Senescent response in inner annulus fibrosus cells in response to TNFα, H2O2, and TNFα-induced nucleus pulposus senescent secretome

Abstract

Senescence, particularly in the nucleus pulposus (NP) cells, has been implicated in the pathogenesis of disc degeneration, however, the mechanism(s) of annulus fibrosus (AF) cell senescence is still not well understood. Both TNFα and H2O2, have been implicated as contributors to the senescence pathways, and their levels are increased in degenerated discs when compared to healthy discs. Thus, the objective of this study is to identify factor(s) that induces inner AF (iAF) cell senescence. Under TNFα exposure, at a concentration previously shown to induce senescence in NP cells, bovine iAF cells did not undergo senescence, indicated by their ability to continue to proliferate as demonstrated by Ki67 staining and growth curves and lack of expression of the senescent markers, p16 and p21. The lack of senescent response occurred even though iAF express higher levels of TNFR1 than NP cells. Interestingly, iAF cells showed no increase in intracellular ROS or secreted H2O2 in response to TNFα which contrasted to NP cells that did. Following TNFα treatment, only iAF cells had increased expression of the superoxide scavengers SOD1 and SOD2 whereas NP cells had increased NOX4 gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2 (50 μM) induced senescence, however unlike TNFα, H2O2 did not induce degenerative-like changes as there was no difference in COL2, ACAN, MMP13, or IL6 gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells may have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling by senescent NP cells, iAF and TNFα-treated NP cells were co-cultured. In contact co-culture the NP cells induced iAF senescence. Thus, senescent NP cells may secrete soluble factors that induce degenerative and senescent changes within the iAF. This may contribute to a positive feedback loop of disc degeneration. It is possible these factors may include H2O2 and cytokines (such as TNFα). Further studies will investigate if human disc cells respond similarly.