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

Summary

Background

Recent work has shown a novel function of angiotensin II (Ang II) in skin wound healing 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 μM) increased the superoxide formation. Ang II (1–100 μM) 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 μM) 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^ω-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.

Introduction

The mechanisms of the angiotensin II (Ang II)-induced alteration in endothelium-dependent vasodilation may have been fully elucidated. Recently, it has become clear that Ang II additionally had mitogenic properties very similar to a growth factor. Following the characterization of two distinct angiotensin receptor subtypes termed AT1 and AT2 [1], it was suggested that the growth promoting effect of Ang II could be attributed to the AT1 subtype [2]. AT2-receptors, on the other hand, were shown to mediate antiproliferation [3]. Finally, a novel function of Ang II in skin wound healing was reported [4].

Ang II exerts some of its cellular effects via AT1 receptors through the production of reactive oxygen species (ROS) by the enzyme NADPH oxidase [5]. Activation of AT1 receptors initiates intracellular signaling that leads to phosphorylation of NADPH cytoplasmic components, which, in turn, results in the production of superoxide [6]. In human keratinocytes, NADPH oxidase is a major source of UVA-induced ROS generation [7]. It has been reported that NADPH oxidase induces resistance against differentiation-induced cell death, suggesting a contribution of NADPH oxidase and its oxidants during the early stage of cell transformation [8].

ROS including hydrogen peroxide are known to activate epidermal growth factor receptor (EGFR) especially in UV irradiated keratinocytes [9], [10]. On the other hand, UVA exposure appears to cause down-regulation of EGFR expression levels without phosphorylation [11]. One reason for these complications may be the duration of observation of activation and expression of EGFR. The activation of EGFR by some stimuli may be a transient response, and the alteration of expression levels by some stimuli presumably takes place in an order of at least hours. Alternatively, the activation and the expression levels of EGFR may be independently modulated with each other under some experimental conditions.

In this study, we investigated whether Ang II-induced ROS enhance the activity and the expression levels of EGFR in HaCaT cells. Apocynin, a NADPH oxidase inhibitor, was used to explore the involvement of NADPH oxidase in Ang II-treated HaCaT cells. As Ang II-induced ROS was considered to react with constitutive nitric oxide (NO) produced by HaCaT cells, we have also examined the effects of N^ω-nitro-l-arginine methyl ester (l-NAME) on the ROS formation and EGFR expression levels in Ang II-treated HaCaT cells.