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1 pathway in GT1–7 hypothalamic cells
Division of Molecular and Life Science, Department of Life Science, Pohang University of Science and Technology, San31 Hyoja-Dong Nam-Gu Pohang, Kyungbuk 790-784, South Korea
1 School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea
(Requests for offprints should be addressed to P-G Suh; Email: pgs{at}postech.ac.kr)
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Abstract |
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1 in an Src-dependent manner in hypothalamic GT1–7 cells, and that pretreatment with either 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazole [3, 4-d] pyrimidine, an Src-kinase inhibitor, or U73122
[GenBank]
, a PLC inhibitor, attenuates the serotonin-induced increase in calcium levels. Also, PLC 1 binds to c-Src through the Src-homology (SH) 223 domain upon serotonin treatment. Moreover, calcium increase is alleviated in the cells transientlyexpressing SH223 domain-deleted PLC 1 or lipase inactive mutant PLC 1, as compared with cells transfected with wild-type PLC 1. Furthermore, the inhibition of the activities of either PLC or Src results in a significant diminution of the serotonin-induced release of gonadotropin-releasing hormone (GnRH). In addition, the results of our small-interfering RNA experiment confirm that endogenous PLC 1 is a prerequisite for serotonin-mediated signaling pathways. Taken together, our findings demonstrate that serotonin stimulates the release of GnRH through the Src-PLC 1 pathway, via the modulation of intracellular calcium levels. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gonadotropin-releasing hormone (GnRH) release is essential for the maintenance of gonadotropin secretion and normal reproductive function. The secretion of GnRH is modulated by neurotransmitters (Moguilevsky & Wuttke 2001), catecholamines (Becu-Villalobos & Libertun 1995), and neuropeptides (Woller et al. 1992). Within the hypothalamus, the auto-regulatory control of GnRH neural activity is integrated with other hormonal inputs to provide a more complex control system. Several mechanisms for the generation of GnRH have been suggested, including the bursts of action potentials (Kusano et al. 1995) and cAMP-dependent cation channel activations (Sakakibara et al. 1998).
Phospholipase C (PLC) 1 possesses two Src-homology 2 (SH2) domains and one SH3 domain, between the X- and Y-catalytic domains (Stahl et al. 1988, Suh et al. 1988). The SH domains have been determined to perform a vital function in the PLC 1-mediated signaling pathway (Chang et al. 1999). The SH2 domains of PLC 1 have been implicated in the association between PLC 1 and the activated receptor tyrosine kinases, and the SH3 domain has been associated with the mitogenic effects of PLC 1 (Huang et al. 1995, Smith et al. 1996).
Following earlier reports that implicated serotonin in the development and migration of GnRH neurons (Pronina et al. 2003), a great deal of attention has become focused on the role of serotonin in GnRH release (Moguilevsky & Wuttke 2001, Wada et al. 2006). The demonstration of synaptic contacts between the serotoninergic system and the GnRH neurons suggested that serotonin could act directly on GnRH release. However, little knowledge is available at present with regard to the molecular mechanisms inherent to the role of serotonin.
In this study, we investigated the effects of serotonin on hypothalamic GT1–7 cells, in order to gain understanding into the molecular mechanisms underlying serotonin-mediated hormone secretion. We have determined that serotonin activates PLC 1 in the hypothalamic GT1–7 cells, and further demonstrated that the activities of PLC and Src affect GnRH secretion. These findings provide novel insights into the manner in which PLC 1 contributes to the serotonin-mediated release of GnRH.
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Materials and Methods |
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Phospho-mitogen-activated protein kinase kinase (MEK)1/2 (Ser217/Ser221), phospho-extracellular signal-regulated kinase (ERK)1/2 (Thr202/Tyr204), MEK1/2, and ERK1/2 antibodies were purchased from Cell Signaling Technology (New England Biolabs, Beverly, MA, USA) and horseradish peroxidase-conjugated secondary antibodies were obtained from Kirkegaard and Perry Lab. (Gaithersburg, MD, USA). PLC 1, phospho-c-Src, c-Src, and actin antibodies were purchased from Sigma-Aldrich. Serotonin, U73122
[GenBank]
, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazole [3, 4-d] pyrimidine (PP2) were obtained from Calbiochem (San Diego, CA, USA).
