possible Involvement of Membrane Glucocorticoid Receptors and Phosphatidylinositol 3‐Kinase Activation

The regulation of transcription of the growth hormone (GH) gene by glucocorticoids was studied in MtT/S cells, a cell line derived from an oestrogen-induced mammotrophic tumour in the rat, and in the primary culture of the anterior pituitary gland of adult mice. The levels of the GH heteronuclear RNA (GH hnRNA), which are mainly determined by the transcription rate, increased by 25-fold during 24 h in response to dexamethasone (DEX; 1 μM) in MtT/S cells that were cultured in the medium containing charcoal-stripped serum for 7 days. The stimulatory effect of DEX on the GH hnRNA levels was detectable as early as 30 min. This rapid effect of DEX did not require on-going protein synthesis, whereas it was considered that DEX requires the presence of unknown cellular proteins produced independently of DEX stimulation. By contrast, on-going protein synthesis was required for DEX action when incubated for 6 h, as has been observed in the previous studies. The specific inhibitor of glucocorticoid receptor, RU486, inhibited both rapid (30 min) and delayed (6 h) the effects of glucocorticoids on GH hnRNA levels. Membrane impermeable glucocorticoid, corticosterone-bovine serum albumin conjugate (CSBSA), was found to have effects similar to those of DEX and free corticosterone (CS), suggesting that glucocorticoids regulate GH gene transcription at least in part through the membrane bound receptors. From pharmacological studies, it was suggested that phosphatidylinositol 3-kinase (PI3K) activation is involved in the rapid effects but not in the delayed effects of glucocorticoids. This also suggests that the delayed effects of glucocorticoids depend on mechanisms other than the activation of PI3-kinase. Finally, both rapid and delayed effects of CS and CSBSA were observed not only in MtT/S cells, but also in the mouse pituitary cells in primary culture. Therefore, it is possible that the membrane initiated action of glucocorticoids is involved in the regulation of GH transcription in normal pituitary cells, as well as in pituitary tumour cells.

Short-Chain Fatty Acids Inhibit Growth Hormone and Prolactin Gene Transcription via cAMP/PKA/CREB Signaling Pathway in Dairy Cow Anterior Pituitary Cells

Short-chain fatty acids (SCFAs) play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH) and prolactin (PRL) gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate) on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs). The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake.

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.

Black carp growth hormone gene transgenic allotetraploid hybrids of Carassius auratus red var. (♀)×Cyprinus carpio (♂)

Ecological safety is a major consideration in the commercialization of transgenic fish. Development of sterile transgenic triploid fish through hybridization of transgenic tetraploid fish and transgenic diploid fish is a feasible way to solve this problem. The "all-fish" transgene, pbcAbcGHc, containing the black carp β-actin gene promoter and the open reading frame (ORF) of the black carp growth hormone (GH) gene was constructed and introduced into fertilized eggs of allotetraploid fish through microinjection. Contrast cultivation results showed that the growth rate of 150 day-old P(0) black carp GH gene transgenic allotetraploid fish was much higher than that of controls. Sixty 150 day-old transgenic allotetraploid fish were assayed by PCR for transgene integration and 90% of fish were positive for the transgene. The transgene was detected in 13 of 20 sperm samples from male transgenic allotetraploid fish. RT-PCR detected transcription of the exogenous black carp GH gene in the muscle, liver, kidney and ovaries of the largest transgenic allotetraploid fish. This study has developed P(0) black carp GH gene transgenic allotetraploid fish with a highly increased growth rate, which provides a solid foundation for the establishment of a pure line of transgenic allotetraploid fish and for the large scale production of sterile transgenic triploid fish.

Role of chicken Pit-1 isoforms in activating growth hormone gene

In the present study, we expressed chicken (ch) Pit-1α (chPit-1α) and chPit-1γ in vitro to compare the roles of chPit-1s in the transcription of the chicken growth hormone (chGH) gene. Both green fluorescence protein (GFP)-fused chPit-1γ and GFP-fused chPit-1α were localized in the nuclei of COS-7 cells. In a luciferase reporter gene assay, both chPit-1α and chPit-1γ transactivated the chGH promoter, and chPit-1α showed a more potent effect than chPit-1γ. On the other hand, an increase of cellular cAMP induced by forskolin promoted transactivation of the chGH gene with chPit-1α and chPit-1γ to similar extents. These results suggest that chPit-1γ may modulate the basal promoter activity of the chGH gene to the same degree as chPit-1α; however, a structural difference observed at the N-terminus transactivation domains in chPit-1α and chPit-1γ could be associated with the efficiency of basal activation of the chGH promoter.

