MAP kinase-independent induction of proto-oncogene c-fos mRNA by hemin in human cells

Treatment of HeLa cells or human skin fibroblast cells with hemin led to a time- and dose-dependent rapid induction of c-fos mRNA. This induction was absent in the cells treated with actinomycin D, indicating that the c-fos i nduction by hemin occurs at the level of transcription. Metalloporphyrins, including zinc-, cobalt-, and tin-protoporphyrin, ferric ion, and protoporp hyrin also induced c-fos mRNA. Transient reporter assay with the reporter c onstructs of the human c-fos gene promoter up to -404 bp connected to the l uciferase gene showed high activity but no induction by hemin, suggesting t hat cis-acting elements, including the serum response element located about -310 bp upstream of the human c-fos gene promoter, may not contribute to t he heme-dependent induction. With in-gel assay of protein kinases, the acti vity of the mitogen-activated protein (MAP) kinases such as extracellular s ignal-regulated kinase 1/2 or p38 MAP kinase in hemin-treated HeLa cells wa s not stimulated. Stimulation of c-Jun N-terminal kinase by hemin was nil. Furthermore, PD58059 and SB203580, inhibitors for MAP kinases, did not affe ct the hemin-dependent c-fos induction. Of the inhibitors for protein kinas es so far tested, KN-62, a specific inhibitor for calmodulin-dependent prot ein kinase II (CaMK II), inhibited the induction of c-fos mRNA by hemin. Ph osphorylation of CaMK II in hemin-treated cells increased. With gel mobilit y assay, the DNA AP-1 binding activity transiently increased when treating HeLa cells with hemin. Therefore, induction of c-fos led to an activation o f AP-1 in the presence of hemin. We suggest that calmodulin-dependent prote in kinase II rather than the MAP kinase family regulates the induction of t he human c-fos gene expression by hemin. (C) 1999 Academic Press.