Modulation of Protein Kinase C-Related Signal Transduction by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exhibits Cell Cycle Dependence

Abstract

The modulation of protein kinase C (PKC)-mediated protein phosphorylation in quiescent vascular smooth muscle cells (SMCs) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exhibits a discrete temporal pattern in which early reductions of kinase activity are followed by marked increases in activity. This profile may be accounted for by transcriptional- and/or cell cycle-related effects of TCDD. To test this hypothesis, experiments were conducted to examine the influence of TCDD on PKC activity during different phases of the cell cycle in vascular (aortic) SMCs. Increased PKC activity was observed in the cytosolic and particulate fractions of randomly cycling SMC cultures derived from female rats treatedin vivowith 10 μg/kg TCDD relative to corn oil. Treatment of cycling naive SMC cultures with TCDD (0.1 to 1000 nM) for 0.5 h caused a concentration-dependent increase of particulate PKC activity and no changes in cytosolic counterparts. Extended challenge of SMCs with TCDD for 24 h increased PKC activity in both cellular fractions. Incubation of SMCs with various concentrations of fetal bovine serum for 72 h to differentially regulate cell cycling followed by challenge with 10 nMTCDD for 24 h reduced cytosolic and particulate PKC activity in quiescent cells, but enhanced activity in cycling cells. To determine if this serum-related profile was strictly dependent upon cell cycle-related events, SMCs were synchronized in the G0phase and then pulsed with 10 nMTCDD during different phases of the cell cycle. Differential profiles were observed where reduced C-kinase activity occurred during the G0/G1transition followed by increases during G1/S and no changes during S. Western blot analysis confirmed the patterns of PKC activity observed during the G0/G1and G1/S transitions. PKCα, βII, and δ isoforms were reduced during G0/G1, while only PKCβII and δ were increased during G1/S. These data show that modulation of PKC by TCDD in vascular SMCs exhibits cell cycle dependence and isoform specificity.