甲状旁腺素非PLC依赖PKC通路激活增强成骨细胞CITED1表达

Activation of phospholipase C-independent protein kinase C signaling pathway of parathyroid hormone enhances CITED1 expression in mouse osteoblasts

目的：通过相关信号通路屏蔽和基因表达分析，探讨甲状旁腺素（PTH）非PLC依赖PKC信号转导途径（PTH/nonPLC/PKC）的功能，分析其对骨代谢的影响。方法取2-3 d C57BL乳鼠颅盖骨分离培养成骨细胞，取贴壁生长的第1代细胞，随机分为4组：100 nmol/L[Gly1, Arg19]hPTH（1-28）（简称GR（1-28））＋10 nmol/L RP-cAMP；10 nmol/L[Gly1, Arg19]hPTH（1-34）（简称GR（1-34））＋10 nmol/L RP-cAMP；10 nmol/L PTH（1-34）及空白对照组加入等体积的0.1%三氟乙酸（trifluoroacetic acid, TFA），作用4 h后，提取各组总RNA，行小鼠全基因组表达谱芯片分析，筛选出可能与nonPLC/PKC信号转导通路相关的差异表达基因，并进行相关通路分析。RT-PCR筛选及验证上述差异表达基因。培养MC3T3-E1细胞，分为4组：GR（1-28）＋RP-cAMP；GR（1-34）＋RP-cAMP；GR（1-34）＋RP-cAMP＋100 nmol/L Go6983及空白对照组，RT-PCR法验证比较GR（1-28）和GR（1-34）引起的基因表达变化情况。结果倒置相差显微镜观察示，培养7 d见细胞排列紧密，为三角形或多边形，呈铺路石样；细胞培养14 d ALP染色可见胞质中出现蓝染颗粒，成骨诱导培养28 d茜素红染色可见红色矿化结节形成。基因芯片分析结果中筛选出与PTH的nonPLC/PKC信号转导通路相关性最大的56个基因，进行RT-PCR验证后发现CITED1的表达量在GR（1-34）＋RP-cAMP组显著高于GR（1-28）＋RP-cAMP组及空白对照组，但小于PTH（1-34）组（P〈0.05）。MC3T3-E1细胞RT-PCR验证的结果与其一致，在阻断cAMP/PKA信号通路后，仅CITED1基因表达量在GR（1-28）和GR（1-34）刺激时存在显著不同，且加入PKC抑制剂（Go6983）后，表达差异消失。结论 PTH的nonPLC/PKC信号转导通路的激活能够使得成骨细胞CITED1的表达量明显升高，介导PTH对成骨代谢的作用。该途径不依赖PLC和PKA信号的激活。

Objective To explore the functions of phospholipase C （PLC）-independent protein kinase C signaling pathway （PTH/nonPLC/PKC） of parathyroid hormone （PTH） and its role in bone metabolism. Methods Osteoblasts isolated from the calvaria of 2-or 3-day-old C57BL mice, identified by alkaline phosphatase staining and Alizarin red staining, were treated for 4 h with 100 nmol/L[Gly1, Arg19]hPTH（1-28） plus 10 nmol/L RP-cAMP, 10 nmol/L[Gly1, Arg19]hPTH（1-34） plus 10 nmol/L RP-cAMP , 10 nmol/L PTH（1-34）, or and 0.1% trifluoroacetic acid （TFA）. The total RNA was then isolated for screening differentially expressed genes related to PTH/nonPLC/PKC pathway using Affymetrix mouse 12x135K gene expression profile microarray, and the identified genes were confirmed by real-time quantitative PCR. MC3T3-E1 cells treated with[Gly1, Arg19]hPTH（1-28）＋RP-cAMP,[Gly1, Arg19]hPTH（1-34）＋RP-cAMP,[Gly1, Arg19]hPTH（1-34）＋RP-cAMP＋100 nmol/L Go6983, or 0.1%TFA were also examined for GR（1-28）-or GR（1-34）-mediated gene expression changes using real-time quantitative PCR. Results Alizarin red staining visualized red mineralized nodules in the osteoblasts at 28 days of culture. According to the genechip results, we selected 56 target genes related to PTH/nonPLC/PKC pathway, among which CITED1 showed higher expressions in[Gly1, Arg19]hPTH（1-34）＋RP-cAMP group than in both the control group and[Gly1, Arg19]hPTH（1-28）＋RP-cAMP group （P〈0.05）, and its expression was the highest in PTH（1-34） group （P〈0.05）. RT-PCR of MC3T3-E1 cells yielded consist results with those in the primary osteoblasts, and the cells treated with Go6983 （a PKC inhibitor） did not show GR（1-28）- or GR（1-34）-mediated differential expression of CITED1. Conclusion The activation of PLC-independent protein kinase C signaling pathway of PTH enhances the expression of CITED1 in mouse osteoblasts to mediate the effect of PTH on bone metabolism, and this pathway is not dependent on the activation of PLC or PKA signaling.