Abstract
AbstractSenescence, 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 that can 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. Unlike iAF cells, NP cells treated with TNFα accumulated more intracellular ROS and secreted more H2O2. Following TNFα treatment, only iAF cells had increased expression of the superoxide scavengersSOD1andSOD2whereas NP cells had increasedNOX4gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2(50 μM) induced senescence, however unlike TNFα, H2O2did not induce degenerative-like changes as there was no difference inCOL2, ACAN, MMP13, orIL6gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling, iAF and TNFα-treated NP cells were co-cultured. In contact co-culture the NP cells did induce 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 H2O2and cytokines (TNFα). Further studies will investigate if human disc cells respond similarly.
Publisher
Cold Spring Harbor Laboratory