Inflammatory Cytokines Associated with Degenerative Disc Disease Control Aggrecanase-1 (ADAMTS-4) Expression in Nucleus Pulposus Cells through MAPK and NF-κB

We investigated TNF-alpha and IL-1 beta regulation of ADAMTS-4 expression in nucleus pulposus (NP) cells and its role in aggrecan degradation. Real-time quantitative RT-PCR, Western blotting, and transient transfections with rat NP cells and Lentiviral silencing with human NP cells were performed to determine the roles of MAPK and NF-kappa B in cytokine-mediated ADAMTS-4 expression and function. ADAMTS4 expression and promoter activity increased in NP cells after TNF-alpha and IL-1 beta treatment. Treatment of cells with MAPK and NF-kappa B inhibitors abolished the inductive effect of the cytokines on ADAMTS4 mRNA and protein expression. Although ERK1, p38 alpha, p38 beta 2, and p38 gamma were involved in induction, ERK2 and p38 delta played no role in TNF-alpha-dependent promoter activity. The inductive effect of p65 on ADAMTS4 promoter was confirmed through gain and loss-of-function studies. Cotransfection of p50 completely blocked p65-mediated induction. Lentiviral transduction with shRNA plasmids shp65, shp52, shIKK-alpha, and shIKK-beta significantly decreased TNF-alpha-dependent increase in ADAMTS-4 and -5 levels and aggrecan degradation. Silencing of either ADAMTS-4 or -5 resulted in reduction in TNF-alpha-dependent aggrecan degradation in NP cells. By controlling activation of MAPK and NF-kappa B signaling, TNF-alpha and IL-1 beta modulate expression of ADAMTS-4 in NP cells. To our knowledge, this is the first study to show nonredundant contribution of both ADAMTS-4 and ADAMTS-5 to aggrecan degradation in human NP cells in vitro.