Human heme oxygenase: cell cycle-dependent expression and DNA microarray identification of multiple gene responses after transduction of endothelial cells

The purpose of the present study was to examine the role of human heme oxygenase (human HO-1) in cell cycle progression following exposure to heme or human HO-1 gene transfer and to identify target genes associated with human HO-1-meditated increases in cell cycle progression using cDNA microarray technology. Heme-induced robust human HO-1 expression in quiescent human microvessel endothelial cells cultured in 1% FBS and the levels of human HO-1 expression progressively declined without a change in the cell cyclin. To identify genes regulated by human HO-1 in the cell cycle, human endothelial cells were transduced with a retroviral vector encoded with human HO-1 gene or an empty vector. Transgene expression and functionality of the recombinant protein were assessed by Western blotting, enzyme activity, carbon monoxide, cGMP production, and cell cycle analysis. Human cDNA gene array and quantitative real-time RT-PCR were used to identify both known and novel differentially expressed genes in cells overexpressing human HO-1. Major findings were upregulation of several genes associated with cell cycle progression, including cyclin E and D; downregulation of cyclin-dependent kinase inhibitors p21 and p27, cyclin-dependent kinases 2, 5, and 6, and monocyte chemoattractant protein-1; and upregulation of growth factors, including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor I (VEGFRI), endothelial growth factor (EGF) and hepatic-derived growth factor (HDGF). These findings identify an array of gene responses to overexpression of human HO-1 and elucidate new aspects of human HO-1 signaling involved in cell growth.