Differential involvement of cAMP/PKA-, PLC/PKC- and Ca2+/calmodulin-dependent pathways in GnRH-induced prolactin secretion and gene expression in grass carp pituitary cells.

Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in vertebrates. In fish models, GnRH can also induce prolactin (PRL) release, but little is known for the corresponding effect on PRL gene expression as well as the post-receptor signalling involved. Using grass carp as a model, the functional role of GnRH and its underlying signal transduction for PRL regulation were examined at the pituitary level. Using laser capture microdissection coupled with RT-PCR, GnRH receptor expression could be located in carp lactotrophs. In primary cell culture prepared from grass carp pituitaries, the native forms of GnRH, GnRH2 and GnRH3, as well as the GnRH agonist [D-Arg6, Pro9, NEt]-sGnRH were all effective in elevating PRL secretion, PRL mRNA level, PRL cell content and total production. In pituitary cells prepared from the rostral pars distalis, the region in the carp pituitary enriched with lactotrophs, GnRH not only increased cAMP synthesis with parallel CREB phosphorylation and nuclear translocation but also induced a rapid rise in cytosolic Ca2+ by Ca2+ influx via L-type voltage-sensitive Ca2+ channel (VSCC) with subsequent CaM expression and NFAT2 dephosphorylation. In carp pituitary cells prepared from whole pituitaries, GnRH-induced PRL secretion was reduced/negated by inhibiting cAMP/PKA, PLC/PKC and Ca2+/CaM/CaMK-II pathways but not the signalling events via IP3 and CaN/NFAT. The corresponding effect on PRL mRNA expression, however, was blocked by inhibiting cAMP/PKA/CREB/CBP and Ca2+/CaM/CaN/NFAT2 signalling but not PLC/IP3/PKC pathway. At the pituitary cell level, activation of cAMP/PKA pathway could also induce CaM expression and Ca2+ influx via VSCC with parallel rises in PRL release and gene expression in a Ca2+/CaM-dependent manner. These findings, as a whole, suggest that the cAMP/PKA-, PLC/PKC- and Ca2+/CaM-dependent cascades are differentially involved in GnRH-induced PRL secretion and PRL transcript expression in carp lactotrophs. During the process, a functional crosstalk between the cAMP/PKA- and Ca2+/CaM-dependent pathways may occur with PRL release linked with CaMK-II and PKC activation and PRL gene transcription caused by nuclear action of CREB/CBP and CaN/NFAT2 signalling.

Differential regulation of hepatic SH3 domain binding kinase 1 (SBK1) expression in mouse and goldfish.

SH3 domain binding kinase 1 (SBK1) is a serine/threonine kinase that belongs to the new kinase family (NFK) with limited information on its function. Previous studies reported that SBK1 plays a role in memory formation, lipid metabolism, and cancer cell progression. Nevertheless, the regulatory mechanism of Sbk1 expression in various tissues remains unknown. We report here that Sbk1 expression in mouse hepatocytes was downregulated by glucocorticoid, whereas saturated and unsaturated fatty acids were stimulators of Sbk1 expression. The regulatory role of glucocorticoid and fatty acid was further confirmed by the Sbk1 promoter assay, which aligned with the presence of several glucocorticoid-response elements (GRE) and peroxisome proliferator responsive elements (PPRE) in the mouse Sbk1 promoter. The inhibitory effect of glucocorticoids on hepatic Sbk1 expression and protein content could also be demonstrated in vivo after prednisolone injection. Moreover, the expression of SBK1 in goldfish (gfSBK1) was also sensitive to glucocorticoid suppression as their mouse orthologues. In contrast, insulin had a differential action on SBK1 expression that it promoted the expression of all SBK1 isoforms in the goldfish hepatocytes but inhibited Sbk1 expression in the mouse hepatocytes. Together, our findings indicate that SBK1 expression is hormone- and nutrient-sensitive with a species-specific response.

Mouse Spexin: (I) NMR Solution Structure, Docking Models for Receptor Binding, and Histological Expression at Tissue Level.

