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 P42/44MAPK 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.

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Signaling mechanisms for α2-adrenergic inhibition of PACAP-induced growth hormone secretion and gene expression grass carp pituitary cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00001.2007 ◽

2007 ◽

Vol 292 (6) ◽

pp. E1750-E1762 ◽

Cited By ~ 6

Author(s):

Xinyan Wang ◽

Mable M. S. Chu ◽

Anderson O. L. Wong

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Adenylate Cyclase ◽

Grass Carp ◽

Promoter Activity ◽

Hormone Secretion ◽

Growth Hormone Secretion ◽

Pituitary Cells ◽

Gh Secretion ◽

Gh Gene

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 α2-adrenoreceptors, NE or the α2-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 α2-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.

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PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00188.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. R1625-R1633 ◽

Cited By ~ 18

Author(s):

Christian Klausen ◽

Takeshi Tsuchiya ◽

John P. Chang ◽

Hamid R. Habibi

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Primary Cultures ◽

Gonadotropin Releasing Hormone ◽

Growth Hormone Gene ◽

Pituitary Cells ◽

Mrna Levels ◽

Releasing Hormone ◽

Gh Secretion ◽

Gh Gene

Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and stimulates the synthesis and secretion of gonadotropin hormones. In addition, GnRH also stimulates the production and secretion of growth hormone (GH) in some fish species and in humans with certain clinical disorders. In the goldfish pituitary, GH secretion and gene expression are regulated by two endogenous forms of GnRH known as salmon GnRH and chicken GnRH-II. It is well established that PKC mediates GnRH-stimulated GH secretion in the goldfish pituitary. In contrast, the signal transduction of GnRH-induced GH gene expression has not been elucidated in any model system. In this study, we demonstrate, for the first time, the presence of novel and atypical PKC isoforms in the pituitary of a fish. Moreover, our results indicate that conventional PKCα is present selectively in GH-producing cells. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators (phorbol ester or diacylglycerol analog) did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (calphostin C and GF109203X) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in the goldfish pituitary. Instead, PD98059 inhibited GnRH-induced GH gene expression, suggesting that the ERK signaling pathway is involved. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.

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Grass carp activin : molecular cloning and functional role in regulating growth hormone gene expression in grass carp pituitary cells

10.5353/th_b3045594 ◽

2004 ◽

Author(s):

Sai-kit Fung

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Molecular Cloning ◽

Grass Carp ◽

Functional Role ◽

Growth Hormone Gene ◽

Pituitary Cells

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Endogenous retinoid X receptors can function as hormone receptors in pituitary cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7105 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7105-7110 ◽

Cited By ~ 25

Author(s):

K D Davis ◽

T J Berrodin ◽

J E Stelmach ◽

J D Winkler ◽

M A Lazar

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Ligand Binding ◽

Hormone Receptors ◽

Nuclear Hormone Receptors ◽

Growth Hormone Gene ◽

Pituitary Cells ◽

Mrna Levels ◽

Retinoid X Receptors ◽

X Receptors

Retinoids regulate gene transcription by interacting with both retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs). Since unliganded RXRs can act as heterodimerization partners for RARs and other nuclear hormone receptors, it is unclear whether ligand binding by RXRs actually regulates the expression of naturally occurring genes. To address this issue, we synthesized the RXR-selective retinoid SR11237 and confirmed its specificity in transient transfection and proteolytic susceptibility assays before using it to assess the contribution of ligand-activated RXRs to retinoid action. Unlike RAR ligands, SR11237 did not increase endogenous RAR beta mRNA levels in F9 embryonal carcinoma cells, even though it activated transcription of an RXR-responsive reporter gene in these cells. Thus, it is likely that RARs mediate the induction of RAR beta gene expression by RA. In contrast, the RXR-specific ligand induced rat growth hormone mRNA in GH3 pituitary cells, indicating that the effects of RA on growth hormone gene expression at least in part involve ligand binding to endogenous RXRs in vivo. Our results indicate that in addition to serving as cofactors for other nuclear hormone receptors, endogenous RXRs can function as ligand-dependent regulators of gene expression, i.e., classical nuclear hormone receptors.