The Insulin-Like Growth Factor I (IGF-I) Within the Bony Fish Pituitary: New Morphofunctional and Phylogenetic Aspects

IGF-I is a major hormonal regulator of differentiation, growth, proliferation, and development. It is mainly produced in the liver, the principal source of endocrine IGF-I. The main stimulus for synthesis and release of liver IGF-I is growth hormone (GH) from the anterior pituitary, and IGF-I specifically inhibits GH gene transcription and secretion via a negative feedback mechanism. As shown for species throughout phylogeny, IGF-I is also produced in extrahepatic sites, including the pituitary, and very recently, new insights have been achieved into the distinct localization of IGF-I. Bony fish pituitary preserves the embryonic compartmentalization throughout life, thus, each endocrine cell type is situated in a distinct region. This makes fish pituitary an excellent tool for morphologic investigations. IGF-I mRNA and/or peptide has been located to subtypes of endocrine cells with similar distribution patterns in lower and higher vertebrates suggesting highly conserved and, thus, important physiological roles of intrapituitary IGF-I. Since a major task of IGF-I is to prevent apoptosis and promote cell proliferation, IGF-I released from the endocrine adenohypophyseal cells may have protective and proliferative autocrine and/or paracrine effects. This is supported by the presence of the type 1 IGF receptor (IGF-1R) at all endocrine subpopulations as has been shown in rat and by the constitutive presence of IGF-I in ACTH cells in bony fish and mammals. ACTH cells probably are challenged in stressful situations by pro-apoptotic cytokines and hormones, and might, thus, have a special demand for IGF-I. The increased transient expression of IGF-I in gonadotrophs during puberty, and in subordinate males of tilapia suggests an impact in sexual differentiation and maturation, a question which has been recently underlined to be of major importance. The pronounced inter-individual differences in the IGF-I content of the GH cells may indicate that synthesis and release of IGF-I from GH cells depend on the physiological status, most likely the serum IGF-I level. Further studies should be performed to elucidate these morphofunctional observations.

Atrazine Binds to the Growth Hormone–Releasing Hormone Receptor and Affects Growth Hormone Gene Expression

BackgroundAtrazine (ATR), a commonly used herbicide in the United States, is widely distributed in water and soil because of its mobility through ecosystems and its persistence in the environment. ATR has been associated with defects in sexual development in animals, but studies on mammalian systems have failed to clearly identify a cellular target.ObjectivesOur goal in this study was to identify a ligand-binding receptor for ATR in pituitary cells that may explain the mechanism of action at the gene expression level.MethodsWe used pituitary cells from postnatal day 7 male rats and pituitary cell lines to study the effect of ATR on gene expression of growth hormone (GH), luteinizing hormone (LH), and prolactin (PRL) at RNA and protein levels. 14C-ATR was used to determine its specific binding to the growth hormone–releasing hormone receptor (GHRHR). The effect of ATR on structural proteins was visualized using immunofluorescent in situ staining.ResultsThe treatment of rat pituitary cells with ATR, at environmentally relevant concentrations (1 ppb and 1 ppm), resulted in a reduction of GH expression. This effect appeared to result from the inhibition of GH gene transcription due to ATR binding to the GHRHR of the pituitary cells.ConclusionsIdentification of GHRHR as the target of ATR is consistent with the myriad effects previously reported for ATR in mammalian systems. These findings may lead to a better understanding of the hazards of environmental ATR contamination and inform efforts to develop guidelines for establishing safe levels in water systems.

