Angiotensin II enhances EGF receptor expression levels via ROS formation in HaCaT cells

Background: Recent work has shown a novel function of angiotensin II (Ang II) in skin wound heating in which reactive oxygen species might be involved. As Ang II is known to increase superoxide production by activating NADPH oxidase in some non-phagocytic cells, we hypothesized that the produced superoxide by NADPH activation could contribute to the regulation of epidermal growth factor receptor (EGFR) in keratinocytes. Objective: We examined whether Ang II could generate superoxide and enhance EGFR expression levels in HaCaT cells. Methods: Superoxide formation was assessed by using hydroethidine. EGFR expression levels were examined by Western blotting. Results: Ang II (1-100 mu M) increased the superoxide formation. Ang II (1-100 mu M) resulted in a dose-dependent increase in cell proliferation in HaCaT cells. Heparin-binding epidermal growth factor activated the EGFR at 5-10 min. Although Ang II did not activate the EGFR, the expression levels of EGFR protein were increased in HaCaT cells treated with Ang II (1 mu M) at 6 h. Apocynin, a NADPH oxidase inhibitor, decreased the expression levels of EGFR. Xanthine/xanthine oxidase system, an exogenous superoxide generating system, enhanced the EGFR protein expression. Although Ang II did not affect the nitric oxide (NO) production, a NO synthase inhibitor N-omega-nitro-L-arginine methyl ester suppressed the Ang II-induced EGFR expression levels in HaCaT cells. Thus, constitutive NO is required for the Ang II-induced EGFR expression in HaCaT cells. Conclusion: These results suggest that Ang II enhances the cell proliferation and EGFR expression via superoxide production under the regulation of NO in HaCaT cells, implying that Ang II may regulate the proliferation, differentiation and tumorigenesis of the epidermis by harmonizing the Superoxide and NO production. (c) 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.