(The) roles of protein kinases on cytochrome P450 expression induced by dioxin in liver cell culture systems간세포 배양시스템에서 디옥신에 의해 유도되는 cytochrome P450 발현에 대한 protein kinase들의 역할에 관한 연구
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...