Reactive oxygen species alter autocrine and paracrine signaling

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.