Role of nuclear factor of activated T-cells in regulating ADAMTS -4 promoter activity in nucleus pulposus cells

Abstract

Objective To observe the regulatory effect of nuclear factor of activated T-cells( NFAT)-1,4,5 on ADAMTS-4 promoter activity in nucleus pulposus cells,so as to discuss the underlying molecular mechanism of intervertebral disc degeneration. Methods The ADAMTS-4-pβ-gal-Basic was inserted into PGL3-Basic vector after double digestion by the restriction enzyme XhoⅠ and HindⅢ. The purified PGL3-ADAMTS-4 plasmid was obtained through screening. Rat nucleus pulposus cells were cultured in vitro and,via Lipofectamine2000 transfection reagent,transfected with NFAT-1,NFAT-4,NFAT-5 or DN-TonEBP expressing plasmids with or without appropriate backbone vector and 175 ng ADAMTS-4 promoter. The normal cells were taken as controls and transfected cells were taken as treatment group. Dual-LuciferaseTMreporter assay system was used to detect the effect of NFAT-1,4,5( TonEBP) on ADAMTS-4 promoter activity. Results We successfully constructed PGL3-ADAMTS-4 plasmid and identified two NFAT-Bind elements in the promoter. Compared with the control group,ADAMTS-4 promoter activity was significantly inhibited in NFAT-1 group( P 0. 05). While the activities of ADAMTS-4 promoter in NFAT-4,NAFT-5 and DN-TonEBP groups showed no significant changes( P 0. 05). Conclusion NFAT-1 can regulate ADAMTS-4 expression,which may play a role in modulating aggrecan content in the intervertebral disc physiology and / or intervertebral disc degeneration,providing a new strategy for biological treatment of intervertebral disc degeneration.