Cell culture
GT1–7 hypothalamus cells, which were donated by Dr Richard Weiner (University of California, San Francisco, CA, USA), were grown in a culture medium consisting of 500 ml Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY, USA), containing 0.584 g/l L-glutamate and 4.5 g/l glucose, mixed with 500 ml F-12 medium containing 0.146 g/l L-glutamate, 1.8 g/l glucose, 100 µg/ml gentamicin, 2.5 g/l sodium carbonate, and 10% heat-inactivated fetal bovine serum.
Immunoprecipitation
The cells, after serotonin stimulation with or without inhibitors, were placed on ice, washed twice in ice-cold PBS, and lysed in lysis buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, 20 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 1% Nonidet P-40, 10 µg/ml aprotonin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstatin, and 1 mM 4-(2-aminoethyl) benzensulfonyl fluoride. The solubilized lysates were then clarified via 10 min centrifugation at 8000 g, pre-cleared with agarose, and incubated with specific antibodies and protein-A agarose. The immunoprecipitates were collected and washed four times in lysis buffer. After 5-min heating at 95 °C, the samples were centrifuged, and the supernatants were analyzed on SDS-PAGE (8–16%) gradient gels.
Immunoblot analysis
The cells were grown on six-well plates and serum-starved for 24 h prior to treatment with the indicated agents. The media were aspirated, and the cells were washed twice in ice-cold PBS and lysed in 100 µl lysis buffer. The samples were then briefly sonicated, heated for 5 min at 95 °C, and centrifuged for 5 min. The supernatants were electrophoresed on SDS-PAGE (8–16%) gradient gels, and transferred to polyvinylidene difluoride membranes. The blots were incubated overnight at 4 °C with primary antibodies, then washed six times in Tris-buffered saline/0.1% Tween 20 (Sigma Aldrich) prior to 1 h probing with horseradish peroxidase-conjugated secondary antibodies at room temperature. The blots were then visualized via enhanced chemiluminescence (Amersham Biosciences). In some cases, the blots were stripped and reprobed using other antibodies.
Transient transfection
To construct and express cDNA encoding whole and mutant forms of PLC 1, rat PLC 1 cDNA was N-terminally tagged with FLAG sequences and sub-cloned into FLAG-cyto-megalovirus (CMV) (Bae et al. 1998). Src wild-type and dominant-negative genes were provided by Dr Kitagawa (Kitagawa et al. 2002). The GT1–7 cells were transfected with FLAG-wild-type PLC 1, FLAG-lipase inactive mutant (LIM) PLC 1, and FLAG-SH223 domain-deleted PLC 1, using LipofectAMINE (Invitrogen). After 5 h, the cells were switched to serum-containing medium for 24 h, followed by replacement with serum-free medium overnight. The cells were then treated with agonists and collected in lysis buffer for immunoblot analysis and immunoprecipitation. DNA transfection was performed using LipofectAMINE, in accordance with the manufacturer’s instructions.
Calcium measurement
Sub-confluent GT1–7 cells cultured in 175-cm2 flasks were collected via trypsinization (0.05% trypsin and 0.02% EDTA) and centrifuged (230 g, 5 min). The cells were then suspended in culture medium at a final concentration of 5 x 106 cells/ml, and incubated for 20 min with fura 2-acetoxymetyl (AM; 10 µM) and 0.02% pluronic F-127 at 30 °C, with continuous agitation. At the end of the incubation, the cells were centrifuged (230 g, 5 min) and washed in culture medium. The cells were washed twice in Hanks’ buffer (in mM: 137 NaCl, 5.4 KCl, 0.34 Na2HPO4, 5.5 glucose, 4.2 NaHCO3, 0.8 MgSO4, and 10 4-(2-hydroxyethyl)-1-piperazine ethane sulfinic acid (HEPES) Sigma Aldrich), with 0.1 mM EGTA for the first wash only, pH 7.4). The cells were resuspended in this buffer at a final concentration of 2.7 x 106 cells/ml, and maintained in darkness at 4 °C until use. Calcium concentrations were measured using an RF-5301PC Spectrofluor-ophotometry (Shimadzu, Kyoto, Japan), after 4-min incubation at 37 °C (excitation at 340 and 380 nm, emission at 510 nm). The levels of free calcium in the cytosol were determined according to a previously described method (Grynkiewicz et al. 1985).
