Effects And Underlying Mechanisms Of GHSR-1a Signaling Pathway On The Proliferation Of Pituitary Somatotrophinoma Cells

Objective The purpose of this study was to investigate the relationship between the ghrelin or GHSR-1a mRNA levels and clinical characteristics and to confirm the effect of Gsp mutations on ghrelin/GHSR-la system in human GH-secreting pituitary adenomas.Methods T The expression levels of ghrelin, GHSR-1a mRNA were determined by SYBR green real-time fluorescent quantitative PCR. The Gsp mutations in 43 cases of human GH-secreting pituitary adenomas were detected using PCR-DNA direct sequencing analysis. Clinical data were obtained from the medical records of 43 acromegalic patients who had GH and IGF-1 assays in the same laboratory.Results The expression of GHSR-la correlated positvely with tumor size (R=0.411, p=0.006) and invasiveness (p<0.05). In contrast, ghrelin mRNA levels correlated positively only with tumor size (R=0.331, p=0.030) but not with tumor invasiveness (p>0.05). The expression level of GHSR-1a mRNA was significantly higher in Gsp positive adenomas than in negative adenomas (p<0.05). Whereas, there was no significant difference in the expression of ghrelin mRNA between Gsp mutation-positive and-negative adenomas (p>0.05). Additionally there was a significant positve correlation between the ghrelin and GHSR-la mRNA expression levels in Gsp positive (R=0.553,p=0.040) or negative adenomas (R=0.489,p=0.007).Conclusions GHSR-1a correlated positively with tumor size and invasiveness, while ghrelin correlated positively only with tumor size. Gsp mutations may upregulate the expression of GHSR-la mRNA and have no effect on ghrelin mRNA levels in human GH-secreting pituitary adenomas. Objective To explore the proliferative effect of Ghrelin-mediated signaling pathways and the detailed signal transduction mechanism in rat GH3 cell line.Methods The effect of Ghrelin/GHSR-la on cell proliferatin was studied using MTT in defferent concentration of Ghrelin; and the activation of ERK1/2 was studied with western blotting method.Results The A490 and the pERK1/2 expression level reached the hightest piont with Ghrelin at the concentration of 10"7M; and the stimulation of pERK1/2 by Ghrelin was in a dose-dependent manner.Conclusions Ghrelin/GHSR-la has a robust dose dependent positive effect on GH3 cell proliferation through the phosphorylation of ERK1/2 Objective To investigate the detailed signal transduction mechanism underlyung the Ghrelin-induced rat pituitary somatotroph GH3 cell proliferation.Methods Inhibitors of the extracelluar signal-regulated kinase kinasel/2(MEK1/2), protein kinase c (PKC), protein kinase A, phosphatidynositol 3-kinase(PI3K) or tyrosine phosphatase pathways was used to investigate the effect of Ghrelin on pERKl/2 activation. Then GH3 cells were exposure to four kind of selective PKC isoform inhibitors respectively, followed by detecting the expression level of pERKl/2 with Western blotting method, and analyzed which isoforms of PKC mediated the activation of pERKl/2 induced by gherlin.Results The elevated pERK1/2 expression level induced by Ghrelin was significantly reduced by the PKC inhibiter GF109203x, but not by PKA inhibitor H-89, PI3K inhibitor Wortmanin or tyrosine phosphatase inhibitor Genistein. The upregulation of pERK1/2 induced by Ghrelin was inhibited by G66983 or PKA, whereas G66976 or BAPTA-AM exert no effect on Ghrelin-induced ERK1/2 activation.Conclusions nPKC isoforms play a key role in the activation of ERK1/2 induced by ghrelin. Objective The aim of the present study was to investigate the potential role of protein kinase C (PKC) isoforms in mediating Ghrelin-induced stimulation of pERK1/2 and further clarify the precise intracellular signal transduction mechanism in rat GH3 pituitary somatotroph cells.Methods The siRNAs of three nPKC isoforms were transfected into GH3 cells and the effects of siRNA depletion of specific PKC isoforms on Ghrelin-induced ERK1/2 phosphorylation were evaluated. Sequently, co-immunoprecipitation was used to investigate the interaction between the screened PKC isoform and raf-1 protein.Results siRNA experiments targeting specific nPKC isoforms indicated that Ghrelin-induced ERK1/2 activation was strongly inhibited by PKCδ-siRNA (p<0.05), whereas PKCε-siRNA or PKCθ-siRNA had no significant effect on this signaling pathway (p>0.05). Increased raf-1 protein interacting with PKCδwas shown by Co-immunoprecipitation experiment upon Ghrelin stimulation.Conclusions The present research confirms that the upregulation pERK1/2 expression in response to Ghrelin requires activation of PKCδ/raf-1-dependent signaling pathway and suggests that reduction of PKCδactivity might be an effective therapeutic target to inhibit the proliferation and differentiation of GH3 cells.