Spexin (SPX), a highly conserved neuropeptide, is known to have diverse functions and has been implicated/associated with pathological conditions, including obesity, diabetes, anorexia nervosa, and anxiety/mood disorders. Although most of the studies on SPX involved the mouse model, the solution structure of mouse SPX, structural aspects for SPX binding with its receptors GalR2/3, and its cellular expression/distribution in mouse tissues are largely unknown. Using CD and NMR spectroscopies, the solution structure of mouse SPX was shown to be in the form of a helical peptide with a random coil from Asn1 to Pro4 in the N-terminal followed by an α-helix from Gln5 to Gln14 in the C-terminus. The molecular surface of mouse SPX is largely hydrophobic with Lys11 as the only charged residue in the α-helix. Based on the NMR structure obtained, docking models of SPX binding with mouse GalR2 and GalR3 were constructed by homology modeling and MD simulation. The models deduced reveal that the amino acids in SPX, especially Asn1, Leu8, and Leu10, could interact with specific residues in ECL1&2 and TMD2&7 of GalR2 and GalR3 by H-bonding/hydrophobic interactions, which provides the structural evidence to support the idea that the two receptors can act as the cognate receptors for SPX. For tissue distribution of SPX, RT-PCR based on 28 tissues/organs harvested from the mouse demonstrated that SPX was ubiquitously expressed at the tissue level with notable signals detected in the brain, GI tract, liver, gonad, and adrenal gland. Using immunohistochemical staining, protein signals of SPX could be located in the liver, pancreas, white adipose tissue, muscle, stomach, kidney, spleen, gonad, adrenal, and hypothalamo-pituitary axis in a cell type-specific manner. Our results, as a whole, not only can provide the structural information for ligand/receptor interaction for SPX but also establish the anatomical basis for our on-going studies to examine the physiological functions of SPX in the mouse model.

Mouse Spexin: (II) Functional Role as a Satiety Factor inhibiting Food Intake by Regulatory Actions Within the Hypothalamus.

Spexin (SPX) is a pleiotropic peptide with highly conserved protein sequence from fish to mammals and its biological actions are mediated by GalR2/GalR3 receptors expressed in target tissues. Recently, SPX has been confirmed to be a novel satiety factor in fish species but whether the peptide has a similar function in mammals is still unclear. Using the mouse as a model, the functional role of SPX in feeding control and the mechanisms involved were investigated. After food intake, serum SPX in mice could be up-regulated with elevations of transcript expression and tissue content of SPX in the glandular stomach but not in other tissues examined. As revealed by immunohistochemical staining, food intake also intensified SPX signals in the major cell types forming the gastric glands (including the foveolar cells, parietal cells, and chief cells) within the gastric mucosa of glandular stomach. Furthermore, IP injection of SPX was effective in reducing food intake with parallel attenuation in transcript expression of NPY, AgRP, NPY type 5 receptor (NPY5R), and ghrelin receptor (GHSR) in the hypothalamus, and these inhibitory effects could be blocked by GalR3 but not GalR2 antagonism. In agreement with the central actions of SPX, similar inhibition on feeding and hypothalamic expression of NPY, AgRP, NPY5R, and GHSR could also be noted with ICV injection of SPX. In the same study, in contrast to the drop in NPY5R and GHSR, SPX treatment could induce parallel rises of transcript expression of leptin receptor (LepR) and melanocortin 4 receptor (MC4R) in the hypothalamus. These findings, as a whole, suggest that the role of SPX as a satiety factor is well conserved in the mouse. Apparently, food intake can induce SPX production in glandular stomach and contribute to the postprandial rise of SPX in circulation. Through GalR3 activation, this SPX signal can act within the hypothalamus to trigger feedback inhibition on feeding by differential modulation of feeding regulators (NPY and AgRP) and their receptors (NPY5R, GHSR, LepR, and MC4R) involved in the feeding circuitry within the CNS.

Mouse Spexin: (III) Differential Regulation by Glucose and Insulin in Glandular Stomach and Functional Implication in Feeding Control.