Association between pituitary adenylate cyclase-activating polypeptide and reproduction in the blue gourami

In order to gain a better understanding of the roles of pituitary adenylate cyclase-activating polypeptide (PACAP) in reproduction and growth, the expression of the PACAP gene during the reproduction cycle and its potential role in regulating gonadotropin and growth hormone (GH) gene transcription in blue gourami were investigated. The cDNA sequences of the full-length blue gourami brain PACAP and that of its related peptide ( PRP) were acquired. PACAP cDNA had two variants, obtainable by alternative splicing: a long form encoding for both PRP and PACAP and a short form encoding only for PACAP. In females, mRNA levels of PACAP were very high only in individuals with oocytes in the maturation stage, as compared to levels in unpaired vitellogenic and non-vitellogenic fish. The PACAP mRNA levels in males were high only in nest builders, as opposed to in non-nest building males and juveniles. In pituitary culture cells from high vitellogenic females, PACAP38 (the 38 amino acid form) only brought about an increase in βFSH levels, without altering GH and βLH mRNA levels. On the other hand, in adult non-reproductive male pituitary cells, PACAP38 decreased the GH mRNA level. Based on these results, we propose that in the blue gourami, PACAP is involved in the final oocyte maturation stage in females, whereas in males, it is associated with sexual behavior. In addition, the effect of PACAP38 on pituitary hormone gene expression is different in females and males, indicating that PACAP38 is potentially a hypophysiotropic regulator of reproduction, which mediates pituitary hormone expression.

In vitro exposure to xenoestrogens induces growth hormone transcription and release via estrogen receptor-dependent pathways in rat pituitary GH3 cells

In this study, we employed an in vitro model to examine the effects of endocrine disruptors (EDs) in the regulation of growth hormone (GH) gene, an important hormone in growth, development and body composition. The rat pituitary cells, GH3, were treated with alkyl-phenols (APs), i.e., 4- tert-octyl-phenol (OP), p-nonyl-phenol (NP) or bisphenol A (BPA) for 24 h in a dose-dependent manner (10 −5, 10 −6 and 10 −7 M) and in a time-dependent fashion (1, 3, 6, 12 and 24 h) at a high concentration (10 −5 M). An anti-estrogen, ICI 182,780, was used to examine the potential involvement of estrogen receptor (ER) in the induction of GH by EDs through an ER-mediated pathway. Treatment with OP, NP and BPA induced a significant increase in GH gene expression at high and medium doses at 24 h. ED-exposure induced a marked increase in GH gene transcription as early as 6 h and peaked at 12 h. Co-treatment with ICI 182,780 significantly attenuated ED-induced GH expression in GH3 cells. Interestingly, the level of in vitro GH release was significantly increased at 24 h in response to OP, NP or BPA, whereas co-treatment with ICI 182,780 significantly reversed ED-induced GH secretion, indicating that ER may take part in both GH gene transcription and its release in these cells. In addition, the activation of extracellular signal-regulated kinases (ERKs), protein kinases B (Akt) or G protein in response to OP, NP or BPA at 24 h was observed in this study. Exposure to these APs resulted in a rapid and significant activation of ERK phosphorylation, reflecting that EDs-induced response may involve both genomic and non-genomic pathways in these cells. Taken together, these results may provide new insight into the mode of ED-induced action in GH gene regulation as well as the biological pathway underlying these molecular events.

Growth hormone and Prolactin-1 gene transcription in natural populations of the black-chinned tilapia Sarotherodon melanotheron acclimatised to different salinities

The effects of salinity on the expression of genes coding for growth hormone (GH) and prolactin-1 (PRL1) were studied in various natural populations of the black-chinned tilapia Sarotherodon melanotheron from West Africa. Individuals were sampled in June 2005 in six locations in Senegal and the Gambia, at various salinities between 0 and 101. The poorest condition factors were recorded in the most saline sampling site and the best growth in the fish from a marine environment. The pituitary GH mRNA levels were significantly higher in fish adapted to seawater, whereas the PRL1 mRNA levels were highest in fish adapted to fresh- and brackish water. These results show that the PRL1 mRNA levels seem to reflect relatively well the differences in environmental salinity, in contrast to those of GH, which would tend instead to reflect the individual growth in each environment. However, no relation could be found between growth in the hypersaline areas and the expression profile of GH. Although the fish analysed were morphologically identical, the expression of genes coding for GH and PRL1 showed large differences between individuals. This inter-individual variation in gene expression remains poorly understood.

