Cellular overexpression of heme oxygenase-1 up-regulates p21 and confers resistance to apoptosis

Background. Induction of heme oxygenase-1 (HO-1) protects against diverse i nsults in the kidney and other tissues. We examined the effect of overexpre ssion of HO-1 on cell growth, expression of p21, and susceptibility td apop tosis. Methods. LLC-PK1 cells were genetically engineered to exhibit stable overex pression of HO-1. The effects of such overexpression on cell growth, the ce ll cycle, and the cell cycle-inhibitory protein, p21, were assessed; additi onally, the susceptibility of these HO-1 overexpressing cells to apoptosis induced by three different stimuli (TNF-alpha /cycloheximide, staurosporine , or serum deprivation) was evaluated by such methods as the quantitation o f caspase-3 activity, phase contrast microscopy, and the TUNEL method. Results. HO-1 overexpressing LLC-PK1 cells demonstrated cellular hypertroph y, decreased hyperplastic growth, and growth arrest in the G(0)/G(1) phase of the cell cycle. HO-1 overexpressing cells were markedly resistant to apo ptosis induced by TNF-alpha /cycloheximide or staurosporine as assessed by the caspase-3 activity assay. Such overexpression also conferred resistance to apoptosis induced by serum deprivation as evaluated by the TUNEL method ; in these studies, inhibition of HO attenuated the resistance to apoptosis . Expression of the cyclin dependent kinase inhibitor, p21(CIP1, WAF1, SDI1 ), as judged by Northern and Western analyses, was significantly increased in HO-1 overexpressing cells, and decreased as HO activity was inhibited. M oreover, this reduction in expression of p21 attendant upon the inhibition of HO activity in HO-1 overexpressing cells paralleled the loss of resistan ce of these cells to apoptosis when HO activity is inhibited, The pharmacol ogic inducer of HO-1, hemin, increased expression of p21 in wild-type cells and decreased apoptosis provoked by TNF-alpha /cycloheximide. Conclusion. Cellular overexpression of HO-1 up-regulates p21, diminishes pr oliferative cell growth, and confers marked resistance to apoptosis. We spe culate that such up-regulation of p21 contributes to the altered pattern of cell growth and resistance to apoptosis. Our studies uncover the capacity of HO-1 to markedly influence the cell cycle in renal epithelial cells. In light of the profound importance of the cell cycle as a determinant of cell fate, we speculate that the inductive effect of HO-1 on p21 and the attend ant inhibitory effect on the cell cycle provide a hitherto unsuspected mech anism underlying the cytoprotective actions of HO-1.