Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells
Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells
复制标题
渗透压和钙调节髓核细胞中结缔组织生长因子 (CTGF/CCN2) 的表达
DOI：
10.1016/j.gene.2019.04.020
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发表时间：
2019-07-01
期刊：
影响因子：
3.5
通讯作者：
Yuan, Wen
中科院分区：
文献类型：
--
作者：
Lin, Wenbo;Shi, Changgui;Yuan, Wen
目的：本研究的目的是验证以下假设：结缔组织生长因子 (CTGF/CCN2) 是维持髓核 (NP) 基质稳态所必需的关键分子，其表达受 NP 细胞中渗透压和细胞内钙的调节。方法：使用定量实时 PCR 和蛋白质印迹评估 CCN2 的基因和蛋白表达水平。通过转染和双荧光素酶测定来测量高渗透压、张力增强剂结合蛋白 (TonEBP) 和 Ca2+-钙调神经磷酸酶 (Cn)-NFAT 信号对 CCN2 启动子活性的影响。结果：在高渗培养基中培养，NP 细胞中 CCN2 启动子活性、基因和蛋白表达水平显着降低。利用JASPAR数据库分析人CCN2启动子的构建，发现保守的TonE和NFAT结合位点。然后我们研究了 TonEBP 是否控制 CCN2 表达。 TonEBP 在 NP 细胞中的强制表达表明，TonEBP 负向调节 CCN2 启动子活性，而抑制 TonEBP 则诱导 CCN2 启动子活性和表达。然后我们检查了 Ca2+-Cn-NFAT 信号传导是否参与了 CCN2 表达的调节。 CCN2 报告基因与单个 NFAT1-4 表达质粒和/或钙调神经磷酸酶 A/B 构建体的共表达表明该信号通路在 NP 细胞中 CCN2 表达的调节中发挥作用。结论：这些研究结果表明，NP 细胞中 CCN2 的表达受到 NFAT 家族通过涉及激活剂（Ca2+-Cn-NFAT 信号传导）和抑制剂（高渗透压-TonEBP）的信号传导通路网络的调节。分子。
Objective: The objective of our study was to verify the hypothesis that the expression of connective tissue growth factor (CTGF/CCN2), a key molecule essential for the maintenance of nucleus pulposus (NP) matrix homeostasis, is regulated by osmolarity and intracellular calcium in NP cells.Methods: Gene and protein expression levels of CCN2 were assessed using quantitative real-time PCR and western blot. Transfections and dual luciferase assays were performed to measure the effect of hyperosmolarity, tonicity enhancer binding protein (TonEBP) and Ca2+-calcineurin (Cn)-NFAT signaling on CCN2 promoter activity.Results: Cultured in hyperosmotic media, there was a significant decrease in the levels of CCN2 promoter activity, gene and protein expression in NP cells. The JASPAR database was used to analyze the construction of human CCN2 promoter, we found conserved TonE and NFAT binding sites. We then investigated whether TonEBP controlled CCN2 expression. Forced expression of TonEBP in NP cells showed that TonEBP negatively regulated CCN2 promoter activity, while suppression of TonEBP induced CCN2 promoter activity and expression. We then examined if Ca2+-Cn-NFAT signaling participated in the regulation of CCN2 expression. Co-expression of CCN2 reporter with individual NFAT1-4 expression plasmids and/or calcineurin A/B constructs suggested this signaling pathway played a role in the regulation of CCN2expression in NP cells.Conclusions: Results of these studies illustrated that the expression of CCN2 in NP cells was regulated by the NFAT family through a signaling pathway network involving both activator (Ca2+-Cn-NFAT signaling) and suppressor (Hyperosmolarity-TonEBP) molecules.