Phosphorylation and hypoxia-induced heme oxygenase-1 gene expression in cardiomyocytes

Heme oxygenase-1 (HO-1) is a stress protein and the rate-limiting enzyme in heme degradation. We sought to examine the notion that protein kinases and phosphatases through phosphorylation and dephosphorylation modulate the HO-1 expression in cardiomyocytes under hypoxic conditions.

Exposure of neonatal rat cardiomyocytes to hypoxia markedly induced the HO-1 expression, as assessed by Northern blot, Western blot, and transfection assay. The hypoxia-induced HO-1 expression was blocked by the kinase inhibitors staurosporine and SB202190 in a dose-dependent manner. Hypoxia decreased the activity of phosphatase-1 (PP-1). To examine the effect of PP-1 inhibition on HO-1 expression we used the phosphatase inhibitor okadaic acid (OA) and an antisense vector. OA treatment or overexpression of the antisense PP-1 transcript markedly induced HO-1 expression. Furthermore, transfection assay using HO-1 promoter constructs revealed the involvement of the nuclear factor kB (NF-kB) and Activator protein-1 (AP-1) in the hypoxia-induced activation of the HO-1 gene. The HO-1 promoter activity was modulated by OA under normoxic conditions or staurosporine under hypoxia.

Our results suggest that activation of protein kinases and downregulation of PP-1 activity contribute to the hypoxia-induced HO-1 gene expression and that the proximal HO-1 promoter region containing NF-kB and AP-1 binding sites is likely to play a role in the transcriptional activation of the HO-1 gene in cardiomyocytes in response to hypoxic stress.