p38 MAPK regulates G1-S transition in hypoxic cardiac fibroblasts

Cardiac fibroblast hyperplasia associated with augmented matrix production is central to wound healing following myocardial injury. Regulation of the cardiac fibroblast cell cycle by factors in the diseased myocardium that can potentially modify the hyperplastic response of cardiac fibroblasts has, however, not been investigated. We examined the regulation of the cardiac fibroblast cell cycle by hypoxia, a major constituent of myocardial ischemia. Significant reductions in DNA synthesis and cell number, and flow cytometry indicated decreased G1/S progression in hypoxic adult rat cardiac fibroblasts. Western blot analysis showed reduced levels of cyclin D and cyclin A, induction of p27 and hypophosphorylation of Rb under hypoxia. Skp2, which targets p27 for degradation, was significantly lower and inversely related to p27 protein levels in hypoxic cells. Marked p38 MAPK activation was observed under hypoxia and its inhibition using SB203580 reversed the effects of hypoxia on DNA synthesis, cell cycle phase distribution, p27, and cyclin D1 but not cyclin A. Interestingly, a 2-fold increase in p27 mRNA in hypoxic cells, demonstrated by real-time PCR, was unaffected by SB203580, which, however, reversed the hypoxic inhibition of Skp2. In summary, p38 MAPK is an important determinant of hypoxia-induced G0/G1 block in cardiac fibroblasts. p27 induction in hypoxic cardiac fibroblasts may involve direct transcriptional regulation, independent of p38 MAPK, and post-translational regulation via p38 MAPK-dependent suppression of its degradation by Skp2. The study identifies Skp2 as a potential downstream target of p38 MAPK, suggesting a novel mechanism of G1-S regulation in cardiac fibroblasts exposed to stress conditions.