PKCδ在GH3细胞ghrelin/pERK1/2信号通路中的作用及其机制

Involvement of PKCδ in the ghrelin/pERK1/2 signaling pathway in rat pituitary somatotroph GH3 cells

目的 观察在垂体腺瘤GH3细胞ghrelin/pERK1/2信号通路中发挥关键作用的蛋白激酶C（PKC）亚型,探讨该途径的详细信号转导机制.方法 4种不同PKC亚型抑制剂分别作用于GH3细胞,Western blot检测pERK1/2表达,初步判定在上述信号通路中发挥重要作用的PKC亚型 然后设计、合成、转染针对初步筛选出的PKC亚型的特异性siRNA,检测pERK1/2表达,从而明确介导该信号途径的关键PKC亚型.免疫共沉淀研究与该亚型可能相互作用的蛋白激酶.结果 综合分析4种抑制剂对pERK1/2表达的影响,发现nPKC在该信号通路中起关键作用.PKCδ沉默后,ghrelin诱导的pERK1/2高表达明显降低（P＜0.05）,而PKCε、PKCθ沉默后对ghrelin诱导的pERK1/2高表达则无明显影响（P＞0.05）.ghrelin作用后,与PKCδ结合的raf-1蛋白明显增加.结论 PKCδ为调控GH3细胞ghrelin/pERK1/2信号通路的关键信号分子,并且可能在raf-1水平与受体酪氨酸激酶/ERK1/2信号通路发生＂交叉通讯＂.

Objective To investigate the potential role of protein kinase C （PKC） isoforms in mediating ghrelin-induced stimulation of pERK1/2 and further elucidate the precise intracellular signal transduction mechanism in rat GH3 pituitary somatotroph cells. Methods GH3 cells were exposed to four kinds of selective PKC isoform inhibitors respectively. The expression level of pERK1/2 was detected by using Western blotting, and isoforms of PKC mediating the activation of pERK1/2 induced by gherlin were analyzed. The effects of small siRNA depletion of specific PKC isoforms on ghrelin-induced ERK1/2 phosphorylation were evaluated. Co-immunoprecipitation was used to investigate the interaction between the screened PKC isoform and raf-1 protein. Results nPKC isoforms were involved in the activation of ERK1/2 induced by ghrelin. siRNA experiments targeting specific PKC 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 PKC was shown by co-immunoprecipitation experiment upon ghrelin stimulation （ P 〈 0. 05 ）. Conclusion The up-regulation of the pERK1/2 expression in response to ghrelin requires activation of PKC/raf-1-dependent signaling pathway.