Spexin (SPX), a neuropeptide with diverse functions, is a novel satiety factor in fish models and its role in feeding control has been recently confirmed in mammals. In mouse, food intake was shown to trigger SPX expression in glandular stomach with parallel rise in serum SPX and these SPX signals could inhibit feeding via central actions within the hypothalamus. However, the mechanisms for SPX regulation by food intake are still unclear. To examine the role of insulin signal caused by glucose uptake in SPX regulation, the mice were IP injected with glucose and insulin, respectively. In this case, serum SPX was elevated by glucose but not altered by insulin. Meanwhile, SPX transcript expression in the glandular stomach was up-regulated by glucose but the opposite was true for insulin treatment. Using in situ hybridization, the differential effects on SPX gene expression were located in the gastric mucosa of glandular stomach. Co-injection experiments also revealed that glucose stimulation on serum SPX and SPX mRNA expressed in glandular stomach could be blocked by insulin. In gastric mucosal cells prepared from glandular stomach, the opposite effects on SPX transcript expression by glucose and insulin could still be noted with similar blockade of the stimulatory effects of glucose by insulin. In this cell model, SPX gene expression induced by glucose was mediated by glucose uptake via GLUT, ATP synthesis by glycolysis/respiratory chain, and subsequent modulation of KATP channel activity, but the voltage-sensitive Ca2+ channels were not involved. The corresponding inhibition by insulin, however, was mediated by PI3K/Akt, MEK1/2/ERK1/2, and P38MAPK cascades coupled to insulin receptor but not IGF-1 receptor. Apparently, glucose uptake in mice can induce SPX expression in the glandular stomach through ATP synthesis via glucose metabolism and subsequent modification of KATP channel activity, which may contribute to SPX release into circulation to act as the satiety signal after food intake. The insulin rise caused by glucose uptake, presumably originated from the pancreas, may serve as a negative feedback to inhibit the SPX response by activating MAPK and PI3K/Akt pathways in the stomach.

Signal Transduction for TNFα-Induced Type II SOCS Expression and Its Functional Implication in Growth Hormone Resistance in Carp Hepatocytes.

In mammals, local production of tumor necrosis factor α (TNFα) inhibits growth hormone (GH)-induced IGF-I expression at tissue level and contributes to GH resistance caused by sepsis/endotoxemia and inflammation. Although the loss of GH responsiveness can be mediated by a parallel rise in SOCS expression, the signaling mechanisms for TNFα-induced SOCS expression at the hepatic level have not been characterized and the comparative aspects of the phenomenon, especially in lower vertebrates, are still unknown. Recently, type II SOCS, including SOCS1-3 and CISH, have been cloned in grass carp and shown to act as the feedback repressors for GH signaling via JAK2/STAT5 pathway. To shed light on the mechanisms for TNFα-induced GH resistance in fish model, grass carp TNFα was cloned and confirmed to be a single-copy gene expressed in various tissues including the liver. In carp hepatocytes, incubation with the endotoxin LPS induced TNFα expression with parallel rises in SOCS1-3 and CISH mRNA levels. Similar to LPS, TNFα treatment could block GH-induced IGF-I/-II mRNA expression and elevate SOCS1, SOCS3, and CISH transcript levels. However, TNFα was not effective in altering SOCS2 expression. In parallel experiment, LPS blockade of IGF-I/-II signals caused by GH could be partially reverted by TNFα receptor antagonism. At hepatocyte level, TNFα induction also triggered rapid phosphorylation of IκBα, MEK1/2, ERK1/2, MKK3/6, P38MAPK, Akt, JAK2, and STAT1,3,5, and TNFα-induced SOCS1, SOCS3, and CISH mRNA expression could be negated by inhibiting the IKK/NFκB, MAPK, PI3K/Akt, and JAK/STAT cascades. Our findings, as a whole, suggest that local production of TNFα may interfere with IGF-I/-II induction by GH in the carp liver by up-regulation of SOCS1, SOCS3, and CISH via IKK/NFκB, MAPK, PI3K/Akt, and JAK/STAT-dependent mechanisms, which may contribute to GH resistance induced by endotoxin in carp species.

Grass Carp Prolactin Gene: Structural Characterization and Signal Transduction for PACAP-induced Prolactin Promoter Activity.

