The Role of Exogenous Stresses on the Proliferation of Intervertebral Disk Cells
Authors: Dimozi A. , Mavrogonatou E. , Pratsinis H. , Kletsas D. 
Published: June 2012

Introduction The native microenvironment of intervertebral disk (IVD) cells is characterized by adverse physicochemical conditions. Low cellularity and proliferation rate is a hallmark of the disk, possibly resulting from the absence of vascularization, which leads to the insufficient nutrition of its cells. Nutrients diffuse from the vessels of the endplates, which may sometimes be several mm away from the IVD and especially from nucleus pulposus cells. On the other hand, the IVD is confronted with several stresses, that is, mechanical load, a constant higher osmolality in comparison to other organs of the body, and oxidative stress. Our aim in the present study was to investigate the effect of the aforementioned exogenous stresses on disk cells’ proliferation and the underlying mechanisms of the observed phenomena. Materials and Methods Primary cultures of IVD cells were established. Cellular proliferation was estimated by tritiated thymidine and bromodeoxyuridine incorporation into DNA, as well as by direct cell counting. Western blot analysis was used to examine the activation of several signaling pathways. Cell cycle analysis was performed by flow cytometry. DNA damage and repair was assessed by comet assay, immunofluorescence for γH2A.X, and the host cell reactivation assay. Finally, reactive oxygen species’ production was measured using DCFH-DA. Results We showed that hyperosmotic treatment reduced nucleus pulposus cells’ proliferation by activating the G2 and G1 cell cycle checkpoints. p38 MAPK was found to participate in the manifestation of the G2 arrest under conditions of increased osmolality, since inhibition of its activity released the cells from G2 phase into mitosis. High osmolality resulted in the ATM-mediated phosphorylation of p53 on Ser15, the upregulation of p21WAF1 and the hypophosphorylation of the retinoblastoma protein in accordance to the observed G1 arrest. Furthermore, comet assay revealed the presence of DNA damage after hyperosmotic treatment, possibly attributed to the abrupt alterations in chromatin configuration observed early after exposure of the cells to this stress. Under these conditions, the histone H2A.X was phosphorylated on Ser139, a classical marker of DNA double strand breaks. In addition, when the DNA repair efficiency of the cells was directly measured by a host cell reactivation of luciferase activity assay, it was found to be significantly increased under hyperosmotic pressure. To shed light in the origin of the response, an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea were used. In contrast to urea, high salt and sorbitol were found to activate the same molecular pathways, indicating that the osmo-regulatory response of nucleus pulposus cells stems rather from cell volume alterations mediated by hypertonicity than from elevated intracellular ionic concentration. The antiproliferative effect of high osmolality was confirmed by the reduced growth factor-mediated novel DNA synthesis and ERK and Akt activation. On the other hand, the recurrent mechanical load of the cells resulted in the activation of MEK/ERK and PI3K/Akt pathways, though no actual effect on cellular proliferative potential was observed. Finally, oxidative stress led to cell cycle arrest through p38MAPK, DNA damage, and the subsequent induction of the ATM-p53-p21WAF1-pRb axis. Overall, oxidative stress was shown to induce similar intracellular mechanisms to those induced by osmotic stress and to also restrain cellular proliferation. Conclusion Our findings indicate that exogenous stresses impose a strict control in IVD cells’ proliferation, thus possibly participating in the maintenance of tissue homeostasis. Understanding disk cells’ physiology—especially after taking into consideration the conditions of their in vivo physicochemical environment—has important implications for targeted treatments against disk degeneration and back pain, as those cells are utterly responsible for the retention of the dynamic equilibrium between synthesis and degradation of the extracellular matrix. Supported by the European Union FP7 (“GENODISC”, grant agreement no. HEALTH-F2-2008-201626) I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared