(The) roles of protein kinases on cytochrome P450 expression induced by dioxin in liver cell culture systems

Abstract

The roles of protein kinases on cytochrome P450 expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated in primary hepatocyte cultures from mice and rats, and in mouse hepatoma Hepa-1 cells. TCDD increased cytochrome P450IA1-specific 7-ethoxyresorufin O-deethylase (EROD) activity both in dose and time-dependent manner. The relationship between protein kinase-dependent signal transduction pathway and TCDD-inducible cytochrome P450IA1 expression were studied using specific inhibitors of the protein kinases. Potent protein kinase C inhibitor, staurosporine, suppressed TCDD-induced cytochrome P450IA1 activity when treated with TCDD for 24 hrs in mouse primary hepatocyte cultures. Pretreatment with staurosporine for 1hr before TCDD (1nM) addition, also suppressed the TCDD-induced EROD activity. Calphostin C, a specific protein kinase C inhibitor, also reduced TCDD-induced EROD activity dose-dependently when pretreated for 1hr as staurosporine treatment. However, tyrphostin AG213, a specific protein tyrosine kinase inhibitor, fail to suppress P450 activity up to 10$\mu$M concentration. Similarly, treatment with genistein, other EGF receptor tyrosine kinase inhibitor, did not affect EROD activity over the range of concentrations employed(1-25$\mu$M range). A possible involvement of Ca$^{2+}$/calmodulin-dependent protein kinases in cytochrome P450 expression was examined by measuring the effect of calmodulin antagonists. Pretreatment with W-7 did not perturb TCDD-induced EROD activity, however, R24571 decreased TCDD-induced EROD activity. But, this effect did not occur in mouse hepatoma cells, and ML-9, specific myosin light chain kinase inhibitor, had little effect on TCDD-induced EROD activity. Data suggests that Ca$^{2+}$/calmodulindependent protein kinase may not modulate the cytochrome P450 expression. To study the mechanism of suppression by protein kinase C inhibitors such as staurosporine and calphostin C, western immunoblot analyses were per...