In this study, structural analysis of grass carp prolactin (PRL) gene was performed and the signaling mechanisms for pituitary adenylate cyclase-activating peptide (PACAP) regulation of PRL promoter activity were investigated. In αT3-1 cells, PRL promoter activity could be induced by oPACAP38 which was blocked by PACAP antagonist but not the VIP antagonist. The stimulatory effect of oPACAP38 was mimicked by activation of AC/cAMP and voltage-sensitive Ca2+ channel (VSCC) signaling, or induction of Ca2+ entry. In parallel, PACAP-induced PRL promoter activity was negated or inhibited by suppressing cAMP production, inhibiting PKA activity, removal of extracellular Ca2+, VSCC blockade, calmodulin (CaM) antagonism, and inactivation of CaM kinase II. Similar sensitivity to L-type VSCC, CaM and CaM kinase II inhibition were also observed by substituting cAMP analog for oPACAP38 as the stimulant for PRL promoter activity. Moreover, PACAP-induced PRL promoter activity was also blocked by inhibition of PLC signaling, attenuation of [Ca2+]i immobilization via IP3 receptors, and blockade of PI3K/P70S6K pathway. The PACAP-induced PRL promoter activation may involve transactivation of the transcription factor CREB. These results suggest that PACAP can stimulate PRL promoter activation by PAC1 mediated functional coupling of the Ca2+/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. Apparently, other signaling pathways, including PLC/IP3 and PI3K/P70S6K cascades, may also be involved in PACAP induction of PRL gene transcription.

Spexin as a neuroendocrine signal with emerging functions.

Spexin (SPX), a novel peptide coevolved with the galanin/kisspeptin family, was first identified by bioinformatics prior to its protein purification/functional studies. Its mature peptide is highly conserved among different vertebrate classes. Based on the studies in mammals and fish models, SPX was found to be widely distributed at tissue level, secreted into systemic circulation, identified at notable levels in central nervous system and peripheral tissues, and has been confirmed/implicated in multiple functions in different tissues/organs, suggesting that SPX may serve as a neuroe​ndocrine signal with pleotropic functions. In this article, different isoforms of SPX and their binding with their cognate receptors GalR2 and GalR3, the biological functions of SPX reported in mammals including GI tract movement, energy balance and weight loss, fatty acid uptake, glucose homeostasis, nociception and cardiovascular/renal functions, as well as the recent findings in fish models regarding the role of SPX in reproduction and feeding control will be reviewed with interesting questions for future investigations.

Dual Role of Insulin in Spexin Regulation: Functional Link Between Food Intake and Spexin Expression in a Fish Model.

Spexin (SPX), a neuropeptide discovered by the bioinformatics approach, has been recently identified as a satiety factor in a fish model. However, the functional link between feeding and SPX expression as well as the signal transduction for SPX regulation are totally unknown. In this study, we used goldfish as a model to examine the functional role of insulin as a postprandial signal for SPX regulation in bony fish. In goldfish, feeding could elevate plasma levels of glucose, insulin, and SPX with concurrent rises in insulin and SPX messenger RNA (mRNA) expression in the liver. Similar elevation in SPX mRNA level was also observed in the liver and brain areas involved in appetite control in goldfish after intraperitoneal injection of glucose and insulin, respectively. In parallel experiments with goldfish hepatocytes and brain cell culture, insulin signal induced by glucose was shown to exert a dual role in SPX regulation, namely (1) acting as an autocrine/paracrine signal to trigger SPX mRNA expression in the liver and (2) serving as an endocrine signal to induce SPX gene expression in the brain. Apparently, the peripheral (in the liver) and central actions of insulin (in the brain) on SPX gene expression were mediated by insulin receptor (to a lesser extent by insulin-like growth factor I receptor) coupled to mitogen-activated protein kinase kinase 3/6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin but not mitogen-activated protein kinase kinase 1/2/extracellular signal-regulated kinase 1/2 cascades. Our findings indicate that an insulin component inducible by glucose is present in the liver of the fish model and may serve as the postprandial signal linking food intake with SPX expression both in the central as well as at the hepatic level.

TAC1 Gene Products Regulate Pituitary Hormone Secretion and Gene Expression in Prepubertal Grass Carp Pituitary Cells.

