Protease-activated receptor-2 activates NQO-1 via Nrf2 stabilization in keratinocytes

https://doi.org/10.1016/j.jdermsci.2013.11.010Get rights and content

Abstract

Background

Protease-activated receptor-2 (PAR-2) mediates inflammation and immune responses by serine proteinases. NF-E2-related factor 2 (Nrf2) confers protection against tissue injury through antioxidant responses to oxidative stress induced by a variety of factors, including electrophilic chemicals, hydrogen peroxide, and ultraviolet irradiation.

Objective

In this study, we investigated if PAR-2 activation can stimulate Nrf2 signaling to preserve homeostasis in keratinocytes.

Methods

We performed western blotting, real-time reverse transcription polymerase chain reaction, and immunocytochemistry of keratinocyte cultures, as well as immunohistochemical labeling of human skin samples. Short interfering RNA (siRNA) was employed to confirm the effects of PAR-2 activation.

Results

PAR-2 activation with a selective PAR-2 agonist peptide increased the nuclear level of Nrf2 protein and subsequently induced phase II enzyme expression. Nrf2 signaling via PAR-2 activation was confirmed with experiments using PAR-2-siRNA-treated keratinocytes. The activation of an Nrf2-targeted gene by PAR-2 activation was not induced by new production of Nrf2 but by prolonged stabilization of Nrf2. Lesional skin samples from vitiligo patients showed significantly lower expression of PAR-2 and Nrf2 than control skin samples.

Conclusion

Collectively, PAR-2 activation enhanced nuclear Nrf2 translocation, and PAR-2-mediated Nrf2 activation was attributable to existing Nrf2 stabilization rather than de novo production. Our findings suggest that in addition to induction of inflammation, PAR-2 can play a chemopreventative role via Nrf2 stabilization in keratinocytes.

Introduction

Protease-activated receptors (PARs) belong to a novel family of G protein-coupled receptors that are activated by proteinases such as thrombin and trypsin. PAR activation plays roles in many tissues, including the gastrointestinal tract, kidney, muscle, nerve, lung, skin, and nervous system [1]. Among the four PAR subtypes (PAR-1 to -4), the expression of functional PAR-2 on human skin and its roles in inflammation, pruritus, skin barrier homeostasis, and pigmentation are well described [2], [3]. In particular, increased inflammation in keratinocytes via PAR-2 activation and its relationship to atopic dermatitis (AD) has been widely studied [2], [4], [5], [6]. Allergens with protease activity can alter epidermal permeability barrier homeostasis through PAR-2 activation [7]. In addition, PAR-2 activation by unrestricted kallikrein 5 in Netherton syndrome can mediate the expression of thymic stromal lymphopoietin, leading to proinflammatory and proallergic signaling as observed in AD [8]. Consequently, PAR-2 is considered to have an important role in AD pathogenesis by acting as a sensor for exogenous proteases from various allergens [2]. The association of PAR-2 with acne has also been investigated; proteases secreted from Propionibacterium acnes can induce expression of inflammatory mediators via PAR-2 activation [9].

Likewise, PAR-2 is involved in inflammation induction; it serves as a gate that recognizes external danger signals. In addition, PAR-2 has been reported to induce ROS formation in several cell types [10]. Human skin is always in contact with external stimuli, but skin diseases are prevented by skin defense mechanisms that balancing oxidant stress with antioxidant defenses. Therefore, we questioned whether PAR-2 activation can activate defense mechanisms, such as NF-E2-related factor 2 (Nrf2), which is a transcription factor to have a role of cellular defense against the cytotoxic effects of oxidative stress, to preserve skin homeostasis.

Different classes of enzymes (phases I–III) are implicated in protecting against oxidative stress. Particularly, phase II detoxification enzymes, including NAD(P)H:quinone oxidase (NQO-1), γ-glutamyl cysteine ligase catalytic subunit, γ-glutamylcystine ligase modulatory subunit, heme oxygenase-1 (HO-1), and glutathione S-transferase are crucial in protecting cells from oxidative insults [11]. Although Nrf2 is known to be enhanced by a variety of activating factors including electrophilic chemicals, hydrogen peroxide, and ultraviolet (UV) irradiation, it is of significant importance to identify other activating factors in keratinocytes.

Investigations of Nrf2 in skin have mainly focused on anti-inflammatory and antioxidant functions against UV irradiation [12], [13]. Vitiligo, a common depigmenting disorder, is related with toxic oxidative stress in pathogenesis [14], [15], [16]. In recent report, Nrf2 and downstream phase II enzymes were upregulated in lesional skin compared with non-lesional skin in vitiligo [16]. Conflicting data reporting decreased nuclear Nrf2 in vitiligo lesions by impaired translocation of cytoplasmic Nrf2 into the nucleus also exist [17]. Moreover, lesional keratinocytes showed lower PAR-2 expression than non-lesional keratinocytes in vitiligo [18], [19]. These series of results raised questions about a close relationship between PAR-2 activation and Nrf2 expression.

Here, we investigated the impact of PAR-2 activation on Nrf2 signaling in keratinocytes. Our results suggest that PAR-2 activation with PAR-2 agonist peptide (AP) may stabilize Nrf2 and subsequently enhance its nuclear translocation, leading to increased expression of NQO-1, a representative phase II enzyme in human keratinocytes.

Section snippets

Reagents

PAR-2 AP (100 μM; SLIGKV-NH2, Peptron, Daejeon, Korea), and PAR-2 agonist control peptide (PAR-2 CP; 100 μM; VKGILS-NH2, Peptron) were used in this experiment. Hydrogen peroxide (H2O2) was used as a positive control of oxidant stress. Curcumin 10 μM (Sigma, St Louis, MO) was used as a positive control for Nrf2 activation. Cycloheximide (CHX), a protein synthesis inhibitor, was purchased from Sigma.

Cell culture

The human immortalized keratinocyte cell HaCaT was used for this experiment. Cells were cultured in

PAR-2 activation increases Nrf2 and NQO-1 protein levels

To determine if curcumin, a well-known Nrf2 activator, induced Nrf2 and NQO-1 protein levels in keratinocytes, HaCaT cells were incubated with 10 μM curcumin for 2 h. This treatment increased the protein levels of nuclear Nrf2 (molecular weight: 57 kDa) and cytosolic NQO-1 in HaCaT cells, and both increases were maintained for approximately 8 h (Fig. 1A).

PAR-2 AP-treated HaCaT cells exhibited increased protein levels of nuclear Nrf2 after 4 h of incubation with PAR-2 AP (Fig. 1B), and the protein

Discussion

PAR-2 activation in the skin amplifies cutaneous inflammation and enhances uptake of melanin from melanocytes to keratinocytes [2], [6], [20], [21]. To date, two unique mechanisms for PAR-2 activation have been characterized. Classically, serine proteases cleave the receptor N-terminal portion of PAR-2 to expose a receptor-activating ligand, named tethered ligand, which binds to the extracellular receptor domain to activate signaling [22], [23]. Alternatively, synthetic PAR-2 AP (SLIGKV-NH2) is

Funding sources

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012-0007581).

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    These authors contributed equally to this work.

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