Growth hormone and Prolactin-1 gene transcription in natural populations of the black-chinned tilapia Sarotherodon melanotheron acclimatised to different salinities

The effects of salinity on the expression of genes coding for growth hormone (GH) and prolactin-1 (PRL1) were studied in various natural populations of the black-chinned tilapia Sarotherodon melanotheron from West Africa. Individuals were sampled in June 2005 in six locations in Senegal and the Gambia, at various salinities between 0 and 101. The poorest condition factors were recorded in the most saline sampling site and the best growth in the fish from a marine environment. The pituitary GH mRNA levels were significantly higher in fish adapted to seawater, whereas the PRL1 mRNA levels were highest in fish adapted to fresh- and brackish water. These results show that the PRL1 mRNA levels seem to reflect relatively well the differences in environmental salinity, in contrast to those of GH, which would tend instead to reflect the individual growth in each environment. However, no relation could be found between growth in the hypersaline areas and the expression profile of GH. Although the fish analysed were morphologically identical, the expression of genes coding for GH and PRL1 showed large differences between individuals. This inter-individual variation in gene expression remains poorly understood.

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.

172 IN VITRO EXPOSURE TO XENOESTROGENS INDUCES GROWTH HORMONE TRANSCRIPTION AND RELEASE VIA AN ESTROGEN RECEPTOR-DEPENDENT PATHWAY IN RAT PITUITARY GH3 CELLS

The term endocrine disruptor (ED) has been used widely to characterize natural and synthetic environmental compounds that may interfere with the endocrine system(s) of humans and wildlife. In previous studies, we demonstrated that in vitro single exposure to EDs induces CaBP-9k expression, a useful biomarker for detecting the estrogenic activities of EDs in rat pituitary GH3 cells. Here we employ the identical model to examine the effects of EDs in the regulation of growth hormone (GH) gene expression, an important hormone in growth, development, and body composition. We measured levels of GH mRNA transcription and GH release using semi-quantitative RT-PCR and EIA kit, respectively. GH3 cells were treated with alkyphenols (APs), i.e., octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA), in a dose-dependent manner (10–5, 10–6, and 10–7 M) and harvested following 24 h of treatment. Cells were also exposed to a high concentration (10–5 M) of OP, NP, or BPA and harvested at various time points (1, 3, 6, 12, and 24 h). An anti-estrogen, ICI 182780 (10–7 M) was used to examine the potential involvement of estrogen receptor (ER) in the induction of GH by EDs through an ER-mediated pathway. The data were analyzed by one-way ANOVA, followed by Tukey's multiple comparison. OP, NP, and BPA induced a significant increase in GH gene expression at high (10–5 M) and medium (10–6 M) doses at 24 h. ED-exposure induced a marked increase in GH gene transcription as early as 6 h and peaked at 12 h. Co-treatment with ICI 182780 significantly attenuated ED-induced GH expression in GH3 cells. Interestingly, the level of in vitro GH release was increased significantly at 24 h in response to OP, NP, or BPA, whereas co-treatment with ICI 182780 significantly diminished ED-induced GH secretion in GH3 cells, indicating that ER may play a part in both GH gene transcription and GH release in these cells. Here we demonstrate for the first time that single in vitro exposure to OP, NP, or BPA results in an increase in GH expression at 24 h in GH3 rat pituitary cells. These results may provide new insight into the mode of ED action in GH gene regulation as well as the biological pathway underlying these molecular events. Furthermore, data showing GH responsiveness evoked by EDs supports the aim to develop an assay for use in predicting adverse health effects of EDs in humans and wildlife.