Tachykinin-1 (TAC1) is known to have diverse functions in mammals, but similar information is scarce in fish species. Using grass carp as a model, the pituitary actions, receptor specificity and postreceptor signaling of TAC1 gene products, namely substance P (SP) and neurokinin A (NKA), were examined. TAC1 encoding SP and NKA as well as tachykinin receptors NK1R and NK2R were cloned in the carp pituitary. The newly cloned receptors were shown to be functional with properties similar to mammalian counterparts. In carp pituitary cells, SP and NKA could trigger luteinizing hormone (LH), prolactin (PRL), and somatolactin α (SLα) secretion, with parallel rises in PRL and SLα transcripts. Short-term SP treatment (3 hours) induced LH release, whereas prolonged induction (24 hours) could attenuate LHß messenger RNA (mRNA) expression. At pituitary cell level, LH, PRL, and SLα regulation by TAC1 gene products were mediated by NK1R, NK2R, and NK3R, respectively. Apparently, SP- and NKA-induced LH and SLα secretion and transcript expression were mediated by adenylyl cyclase/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholiphase C (PLC)/inositol 1,4,5-triphosphate/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/CaM-dependent protein kinase-II pathways. The signal transduction for PRL responses was similar, except for the absence of a PKC component. Regarding SP inhibition of LHß mRNA expression, the cAMP/PKA- and PLC/PKC-dependent (but not Ca2+/CaM-dependent) cascades were involved. These results, as a whole, suggest that TAC1 gene products play a role in LH, PRL, and SLα regulation via overlapping postreceptor signaling coupled to different subtypes of tachykinin receptor expressed in the carp pituitary.

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

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.

Kisspeptin induction of somatolactin-α release in goldfish pituitary cells: functional role of cAMP/PKA-, PLC/PKC-, and Ca(2+)/calmodulin-dependent cascades.

Although the importance of kisspeptin in the pituitary is firmly established, the signaling mechanisms for the pituitary actions of kisspeptin are still largely unknown. Somatolactin (SL), a member of the growth hormone (GH)/prolactin (PRL) family, is a pituitary hormone with pleiotropic functions in fish, but its regulation by kisspeptin has not been examined. To investigate the functional role of kisspeptin in SL regulation, expression of two paralogues of goldfish Kiss1 receptors (Kiss1ra and Kiss1rb) were confirmed in immunoidentified SLα but not SLß cells isolated by RT-PCR coupled with laser capture microdissection. In goldfish pituitary cells prepared from neurointermediate lobe (NIL), synthetic goldfish Kiss decapeptides (gKiss1-10 and gKiss2-10) could increase SLα release. Consistent with the lack of Kiss1r expression in SLß cells, SLß release was not altered by kisspeptin stimulation. In parallel experiments, goldfish gKiss1-10 could elevate cyclic adenosine monophosphate (cAMP) production, upregulate protein kinase A (PKA) and protein kinase C (PKC) activities, and trigger a rapid rise in intracellular Ca(2+) levels in goldfish NIL cells. Using a pharmacological approach, cAMP/PKA and phospholipase C (PLC)/PKC pathways and subsequent activation of Ca(2+)/calmodulin (CaM)-dependent cascades were shown to be involved in SLα release induced by gKiss1-10. Apparently, the Ca(2+)-dependent cascades were triggered by extracellular Ca(2+) entry via voltage-sensitive Ca(2+) channels and mobilization of inositol trisphosphate-sensitive intracellular Ca(2+) stores. Our results demonstrate that gKiss1-10 can act directly at the pituitary level to trigger SLα release via a complex network of post-receptor signaling mechanisms.

Somatostatin-28 inhibitory action on somatolactin-α and -ß gene expression in goldfish.