Small-interfering RNA (siRNA) for PLC 1
siRNA of PLC 1 (PLC 1 siRNA) was purchased from Dharmacon (Lafayette, CO, USA). The sequences of siRNA for luciferase (control siRNA) were as follows: sense, 5'-CUUACGCUGAGUACUUCGAdTdT-3'; antisense, 5'-UCGAAGUACUCAGCGUAAGdTdT-3'. Double-strand RNA was produced using the conditions described previously (Patterson et al. 2002). SiRNA was transfected into GT1–7 cells using LipofectAMINE.
GnRH measurement via the RIA method
Static incubation and GnRH RIA were conducted as previously described, with some modifications (Cho et al. 1998). In brief, the hypothalamic fragments were rapidly isolated from 10-week-old male Institute of Cancer Research (ICR) mice and cultured in 96-well tissue culture plates (one fragment per well) in 0.3 ml DMEM supplemented with an F12 nutrient mixture. The boundaries of the hypothalamic fragments were caudally the anterior border of the mamilliary body, rostrally the apex of the isosceles triangle-shaped region and laterally the lateral hypothalamic sulci. Hypothalamic fragments were sliced at a depth of approximately 1 mm. For equilibration, the tissues were pre-incubated for 1 h at 37 °C in a humidified incubator, under 5% CO2 tension. After 1 h, the tissues were washed twice in PBS, and incubated for an additional 1 h in fresh medium with the appropriate vehicle or drugs. The media were then collected and centrifuged for 5 min at 10 000 g, in order to prevent any possible contamination of the cells. GnRH RIA was conducted using the Chen-Ramirez GnRH antiserum, CRR13B73 (generously provided by Dr V D Ramirez, University of Illinois, Urbana, IL, USA) at a final dilution of 1:180 000. Sensitivity at 80% binding was approximately 0.5 pg/tube. The intra- and interassay coefficients of variation were determined to be 4–5 and 5–10% respectively for a 5 pg dose of synthetic GnRH.
Band intensity analysis
The band intensity signal was quantified using Image Gauge software (version 3.12; Fujifilm).
Statistical analysis
Data were expressed as means ± S.E.M. Statistical analyses were conducted using SigmaStat (SPSS, Inc., Chicago, IL, USA). The differences were considered to be significant at a P value of <0.05. We used one-way ANOVA with Holm–Sidak comparisons to determine the effects of PP2 and U73122 [GenBank] on GnRH secretion, and used one-way ANOVA with a post-hoc Fisher’s test in order to analyze band intensity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 in GT1–7 cells in a time-dependent manner
In order to determine the role of PLC 1 in serotonin signaling, we conducted an immunoprecipitation experiment to ascertain whether PLC 1 was activated by serotonin in the hypothalamic GT1–7 cells, which have been shown to express the serotonin receptor (Doucet et al. 1995). The cells were initially serum-starved for 24 h to remove any stimulants from the media, and were then treated with either vehicle alone or with serotonin (1 µM) for the indicated times. The phosphorylation of endogenous PLC 1 increased after stimulation with serotonin (Fig. 1A
). A previous report suggested that PLC 1 phosphorylation on Tyr783 residue was essential for the activity of PLC 1 (Kim et al. 1991). Therefore, we used a phospho-specific antibody, which could recognize the phosphotyrosine residue of PLC 1 (Tyr783) to detect tyrosine phosphorylated PLC 1 (Choi et al. 2005). Phosphorylation reached maximum levels at 10 min, and began to decrease at 30 min. Then, in order to ascertain whether the activation of PLC 1 could be correlated with the activation of downstream signaling molecules, we employed western blot analysis to determine whether serotonin induced the activation of MEK1/2 and ERK1/2. MEK1/2 and ERK1/2 were phosphorylated in a time-dependent manner upon serotonin stimulation, whereas total MEK1/2 and ERK1/2 levels remained unaffected (Fig. 1B). Taken together, these findings indicate that PLC 1 may play a role in serotonin signaling.
