Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION

The objective of the study was to investigate how inflammatory cytokines, IL-1 beta, and TNF-alpha control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-kappa B and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKK beta significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38 alpha/beta 2/delta, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38 gamma played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1 beta as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1 beta and TNF-alpha activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.