Somatostain (SS) is known to inhibit growth hormone (GH) and prolactin (PRL) secretion. Somatolactin (SL) is a member of the GH/PRL family, but its regulation by goldfish brain somatostatin-28 (gbSS-28) has not been examined. To this end, the structural identity of goldfish SLα was established by 5'/3'-rapid amplification of cDNA ends. As revealed by in situ hybridization and immunohistochemical staining, the expression of SL isoforms was detected in pituitary cells located in the neurointermediate lobe (NIL). The transcripts of goldfish SS receptor 5a (Sst5a) but not Sst1b, Sst2, or Sst3a were detected in the goldfish NIL cells by RT-PCR. In goldfish pituitary cells, gbSS-28 not only had an inhibitory effect on basal SLα and SLß mRNA levels but also could abolish insulin-like growth factor-stimulated SL gene expression. In primary cultures of goldfish NIL cells, gbSS-28 reduced forskolin-stimulated total cAMP production. With the use of a pharmacological approach, the adenylate cyclase (AC)/cAMP and phospholipase C (PLC)/inositol trisphosphate (IP3)/protein kinase C (PKC) cascades were shown to be involved in gbSS-28-inhibited SLα mRNA expression. Similar postreceptor signaling cascades were also observed for gbSS-28-reduced SLß mRNA expression, except that PKC coupling to PLC was not involved. These results provide evidence that gbSS-28 can inhibit SLα and SLß gene expression at the goldfish pituitary level via Sst5 through differential coupling of AC/cAMP and PLC/IP3/PKC cascades.

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, P(38) 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.

Gene expression of luteinizing hormone receptor in carp somatotrophs differentially regulated by local action of gonadotropin and dopamine D1 receptor activation.

In grass carp, luteinizing hormone (LH) can act locally within the pituitary to regulate growth hormone expression. To test if LH receptor (LHR) expression in the carp pituitary can also serve as a target of modulation for LH actions, grass carp LHR was cloned and characterized by functional expression. In carp pituitary cells, LHR mRNA (lhr) level could be reduced by LH or human chorionic gonadotropin (hCG) but up-regulated by dopamine treatment. Dopamine-induced lhr expression occurred mainly in carp somatotrophs via the cAMP/PKA pathway coupled to pituitary D1 receptors. This stimulatory effect could be blocked by LHR activation by hCG, presumably through phosphodiesterase III activation. These findings provide evidence that lhr expression in the carp pituitary is under the differential control of LH and dopamine via modification of cAMP-dependent signaling mechanisms, which may play a role in regulating somatotroph responsiveness to the paracrine action of LH in carp species.

Kisspeptin-1 directly stimulates LH and GH secretion from goldfish pituitary cells in a Ca(2+)-dependent manner.

It has been established that kisspeptin regulates reproduction via stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion, which then induces pituitary luteinizing hormone (LH) release. Kisspeptin also directly stimulates pituitary hormone release in some mammals. However, in goldfish, whether kisspeptin directly affects pituitary hormone release is controversial. In this study, synthetic goldfish kisspeptin-1((1-10)) (gKiss1) enhances LH and growth hormone (GH) release from primary cultures of goldfish pituitary cells in column perifusion. gKiss1 stimulation of LH and GH secretion were still manifested in the presence of the two native goldfish GnRHs, salmon (s)GnRH (goldfish GnRH-3) and chicken (c)GnRH-II (goldfish GnRH-2), but were attenuated by two voltage-sensitive calcium channel blockers, verapamil and nifedipine. gKiss-induced increases in intracellular Ca(2+) in Fura-2AM pre-loaded goldfish pars distalis cells were also inhibited by nifedipine. These results indicate that, in goldfish, (1) direct gKiss1 actions on pituitary LH and GH secretion exist, (2) these actions are independent of GnRH and (3) they involve Ca(2+) signalling.

Goldfish kisspeptin: molecular cloning, tissue distribution of transcript expression, and stimulatory effects on prolactin, growth hormone and luteinizing hormone secretion and gene expression via direct actions at the pituitary level.

Kisspeptin, the product of Kiss1 gene, is a novel regulator of the gonadotropic axis. In mammals, its stimulatory effect on gonadotropin secretion is well documented and mediated mainly by hypothalamic release of gonadotropin-releasing hormone. Although the pituitary actions of kisspeptin have been reported, the effects of kisspeptin on gonadotropin release via direct action on pituitary cells are still controversial. Using goldfish as a model, here we examined the direct actions of kisspeptin on pituitary functions in modern-day bony fish. As a first step, the structural identity of goldfish Kiss1 was established by 5'/3'RACE and Kiss1 transcript was shown to be widely expressed in various tissues in goldfish. At the pituitary level, Kiss1 receptor (Kiss1r) expression was detected in immuno-identified gonadotrophs, lactotrophs, and somatotrophs. Kiss1 transcript was also located in goldfish somatotrophs but not in lactotrophs or gonadotrophs. In parallel studies, goldfish kisspeptin-10 was synthesized and used to test the pituitary actions of kisspeptin in vitro. In goldfish pituitary cell cultures, 30-min incubation with kisspeptin-10 increased basal release of luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH). Transcript expression of LH, PRL, and GH were also elevated by prolonging kisspeptin-10 treatment to 24h. These results taken together suggest that kisspeptin via Kiss1r activation can act directly at the pituitary level to trigger LH, PRL, and GH secretion and gene expression in goldfish. Our finding of Kiss1 expression in somatotrophs also rises the possibility that kisspeptin may be produced locally in the fish pituitary and serve as an autocrine/paracrine regulator.

