ERK and p38 MAPK, but not NF-κB, Are Critically Involved in Reactive Oxygen Species–Mediated Induction of IL-6 by Angiotensin II in Cardiac Fibroblasts

Abstract

We recently reported that angiotensin II (Ang II) induced IL-6 mRNA expression in cardiac fibroblasts, which played an important role in Ang II–induced cardiac hypertrophy in paracrine fashion. The present study investigated the regulatory mechanism of Ang II–induced IL-6 gene expression, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. Ang II increased intracellular ROS in cardiac fibroblasts, and the increase was completely inhibited by the AT-1 blocker candesartan and the NADH/NADPH oxidase inhibitor diphenyleneiodonium (DPI). We first confirmed that antioxidant N -acetylcysteine, superoxide scavenger Tiron, and DPI suppressed Ang II–induced IL-6 expression. Because we observed that exogenous H 2 O 2 also increased IL-6 mRNA, the signaling pathways downstream of Ang II and exogenous H 2 O 2 were compared. Ang II, as well as exogenous H 2 O 2 , activated ERK, p38 MAPK, and JNK, which were significantly inhibited by N -acetylcysteine and DPI. In contrast with exogenous H 2 O 2 , however, Ang II did not influence phosphorylation and degradation of IκB-α/β or nuclear translocation of p65, nor did it increase NF-κB promoter activity. PD98059 and SB203580 inhibited Ang II–induced IL-6 expression. Truncation and mutational analysis of the IL-6 gene promoter showed that CRE was an important cis -element in Ang II–induced IL-6 gene expression. NF-κB–binding site was important for the basal expression of IL-6, but was not activated by Ang II. Ang II phosphorylated CREB through the ERK and p38 MAPK pathway in a ROS-sensitive manner. Collectively, these data indicated that Ang II stimulated ROS production via the AT1 receptor and NADH/NADPH oxidase, and that these ROS mediated activation of MAPKs, which culminated in IL-6 gene expression through a CRE-dependent, but not NF-κB–dependent, pathway in cardiac fibroblasts.