The effects of the members of growth hormone family knockdown in zebrafish development

Growth hormone (GH), prolactin (PRL), and somatolactin (SL) are members of GH/PRL superfamily. These hormones are involved in the regulation of an array of physiological processes, including growth, lactation, and osmoregulation. While recent evidence has shown the GH, PRL, and SL gene transcripts and protein products are expressed during early zebrafish development, their functions at this time of embryogenesis remain unknown. In the current study, antisense morpholino oligonucleotide (MO) inhibition of gh, prl, and sl gene translation was used to examine the effects of gene knockdown on hormone function in zebrafish development. We observed that PRL, SLα and SLβ MO treatment all affected development. PRL MO-treated embryos showed defects in gas bladder inflation, reduced head and eye size, shorter body length and fewer melanophores than untreated controls, whereas SLα and SLβ MO-treated embryos were only defective in gas bladder inflation, GH MO-inhibition of GH specific translation did not lead to any discernable morphological changes within 10 days post fertilization (dpf). The effects of PRL knockdown were further verified using a second PRL morpholino antisense and by a rescue experiment with in vitro transcribed prl mRNA containing 5 nucleotide mismatch within the PRL–MO binding region. These results provide the first evidence that members of the GH/PRL superfamily play a role in proper development of various structures including the head, eyes, melanophores and the gas bladder in zebrafish.

Tumor necrosis factor inhibits growth hormone-mediated gene expression in hepatocytes

Growth hormone (GH) stimulates STAT5 phosphorylation by JAK2, which activates IGF-I and serine protease inhibitor 2.1 (Spi 2.1) transcription, whereas STAT5 dephosphorylation by protein tyrosine phosphatases (PTPs) terminates this signal. We hypothesized that the inhibitory effects of TNF on GH signaling and gene transcription were responsible for hepatic GH resistance. CWSV-1 hepatocytes were treated with TNF, pervanadate (a PTP inhibitor), or both, before GH stimulation. Total and tyrosine-phosphorylated JAK2, STAT5, ERK1/2, SHP-1 and SHP-2, IGF-I, and Spi 2.1 mRNA levels were measured. GH stimulated STAT5 and ERK1/2 phosphorylation, IGF-I, and Spi 2.1 mRNA expression. TNF attenuated JAK2/STAT5 and ERK1/2 phosphorylation and IGF-I and Spi 2.1 mRNA expression following GH stimulation. SHP-1 and SHP-2 protein levels were unaltered by TNF or GH, and the GH-induced increase in SHP-1 PTP activity was not further increased by TNF. In TNF-treated cells, pervanadate restored STAT5 and ERK1/2 phosphorylation to control levels following GH stimulation but did not restore IGF-I or Spi 2.1 mRNA induction. Cells transfected with a Spi 2.1 promoter-luciferase vector demonstrate a 50-fold induction in luciferase activity following GH stimulation or cotransfection with a constitutively active STAT5 vector. TNF prevented the induction of Spi 2.1 promoter activity by GH and the STAT5 construct. We conclude that TNF does not inhibit GH activity by inducing SHP-1 or -2 expression and that correction of GH signaling defects in TNF-treated cells by pervanadate does not restore GH-induced gene expression. The inhibitory effects of TNF on GH-mediated gene transcription appear independent of STAT5 activity and previously identified abnormalities in JAK2/STAT5 signaling.

Expression of insulin-like growth factor I gene is involved in enhanced growth of juvenile four-spine sculpin Cottus kazika in seawater

To study the mechanism of faster growth of the sculpin Cottus kazika in seawater (SW) than in fresh water (FW), we transferred 120-day-old juveniles from FW to SW, 1/3 SW or FW, and survival, growth and levels of transcripts of growth hormone (GH) gene in the pituitary and insulin-like growth factor I (IGF-I) gene in the liver were examined 20 and 40 days after transfer. The survival rates of fish reared in SW and 1/3 SW were higher than the FW controls. The growth was fastest in SW and slowest in the controls. IGF-I gene transcripts increased in SW-reared fish 40 days after transfer compared with the controls, while GH gene transcripts did not. Juveniles of 1 year of age were transferred from FW to SW or 1/3 SW, and GH and IGF-I gene transcripts were compared after 48 h. In this shortterm experiment, the level of GH gene transcripts was higher in fish reared in SW and 1/3 SW, while no difference was found in the IGF-I gene transcripts. Thus, the GH gene transcripts increased only transiently after SW transfer but the IGF-I gene transcripts increased gradually for 40 days, suggesting that the long-term increase in IGF-I gene expression is involved in the enhanced growth in SW and GH may play an initiation role for growth enhancement.

Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: functional implications, molecular mechanisms and signal transduction

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased 'steady-state' GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44(MAPK) and P38 (MAPK). These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.