p38 MAPK Signalling in Endothelial Apoptosis

University dissertation from Department of Laboratory Medicine, Lund University

Abstract: Endothelial apoptosis plays an important role in atherosclerosis, and a direct proapoptotic effect of chemotherapeutics on the tumour vasculature, emphasizes the great potency of antiangiogenic therapy in the treatment of cancer. We investigated signalling pathways in endothelial apoptosis induced by the inflammatory cytokine tumour necrosis factor alpha (TNF), the main target of which is the endothelium, and by the anticancer drug doxorubicin. Doxorubicin (also called Adriamycin) is a widely used anthracycline against a broad range of tumours and has been shown to exert its antitumoural effect via directly targeting the tumour vasculature. However, endothelial apoptosis is also implicated in doxorubicin-mediated cardiotoxicity, an undesirable side effect of the cancer therapy.

The main objective was to elucidate the role of p38 mitogen activated kinase (p38) in such systems, using the endothelial cell line EA.hy926. We found that p38 plays an important proapoptotic role in both, TNF- and doxorubicin-induced apoptosis. In TNF-induced cell death, p38 mediates phosphorylation of Bcl-xL, which is followed by Bcl-xL degradation in the proteasomes. Furthermore, we observed that p38 signalling inhibits the MEK/ERK survival pathway and the phosphorylation of its downstream target Bad, which ocurris through an increased activatity of the protein phosphatase 2A (PP2A). In addition to pharmacological inhibition, we used lentiviral vector transfection of EA.hy926 cells to express a dominant negative mutant Flag-p38 MAPK harboring T180A and Y182 F amino acid substitutions. Similarly to the TNF- induced cell death, we found a p38-mediated downregulation of Bcl-xL in cells undergoing doxorubicin-induced apoptosis. In contrast, MEK/ERK signalling appeared to be proapoptotic in this system. Interestingly, p38 signalling inhibited the PI3-K/Akt survival pathway and the phosphorylation of Bad. Results from a phosphatase assay showed that doxorubicin-induced p38 activity in endothelial cells could maintain PP2A activity at a normal level and thereby prevents phosphorylation of Akt and Bad. In summary, p38 exerts a strong proapoptotic action in endothelial cells exposed to TNF or doxorubicin by simultaneously decreasing the level of antiapoptotic Bcl-xL and increasing the level of proapoptotic Bad.