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1 phosphorylation
PLC has been reported to function as a substrate for members of the Src-kinase family (Haendeler et al. 2003, Stope et al. 2004). It has also been demonstrated that c-Src plays a crucial role in serotonin-mediated calcium responses (Giusti et al. 1999, Tolloczko et al. 2002, Chang et al. 2004), thereby raising the possibility that c-Src may play some role in serotonin signaling. In order to better understand the signaling pathway involved in the serotonin-mediated activation of PLC 1, we pretreated the cells with PP2 (10 µM), an Src-kinase inhibitor, prior to serotonin (1 µM) administration, and then assessed PLC 1 phosphorylation via western blot analysis. The tyrosine phosphorylation of PLC 1 (Tyr783) was impaired in the presence of PP2 (Fig. 2A). Subsequently, we attempted to determine whether the activation of downstream signaling molecules was also affected by treatment with the Src-kinase inhibitor. The potency of MEK1/2 and ERK1/2 phosphorylation was attenuated substantially as a result of pretreatment with PP2 (Fig. 2B). For further confirmation, we conducted an additional investigation into the effects of serotonin on the phosphorylation of c-Src. c-Src was phosphorylated directly upon stimulation with serotonin (Fig. 2C). In order to clarify the hierarchy inherent to Src and PLC 1, we have investigated the effects of an Src dominant-negative mutant on serotonin-mediated PLC 1 phosphorylation. PLC 1 phosphorylation (Tyr783) due to serotonin was completely suppressed in the Src mutant-transfected cells (Fig. 2D). To delineate, we have investigated the effects of U73122
[GenBank]
, a PLC inhibitor, on serotonin-induced mitogen-activated protein kinase (MAPK) activity. As MAPK activity was completely suppressed by U73122
[GenBank]
, PLC 1 appears to function as an upstream regulator of the MAPK pathway (Fig. 2E). Collectively, these results suggest that Src is involved in the serotonin-mediated activation of PLC 1.
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Calcium-mobilizing stimuli have been shown to control GnRH release within the hypothalamus (Senthilkumaran et al. 2001, Beauvillain & Prevot 2004). Thus, hypothalamic tissue or cell lines are considered to constitute a good model for the study of the relationship between calcium and hormone secretion. On the basis of our biochemical results, we hypothesized that the serotonin-mediated activation of PLC 1 might result in GnRH release via the modulation of intracellular calcium levels. First, in order to gain some insight into the role of PLC 1 in the process of hormone secretion, we pretreated the cells with U73122
[GenBank]
(2 µM) prior to serotonin (1 µM) administration, and then measured calcium levels in the GT1–7 cells. Calcium levels were increased upon stimulation with serotonin (Fig. 3A), and this increase was blocked completely by the administration of U73122
[GenBank]
(Fig. 3B). To confirm the involvement of Src kinase in this process, we pretreated the cells with PP2 (10 µM), and then evaluated the amounts of calcium released. We noted that PP2 pretreatment significantly suppressed the serotonin-induced calcium increase (Fig. 3C). These findings show that both PLC and Src can be associated with serotonin-mediated calcium-related physiological phenomena within the hypothalamus.
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1 binds to c-Src through the SH223 domain
The primary structures of c-Src and PLC 1 are provided in Fig. 4A. The linker region between the X and Y domains in PLC 1 is extended, and harbors two SH2 domains and one SH3 domain. However, Src kinase is comprised of one SH2, one SH3, and one Tyk domain. Tyrosine phosphorylation is considered a crucial step in the activation of PLC 1, and this tyrosine phosphorylation is mediated via the recognition of phosphorylation sites by the SH2 domain. This implies that the protein–protein interactions may be involved in the Src-mediated activation of PLC 1. In order to determine whether c-Src was associated with PLC 1, we performed an immunoprecipitation experiment using the anti-c-Src antibody, followed by blotting with the anti-PLC 1 antibody. Endogenous PLC 1 was first co-immunoprecipitated with c-Src upon serotonin stimulation (Fig. 4B). Then, to determine which PLC 1 domains were responsible for this binding, we conducted an immunoprecipitation experiment following transient transfection with several forms of PLC 1. c-Src was co-immunoprecipitated with wild-type PLC 1 in a serotonin-dependent manner, but this was not observed with the LIM or SH223 domain-deleted PLC 1 mutants (Fig. 4C). These findings indicated that the activation of PLC 1 was mediated by direct interaction with c-Src via the SH223 domain, and also showed that the enzyme activity of PLC 1 was critical with regard to this binding. Focusing on this result, we attempted to determine whether calcium response was affected by transient transfection with several forms of PLC 1. Accordingly, serotonin induced a profound increase in the concentration of intracellular calcium in the cells that had been transfected with wild-type PLC 1. However, this increase was dramatically attenuated in the cells transfected with either LIM or SH223 domain-deleted PLC 1 mutants (Fig. 4D). Plasmid transfection had no significant effect. The expression of PLC 1 was confirmed via western blotting with anti-FLAG antibody (Fig. 4E). These findings support our hypothesis that both the interaction of PLC 1 with c-Src and the enzyme activity of PLC 1 are critical factors in serotonin-mediated calcium release.
