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 insults in the kidney and other tissues. We examined the effect of overexpression of HO-1 on cell growth, expression of p21, and susceptibility td apoptosis. Methods. LLC-PK1 cells were genetically engineered to exhibit stable overexpression of HO-1. The effects of such overexpression on cell growth, the cell cycle, and the cell cycle-inhibitory protein, p21, were assessed; additionally, 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 of caspase-3 activity, phase contrast microscopy, and the TUNEL method. Results. HO-1 overexpressing LLC-PK1 cells demonstrated cellular hypertrophy, 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 apoptosis 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. Moreover, this reduction in expression of p21 attendant upon the inhibition of HO activity in HO-1 overexpressing cells paralleled the loss of resistance of these cells to apoptosis when HO activity is inhibited, The pharmacologic 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 proliferative cell growth, and confers marked resistance to apoptosis. We speculate 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 attendant inhibitory effect on the cell cycle provide a hitherto unsuspected mechanism underlying the cytoprotective actions of HO-1.