Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: Role of PKC and p38 MAPK

Abstract

We previously reported that hypoxia induces the proliferation of cultured mesangial cells mediated by the stimulation of intracellular calcium and the activation of protein kinase C (PKC). In the present study, we examined the roles of mesangial cell specific growth factors (platelet-derived growth factor and endothelin-1) and osteopontin (OPN) in hypoxia-induced proliferation of mesangial cells. In addition, we determined the effect of hypoxia on p38 mitogen-activated protein (MAP) kinase activity and the roles of both PKC and p38 MAP kinase in hypoxia-induced alterations in OPN and mesangial cell growth. Quiescent cultures of mesangial cells were exposed to hypoxia (3% O2) or normoxia (18% O2) in a serum-free medium, and [3H]-thymidine incorporation, OPN protein and mRNA expression, and p38 MAP kinase activity were assessed. Hypoxic-conditioned medium mimicked the effect of hypoxia on thymidine incorporation, suggesting the release of diffusable growth promoting factor(s) by hypoxia. Neither anti-endothelin-1 nor anti-platelet-derived growth factor-neutralizing antibodies had an effect on increased thymidine incorporation induced by hypoxia. However, blocking the effects of OPN either with anti-OPN antibody or its beta3 integrin receptor antibody completely prevented the hypoxia-induced increase in thymidine incorporation. Hypoxia also stimulated OPN protein and mRNA levels. Hypoxia caused an acute activation of p38 MAP kinase, which was inhibited by both verapamil and an inhibitor of PKC (calph C). PKC inhibitor and an inhibitor of p38 MAP kinase (SB203580) reduced the hypoxia-induced stimulation of both OPN and cell growth. These studies provide, to our knowledge, the first evidence demonstrating the role of OPN in hypoxia-induced proliferation of mesangial cells. In addition, hypoxia causes an activation of p38 MAP kinase in a calcium- and PKC-dependent manner, and the activation of PKC and p38 MAP kinase appears to be involved in the stimulation of both OPN and mesangial cell proliferation induced by hypoxia.