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The spontaneous firing of exocytosis vesicles appears to be fundamental to hormone secretion. Its absence in the neurons of 5-hydroxy tryptophan (5-HT) 1B knockout mice (Evrard et al. 1999) indicates that serotonin may be involved in this process. In order to determine whether Src-mediated PLC 1 activation is involved in hormone secretion, we employed an RIA to characterize the effects of PLC on serotonin-mediated GnRH release. Hypothalamic fragments were rapidly isolated from 10-week-old male mice (n=6) and cultured on 96-well tissue culture plates. After equilibration, the tissues were incubated for an additional 1 h in fresh medium containing either vehicle or serotonin (1 µM). The media were then collected and centrifuged. The amounts of GnRH were increased by nearly fourfold upon stimulation with serotonin. Next, to delineate the signaling pathways involved in the serotonin-mediated release of GnRH, we pretreated the hypothalamic fragments with either PP2 (10 µM) or U73122
[GenBank]
(2 µM), and then measured the levels of GnRH. The GnRH levels were substantially reduced in the presence of these two inhibitors (Fig. 5A). To determine whether serotonin-mediated GnRH release was correlated with the activation of PLC 1, we then attempted to determine if the phosphorylation of PLC 1 would be affected by serotonin administration. Although PLC 1 was basally phosphorylated in the hypothalamic tissue, its phosphorylation (Tyr783) increased further as a result of stimulation with serotonin (Fig. 5B). The phosphorylation of Src, MEK1/2, and ERK1/2 was also increased in this experimental condition and then suppressed as a result of pretreatment with PP2. Therefore, even though the serotoninergic system is just one of several neural inputs that innervate the hypothalamus, our findings indicate that the relation of PLC 1 activity with GnRH release in the hypothalamus.
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1 knockdown attenuated the serotonin-induced phosphorylation of ERK1/2 and increase in calcium levels
To determine whether PLC 1 was, indeed, a prerequisite for the serotonin-mediated signal pathway, we employed RNA interference techniques. The knockdown of endogenous PLC 1 resulted in a noticeable inhibition of serotonin-mediated ERK1/2 phosphorylation (Fig. 6A). Src phosphorylation remained unaffected by PLC 1 knockdown, thereby confirming that PLC 1 was a downstream event in serotonin-mediated signaling. Specific PLC 1 suppression was verified via immunoblotting, whereas actin expression remained unaffected. To confirm this in separate experiments, we compared the calcium response in the PLC 1 wild-type cells with that in the PLC 1 knockdown cells. The calcium concentration increased by approximately twofold in the wild-type cells, but was negligible in the knockdown cells (Fig. 6B). These findings, then, provide evidence that endogenous PLC 1 is crucial for serotonin-mediated signaling events.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 is involved in the serotonin-mediated secretion of GnRH. In this study, we determined that the Src-mediated activation of PLC 1 and calcium are instrumental in serotonin-mediated GnRH hormone release.
Src kinase is considered to be a common mediator of both G protein-coupled receptors and tyrosine kinase-coupled receptors (Haendeler et al. 2003, Stope et al. 2004). It has also been reported that Src cascades exploit the Gq-PLC pathway for their activation in the process of calcium-dependent pancreatic exocytosis (Tsunoda et al. 2004), thereby indicating that Src operates downstream of PLC 1. However, others have asserted that Src is upstream of PLC 1 (Bivona et al. 2003). Therefore, there remains some controversy regarding the relationship between Src and PLC 1. In our study, we have determined that c-Src undergoes tyrosine phosphorylation, and is also associated with PLC 1 in the responses of GT1–7 cells to serotonin. Thus, we suggest that c-Src is a novel regulator of PLC 1 function, which appears to mediate serotonin-mediated hormone secretion.