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Modulation of Calmodulin Gene Expression as a Novel Mechanism for Growth Hormone Feedback Control by Insulin-like Growth Factor in Grass Carp Pituitary Cells

Endocrinology ◽

10.1210/en.2004-1508 ◽

2005 ◽

Vol 146 (9) ◽

pp. 3821-3835 ◽

Cited By ~ 19

Author(s):

Longfei Huo ◽

Guodong Fu ◽

Xinyan Wang ◽

Wendy K. W. Ko ◽

Anderson O. L. Wong

Keyword(s):

Gene Expression ◽

Feedback Control ◽

Grass Carp ◽

Cell Model ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Mrna Levels ◽

Hormone Synthesis ◽

Igf I ◽

Gh Gene

Abstract Calmodulin (CaM), the Ca2+ sensor in living cells, is essential for biological functions mediated by Ca2+-dependent mechanisms. However, modulation of CaM gene expression at the pituitary level as a means to regulate pituitary hormone synthesis has not been characterized. In this study we examined the functional role of CaM in the feedback control of GH by IGF using grass carp pituitary cells as a cell model. To establish the structural identity of CaM expressed in the grass carp, a CaM cDNA, CaM-L, was isolated from the carp pituitary using 3′/5′ rapid amplification of cDNA ends. The open reading frame of this cDNA encodes a 149-amino acid protein sharing the same primary structure with CaMs reported in mammals, birds, and amphibians. This CaM cDNA is phylogenetically related to the CaM I gene family, and its transcripts are ubiquitously expressed in the grass carp. In carp pituitary cells, IGF-I and IGF-II induced CaM mRNA expression with a concurrent drop in GH transcript levels. These stimulatory effects on CaM mRNA levels were not mimicked by insulin and appeared to be a direct consequence of IGF activation of CaM gene transcription without altering CaM transcript stability. CaM antagonism and inactivation of calcineurin blocked the inhibitory effects of IGF-I and IGF-II on GH gene expression, and CaM overexpression also suppressed the 5′ promoter activity of the grass carp GH gene. These results, as a whole, provide evidence for the first time that IGF feedback on GH gene expression is mediated by activation of CaM gene expression at the pituitary level.

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Endogenous retinoid X receptors can function as hormone receptors in pituitary cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7105-7110.1994 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7105-7110

Author(s):

K D Davis ◽

T J Berrodin ◽

J E Stelmach ◽

J D Winkler ◽

M A Lazar

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Ligand Binding ◽

Hormone Receptors ◽

Nuclear Hormone Receptors ◽

Growth Hormone Gene ◽

Pituitary Cells ◽

Mrna Levels ◽

Retinoid X Receptors ◽

X Receptors

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Mechanisms for PACAP-induced prolactin gene expression in grass carp pituitary cells

Journal of Endocrinology ◽

10.1530/joe-16-0433 ◽

2017 ◽

Vol 233 (1) ◽

pp. 37-51 ◽

Cited By ~ 4

Author(s):

Chengyuan Lin ◽

Xue Jiang ◽

Mulan He ◽

Ling Zhao ◽

Tao Huang ◽

...

Keyword(s):

Gene Expression ◽

Adenylate Cyclase ◽

Mrna Expression ◽

Grass Carp ◽

Functional Coupling ◽

Pituitary Cells ◽

Pac1 Receptor ◽

Fish Model ◽

Camp Analog ◽

Model Grass

In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone with diverse functions but its role in prolactin (PRL) regulation is highly controversial. To shed light on Prl regulation by PACAP in fish model, grass carp pituitary cells was used as a model to examine the receptor specificity and signal transduction for PACAP modulation of prl gene expression in the carp pituitary. Using RT-PCR, PACAP-selective PAC1 receptor was detected in carp lactotrophs. In carp pituitary cells, nanomolar doses of PACAP, but not VIP, could elevate Prl secretion and protein production with concurrent rise in prl mRNA and these stimulatory effects were blocked by PACAP antagonist but not VIP antagonist. PACAP-induced prl mRNA expression could be mimicked by activating adenylate cyclase (AC), increasing cAMP level by cAMP analog, or increasing intracellular Ca2+ ([Ca2+]i) by Ca2+ ionophore/voltage-sensitive Ca2+ channel (VSCC) activator. PACAP-induced prl gene expression, however, was attenuated/abolished by suppressing cAMP production, inhibiting PKA activity, blocking [Ca2+]i mobilization and VSCC activation, calmodulin (CaM) antagonism, and inactivation of JNK and CaM Kinase II (CaMK-II). Similar sensitivity to CaM, JNK, and CaMK-II blockade was also noted by substituting cAMP analog for PACAP as the stimulant for prl mRNA expression. These results, as a whole, provide evidence for the first time that (i) PACAP activation of PAC1 receptor expressed in carp lactotrophs could induce Prl synthesis and secretion, and (ii) Prl production induced by PACAP was mediated by upregulation of prl gene expression, presumably via functional coupling of cAMP/PKA-, Ca2+/CaM-, and MAPK-dependent cascades.