Genomic structure and transcriptional regulation of grass carp calmodulin gene.

A fish calmodulin (CaM) gene was characterized for the first time in grass carp. The CaM gene is about 12-Kb in size with identical intron/exon organization as that of mammalian CaM genes. When compared to mammalian counterparts, the 5'-promoter region of grass carp CaM gene contains a TATA box and has a much lower GC content and CpG dinucleotide frequency. Interestingly, the 5'-promoter of carp CaM gene is AT-rich with multiple IRS elements and putative binding sites for Pit-1, Sp1/Sp3 and AP1. Using luciferase reporter assay, a potent silencer region was identified in the distal region of grass carp CaM promoter. Besides, the CaM promoter activity could be upregulated by IGF but suppressed by PACAP, forskolin and over-expression of Sp1 and Sp3. These findings, taken together, indicate that grass carp CaM gene does not exhibit the typical features of housekeeping genes and its expression is under the control of hormone factors, presumably by coupling with the appropriate signaling pathways/transcription factors.

Serotonin interferes with Ca2+ and PKC signaling to reduce gonadotropin-releasing hormone-stimulated GH secretion in goldfish pituitary cells.

In goldfish, two endogenous gonadotropin-releasing hormones (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), are thought to stimulate growth hormone (GH) release via protein kinase C (PKC) and subsequent increases in intracellular Ca(2+) levels ([Ca(2+)](i)). In contrast, the signaling mechanism for serotonin (5-HT) inhibition of GH secretion is still unknown. In this study, whether 5-HT inhibits GH release by actions at sites along the PKC and Ca(2+) signal transduction pathways leading to hormone release were examined in primary cultures of goldfish pituitary cells. Under static incubation and column perifusion conditions, 5-HT reduced basal, as well as sGnRH- and cGnRH-II-stimulated, GH secretion. 5-HT also suppressed GH responses to two PKC activators but had no effect on the GH-releasing action of the Ca(2+) ionophore ionomycin. Ca(2+)-imaging studies with identified somatotropes revealed that 5-HT did not alter basal [Ca(2+)](i) but attenuated the magnitude of the [Ca(2+)](i) responses to the two GnRHs. Prior treatment with 5-HT and cGnRH-II reduced the magnitude of the [Ca(2+)](i) responses induced by depolarizing levels of K(+). Similar inhibition, however, was not observed with prior treatment of 5-HT and sGnRH. These results suggest that 5-HT, by direct actions at the somatotrope level, interferes with PKC and Ca(2+) signaling pathways to reduce the GH-releasing effect of GnRH. 5-HT action may occur at the level of PKC activation or its downstream signaling events prior to the subsequent rise in [Ca(2+)](i.). The differential Ca(2+) responses by depolarizing doses of K(+) is consistent with our previous findings that sGnRH and cGnRH-II are coupled to overlapping and yet distinct Ca(2+)-dependent mechanisms.

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

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, SLalpha and SLbeta 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 (IP(3))/PKC pathways and subsequent activation of the Ca(2+)/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLalpha mRNA expression. Apparently, the downstream Ca(2+)/CaM-dependent cascades were triggered by extracellular Ca(2+) ([Ca(2+)](e)) entry via L-type voltage-sensitive Ca(2+) channels (VSCC) and Ca(2+) release from IP(3)-sensitive intracellular Ca(2+) 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 SLbeta mRNA expression, except that [Ca(2+)](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 SLalpha and SLbeta gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.