The primary assertion of this study is that PLC 1 mediates some of the effects of serotonin. The role of PLC has previously been evaluated in conjunction with adrenocorticotropic hormone, follicle-stimulating hormone, and pro-lactin (Yajima et al. 1997, Rabadan-Diehl & Aguilera 1998). PLCs appear to be responsible for the release of diacylglycerol and inositol triphosphate, and may also contribute to the observed increases in intracellular calcium levels. The contribution of the PLCs to hormone secretion has raised several questions as to which of the characteristics of PLC are relevant to its participation in hormone secretion. Protein–protein interactions and PLC enzyme activity have been considered to be the primary candidates for this crucial attribute. To date, however, the role of the domains in the process of hormone secretion, that of the enzyme activity of PLC 1 in this process, have not been elucidated adequately. In the present study, we have established that the serotonin-induced release of intracellular calcium occurred to a less profound degree in the cells transfected with SH223 domain-deleted or LIM PLC 1 mutants than in the wild-type PLC 1-transfected cells. Additionally, GnRH release was also suppressed by either the Src or the PLC inhibitor. Collectively, our results suggested that both protein–protein interactions and the enzyme activity of PLC 1 may play crucial roles in serotonin-mediated GnRH secretion.
Many neurotransmitters, including serotonin, gamma-amino butyric acid (GABA), and neuropeptide Y, have been shown to modulate GnRH release (Senthilkumaran et al. 2001). However, it remains unclear as to whether the effects of these neurotransmitters on GnRH secretion are directly exerted on the GnRH neurosecretory neurons, or indirectly exerted, via multisynaptic pathways. GnRH neurons are assumed to be located in the hypothalamic pre-optic area, and distributed in a scattered fashion. The total number of cells expressing luteinizing hormone-releasing hormone mRNA is approximately 800 in postnatal animals (Hoffman & Finch 1986, Wray et al. 1989). These findings indicated that our results might be due to the indirect effects of serotonin on GnRH neurons. Although we were able to demonstrate that PLC 1 phosphorylation was increased by exposure to serotonin in the hypothalamic cultures, we were unable to determine precisely whether the data from the hypothalamic cultures could be used to distinguish between direct and indirect serotonin effects on the GT1–7 cells.
The objective of the present study was to ascertain whether or not the secretion of GnRH from hypothalamic tissue was directly regulated by serotonin and, if so, to determine which molecules were actually involved in this process. Our data showed that serotonin evoked GnRH secretion in the hypothalamus in a PLC-dependent manner, which implies that serotonin may directly stimulate the hypothalamus to release GnRH through the PLC pathway, via the modulation of calcium concentrations. We further demonstrated that serotonin-mediated signaling events were attenuated as the result of PLC 1 knockdown. Overall, these findings implicated PLC 1 in the process of serotonin-mediated GnRH secretion.
In conclusion, we have determined that PLC 1 is activated by serotonin in hypothalamic cells, and serotonin-associated GnRH release can be substantially suppressed by inhibitors of either Src or PLC. These results demonstrate that the Src-PLC 1 pathway exerts a profound influence on serotonin-mediated GnRH secretion. Future studies should focus on the elucidation of the specificity of the PLC isozymes within the context of serotonin-mediated signaling.
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Acknowledgements |
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References |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 inhibit nerve growth factor-induced differentiation of PC12 cells. Journal of Neurochemistry 71 178–185.[Web of Science][Medline]Beauvillain JC & Prevot V 2004 Hypothalamic glial cells and endothelial cells as key regulators of GnRH secretion. Journal de la Societe de Biologie 198 68–72.[Medline]
Becu-Villalobos D & Libertun C 1995 Development of gonadotropin-releasing hormone (GnRH) neuron regulation in the female rat. Cellular and Molecular Neurobiology 15 165–176.[CrossRef][Web of Science][Medline]
Bivona TG, Perez De Castro I, Ahearn IM, Grana TM, Chiu VK, Lockyer PJ, Cullen PJ, Pellicer A, Cox AD & Philips MR 2003 PLC 1 activates Ras on the Golgi apparatus by means of RasGRP1. Nature 424 694–698.[CrossRef][Medline]
Chang JS, Iwashita S, Lee YH, Kim MJ, Ryu SH & Suh PG 1999 Transformation of rat fibroblasts by phospholipase C 1 overexpression is accompanied by tyrosine dephosphorylation of paxillin. FEBS Letters 460 161–165.[CrossRef][Web of Science][Medline]
Chang YJ, Holtzman MJ & Chen CC 2004 Differential role of Janus family kinases (JAKs) in interferon gamma-induced lung epithelial ICAM-1 expression: involving protein interactions between JAKs, phospholipase C , c-src, and STAT1. Molecular Pharmacology 65 589–598.