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Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-α and -β gene expression

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90386.2008 ◽

2008 ◽

Vol 295 (2) ◽

pp. E477-E490 ◽

Cited By ~ 21

Author(s):

Quan Jiang ◽

Mulan He ◽

Xinyan Wang ◽

Anderson O. L. Wong

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Grass Carp ◽

Pharmacological Study ◽

Primary Cultures ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Mrna Levels ◽

Fish Model ◽

Subsequent Activation

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SLα and SLβ mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP3)/PKC pathways and subsequent activation of the Ca2+/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLα mRNA expression. Apparently, the downstream Ca2+/CaM-dependent cascades were triggered by extracellular Ca2+ ([Ca2+]e) entry via L-type voltage-sensitive Ca2+ channels (VSCC) and Ca2+ release from IP3-sensitive intracellular Ca2+ stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLβ mRNA expression, except that [Ca2+]e entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SLα and SLβ gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.

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IDENTIFIKASI IKAN CUPANG (Betta imbelis) TRANSGENIK FOUNDER MEMBAWA GEN PENYANDI HORMON PERTUMBUHAN; Identification of Transgenic Founder Betta Fish (Betta imbelis) Carry Growth Hormone Gene.

Jurnal Riset Akuakultur ◽

10.15578/jra.11.3.2016.197-205 ◽

2017 ◽

Vol 11 (3) ◽

pp. 197

Author(s):

Eni Kusrini ◽

Alimuddin Alimuddin ◽

Mohammad Zairin ◽

Dinar Tri Sulistyowati

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Mrna Expression ◽

Transgenic Fish ◽

Pcr Analysis ◽

Growth Hormone Gene ◽

Rt Pcr ◽

Total Rna ◽

Gh Gene ◽

Dna Template

Penelitian dilakukan untuk mengidentifikasi keberhasilan introduksi gen penyandi hormon pertumbuhan (Growth Hormone, GH) pada induk F-0 ikan Betta imbellis. Ikan transgenik F-0 dibuat dengan menggunakan metode transfeksi. Identifikasi dilakukan menggunakan metode RT-PCR. RNA total diekstraksi dari embrio pooled sample hasil persilangan induk transgenik dan non-transgenik. Berdasarkan analisis ekspresi gen pada embrio juga menunjukkan adanya aktivitas ekspresi gen GH pada semua perlakuan dibandingkan dengan kontrol (embrio hasil persilangan non-transgenik x non-transgenik). Jumlah individu induk F-0 yang membawa gen GH eksogen berdasarkan analisis PCR dengan DNA template dari sirip ekor adalah sebanyak 16%. Individu positif membawa gen GH eksogen tersebut dibesarkan lebih lanjut untuk memproduksi Betta imbellis transgenik F-1. Kandidat ikan transgenik jantan F-0 dikawinkan dengan ikan non-transgenik betina, sedangkan transgenik F-0 betina dikawinkan dengan non-transgenik jantan. Sebanyak 30-50 butir embrio hasil pemijahan F-0 digabung, kemudian DNA genom diekstrak. Sebagian embrio digunakan untuk ekstraksi RNA total untuk analisis ekspresi mRNA GH eksogen. Hasil analisis PCR menunjukkan bahwa semua sampel embrio dari induk transgenik F-0 dapat terdeteksi gen GH eksogen, sedangkan untuk kontrol (non-transgenik) tidak terdeteksi. Ekspresi mRNA juga terdeteksi pada embrio F-1. Dengan demikian, metode transfeksi embrio Betta imbellis efektif digunakan untuk menghasilkan ikan transgenik, dan sangat berpotensi menghasilkan individu F-1 Betta imbellis dengan pertumbuhan lebih cepat.The study was conducted to identify the successful introduction of the growth hormone gene (Growth Hormone, GH) on the F-0 Betta imbellis broodstock. The F-0 transgenic fish was made through transfection methods. Identification was done using RT-PCR method. Total RNA was extracted from pooled embryos sample. Based on the analysis of gene expression in embryos also showed activity GH gene expression in all treatments compared to the control (non-transgenic x non-transgenic). The number of individuals F-0 which carried exogenous GH gene by PCR analysis of the DNA template of the tail fin was as much as 16%. Positive individuals carried the exogenous GH gene raised further to produce transgenic F-1 B. imbellis. Candidate of transgenic F-0 males fish were mated with non-transgenic female fish, whereas the transgenic F-0 females were mated with non-transgenic males. The 30-50 embryos obtained were combined, then their genomic DNA were extracted. Some of the embryos was used for the extraction of total RNA for analysis of mRNA expression of GH exogenous. The PCR analysis showed that all samples of embryos from the transgenic F-0 broodstock could be detected, whereas for the control (non-transgenic) was not detected. mRNA expression was also detected in embryos of F-1. The average weight of the F-0 broodstocks were 1.55 g and a total length was 12.97 cm. Thus, the transfection methods through betta embryos peaceful effectively generated transgenic fish, and potentially produced fast growth of individuals F-1 Betta imbellis.

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