Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells

• Published in: Gene 2019-07, Vol.704, p.15-24
• Main Authors: Lin, Wenbo, Shi, Changgui, Wang, Weiheng, Wu, Huiqiao, Yang, Chen, Wang, An, Shen, Xiaolong, Tian, Ye, Cao, Peng, Yuan, Wen
• Format: Article
• Language: English
• Subjects: Animals; Calcineurin; Calcium - pharmacology; CCN2; Cells, Cultured; Connective Tissue Growth Factor - genetics; Connective Tissue Growth Factor - metabolism; Gene Expression Regulation - drug effects; Homeostasis - drug effects; Homeostasis - genetics; Male; NFAT; NFATC Transcription Factors - physiology; Nucleus Pulposus - drug effects; Nucleus Pulposus - metabolism; Nucleus pulposus cells; Osmolar Concentration; Osmolarity; Rats; Rats, Sprague-Dawley; Signal Transduction - drug effects; Signal Transduction - genetics; TonEBP; Water-Electrolyte Balance - drug effects; Water-Electrolyte Balance - physiology; Water-Electrolyte Imbalance - genetics; Water-Electrolyte Imbalance - metabolism
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2019.04.020

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. 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. 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. 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. •Hyperosmolarity decreases CCN2 expression in nucleus pulposus cells via TonEBP.•Ca2+-calcineurin signaling increased CCN2 expression in nucleus pulposus cells.•NFATs regulate CCN2 promoter activity in nucleus pulposus cells.•Expression of CCN2 is tightly regulated in nucleus pulposus cells.