Chaouloff F 2000 Serotonin, stress and corticoids. Journal of Psychopharmacology 14 139–151.
Cho S, Cho H, Geum D & Kim K 1998 Retinoic acid regulates gonadotropin-releasing hormone (GnRH) release and gene expression in the rat hypothalamic fragments and GT1-1 neuronal cells in vitro. Molecular Brain Research 54 74–84.[Medline]
Choi JH, Hong WP, Yun S, Kim HS, Lee JR, Park JB, Bae YS, Ryu SH & Suh PG 2005 Grb2 negatively regulates epidermal growth factor-induced PLC 1 activity through the direct interaction with tyrosine-phosphorylated PLC 1. Cellular Signalling 17 1289–1299.[CrossRef][Web of Science][Medline]
Doucet E, Pohl M, Fattaccini CM, Adrien J, Mestikawy SE & Hamon M 1995 In situ hybridization evidence for the synthesis of 5-HT1B receptor in serotoninergic neurons of anterior raphe nuclei in the rat brain. Synapse 19 18–28.[CrossRef][Web of Science][Medline]
Evrard A, Laporte AM, Chastanet M, Hen R, Hamon M & Adrien J 1999 5-HT1A and 5-HT1B receptors control the firing of serotoninergic neurons in the dorsal raphe nucleus of the mouse: studies in 5-HT1B knock-out mice. European Journal of Neuroscience 11 3823–3831.[CrossRef][Web of Science][Medline]
Ferris CF, Melloni RH Jr, Koppel G, Perry KW, Fuller RW & Delville Y 1997 Vasopressin/serotonin interactions in the anterior hypothalamus control aggressive behavior in golden hamsters. Journal of Neuroscience 17 4331–4340.
Giusti AF, Carroll DJ, Abassi YA & Foltz KR 1999 Evidence that a starfish egg src family tyrosine kinase associates with PLC 1 SH2 domain at fertilization. Developmental Biology 208 189–199.[CrossRef][Web of Science][Medline]
Grynkiewicz G, Poenie M & Tsien RY 1985 A new generation of calcium indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260 3440–3450.
Haendeler J, Yin G, Hojo Y, Saito Y, Melaragno M, Yan C, Sharma VK, Heller M, Aebersold R & Berk BC 2003 GITI mediates src-dependent activation of phospholipase C by angiotensin II and epidermal growth factor. Journal of Biological Chemistry 278 49936–49944.
Hoffman GE & Finch CE 1986 LHRH neurons in the female C57BL/6J mouse brain during reproductive aging: no loss up to middle age. Neurobiology of Aging 7 45–48.[CrossRef][Web of Science][Medline]
Huang PS, Davis L, Huber H, Goodhart PJ, Wegrzyn RE, Oliff A & Heimbrook DC 1995 An SH3 domain is required for the mitogenic activity of microinjected phospholipase C 1. FEBS Letters 358 287–292.[CrossRef][Web of Science][Medline]
Kang YM, Chen JY, Ouyang W, Qiao JT & Reyes-Vazquez C 2004 Serotonin modulates hypothalamic neuronal activity. International Journal of Neuroscience 114 293–319.
Kim HK, Kim JW, Zilberstein A, Margolis B, Kim JG, Schlessinger J & Rhee SG 1991 PDGF stimulation of inositol phospholipid hydrolysis requires PLC 1 phosphorylation on tyrosine residues 783 and 1254. Cell 65 435–441.[CrossRef][Web of Science][Medline]
Kitagawa D, Tanemura S, Ohata S, Shimizu N, Seo J, Nishitai G, Watanabe T, Nakagawa K, Kishimoto H, Wada T et al. 2002 Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function. Journal of Biological Chemistry 277 366–371.
Kusano K, Fueshko S, Gainer H & Wray S 1995 Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures. PNAS 92 3918–3922.
Moguilevsky JA & Wuttke W 2001 Changes in the control of gonadotropin secretion by neurotransmitters during sexual development in rats. Experimental and Clinical Endocrinology and Diabetes 109 188–195.
