INTRODUCTION: Osmotic fluctuations resulting from intervertebral disc's specific physicochemical environment, daily activities or degeneration are an every day experience for nucleus pulposus cells.
METHODS: Proliferative potential was evaluated by growth curves and 3H‐thymidine incorporation, while cell cycle distribution was assessed using one‐ and two‐color flow cytometry. Western blot analysis was used for the estimation of proteins' expression levels and phosphorylation status. In order to investigate the possible genotoxic effect of high osmolality, we performed single‐cell gel electrophoresis and immunofluorescence experiments for lamin A/C, whereas the capacity of the cells for DNA repair was directly measured through a host cell reactivation assay. Finally, siRNA‐mediated knocking down of p53 was carried out.
RESULTS AND DISCUSSION: Hyperosmotic treatment reduces cellular proliferation by activating the G2 and G1 cell cycle checkpoints. The G2 arrest was shown to be p38‐dependent, while the G1 delay was established by the phosphorylation of p53, the up‐regulation of p21WAF1 and the hypophosphorylation of the retinoblastoma protein (pRb). High osmolality also decreased the response of the cells to growth factors (PDGF and IGF‐I) and the activation of intracellular signaling pathways. Hyperosmolality‐induced DNA damage was evidenced by the existence of comet tails and was verified by the phosphorylation and accumulation of H2A.X. In parallel, the cells' DNA repair efficiency was significantly increased under hyperosmotic pressure. p53 was necessary for the activation of the G1 checkpoint, since its repression abolished the hypophosphorylation of pRb and abrogated the G1 arrest provoked by hyperosmotic stress. Most importantly, p53 and the consequent G1 arrest were found to participate in the DNA repair pathway, as their inhibition led to an enhanced and prolonged H2A.X phosphorylation. Supported by the EU (“Genodisc”) and the AO Foundation