Fig. 3. Ang II induces CF migration via Nox4-dependent ERK activation.
A, Ang II induces ERK activation. The quiescent CF incubated with Ang II (10−7M) were analyzed for ERK activation by an in vitro immune complex kinase assay using Elk-1 as a substrate. A representative of three independent experiments is shown. B, Ang II stimulates Nox4-dependent H2O2 generation. CF infected with Ad.siNox4 (moi 100 for 48 h) or treated with DPI (10 µM for 30 min) prior to Ang II addition (10−7M) for 30 min were analyzed for H2O2 production using the Amplex® Red assay. *P < 0.01 vs. untreated; †P < at least 0.05 vs. Ang II ± DMSO or Ad.siGFP (n=6). C, Ang II induces ERK activation via Nox4 and ROS. CF transduced with Ad.siNox4 (moi 100 for 48 h) or pretreated with DPI (10 µM for 30 min) were incubated with Ang II (10−7M) for 30 min. ERK activation was analyzed as in A (n=3). Knockdown of Nox4 was confirmed by immunoblotting (right hand panel). D, PD98059 inhibits Ang II-induced ERK activation. The quiescent CF were treated with the ERK inhibitor PD98059 (10 µM for 1 h), p38MAPK inhibitor SB203580 (1 µM for 30 min) or JNK inhibitor SP600125 (20 µM for 30 min) prior to Ang II addition (10−7M for 30 min). ERK activation was analyzed as in A (n=3). E, Ang II stimulates CF migration via ERK. The quiescent CF were layered on Matrigel™ basement membrane matrix-coated filters, incubated with PD98059 (10 µM for 1 h) followed by Ang II (10−7M for 12 h). Cell migration was analyzed by MTT assay. *P < 0.001 vs. untreated; †P < 0.01 vs. Ang II (n=6). F, Knockdown of Nox4 attenuates Ang II-induced CF migration. CF transduced with Ad.siNox4 (moi 100 for 48 h) were incubated with Ang II (10−7M) for 12 h were analyzed for migration as in A. A–D *P < at least 0.01 vs. untreated; †P < at least 0.05 vs. Ang II (n=6).