Patterson RL, Rossum DB, Ford DL, Hurt KJ, Bae SS, Suh PG, Kurosaki T, Snyder SH & Gill DL 2002 Phospholipase C gamma is required for agonist-induced Ca2+ entry. Cell 111 529–541.[CrossRef][Web of Science][Medline]
Pronina T, Ugrumov M, Adamskaya E, Kuznetsova T, Shishkina I, Babichev V, Calas A, Tramu G, Mailly P & Makarenko I 2003 Influence of serotonin on the development and migration of gonadotropin-releasing hormone neurons in rat fetuses. Journal of Neuroimmunology 15 549–558.
Rabadan-Diehl C & Aguilera G 1998 Glucocorticoids increase vasopressin V1b receptor coupling to phospholipase C. Endocrinology 139 3220–3226.
Sakakibara H, Conti M & Weiner RI 1998 Role of phosphodiesterases in the regulation of gonadotropin-releasing hormone secretion in GT1 cells. Neuroendocrinology 68 365–373.[CrossRef][Web of Science][Medline]
Sawchenko PE, Swanson LW, Steinbusch HW & Verhofstd AA 1983 The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat. Brain Research 277 355–360.[CrossRef][Web of Science][Medline]
Senthilkumaran B, Okuzawa K, Gen K & Kagawa H 2001 Effects of serotonin, GABA and neuropeptide Yon seabream gonadotropin releasing hormone in vitro from preoptic-anterior hypothalamus and pituitary of red seabream, Pagrus major. Journal of Neuroendocrinology 13 395–400.[CrossRef][Web of Science][Medline]
Smith MR, Liu YL, Kim SR, Bae YS, Kim CG, Kwon KS, Rhee SG & Kung HF 1996 PLC 1 src homology domain induces mitogenesis in quiescent NIH 3T3 fibroblasts. Biochemical and Biophysical Research Communications 222 186–193.[CrossRef][Web of Science][Medline]
Stahl ML, Ferenz CR, Kelleher KL, Kriz RW & Knopf JL 1988 Sequence similarity of phospholipase C with the non-catalytic region of src. Nature 332 269–272.[CrossRef][Medline]
Stope MB, vom Dorp F, Szatkowski D, Bohm A, Keiper M, Nolte J, Oude Weernink PA, Rosskopf D, Evellin S, Jakobs KH et al. 2004 Rap2B-dependent stimulation of phospholipase C- by epidermal growth factor receptor mediated by c-src phosphorylation of RasGRP3. Molecular and Cellular Biology 24 4664–4676.
Suh PG, Ryu SH, Moon KH, Suh HW & Rhee SG 1988 Cloning and sequence of multiple forms of phospholipase C. Cell 54 161–169.[CrossRef][Web of Science][Medline]
Tolloczko B, Turkewitsch P, Choudry S, Bisotto S, Fixman ED & Martin JG 2002 Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4, 5-bisphosphate. American Journal of Physiology. Lung Cellular and Molecular Physiology 282 L1305–L1313.
Tsunoda Y, Yoshida H & Nozu F 2004 Receptor-operated calcium influx and its association with the src family in secretagogues-stimulated pancreatic acini. Biochemical and Biophysical Research Communications 314 916–924.[CrossRef][Web of Science][Medline]
Wada K, Hu L, Mores N, Navarro CE, Fuda H, Krsmanovic LZ & Catt KJ 2006 Serotonin (5-HT) receptor subtypes mediate specific modes of 5-HT-induced signaling and regulation of neurosecretion in gonadotropin-releasing hormone neurons. Molecular Endocrinology 20 125–135.
Woller MJ, McDonald JK, Reboussin DM & Terasawa E 1992 Neuropeptide Y is a neuromodulator of pulsatile luteinizing hormone-releasing hormone release in the gonadectomized rhesus monkey. Endocrinology 130 2333–2342.
Wray S, Grant P & Gainer H 1989 Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. PNAS 86 8132–8136.
Yajima Y, Uchino K, Ito H & Kawashima S 1997 Mastoparan-stimulated prolactin secretion in rat pituitary GH3 cells involves activation of Gq/11 proteins. Endocrinology 138 1949–1958.
Received 4 January 2006
Received in final form 4 May 2006
Accepted 26 May 2006
Made available online as an Accepted Preprint 23 May 2006
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223), and c-Src. (B) The binding of endogenous c-Src with PLC 
