Celastrol induces expression of heme oxygenase-1 through ROS/Nrf2/ARE signaling in the HaCaT cells

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Abstract

We previously demonstrated that celastrol, a quinone methide triterpenoid derived from the medicinal plant Tripterygium wilfordii, exerts its anti-inflammatory activity through up-regulation of heme oxygenase-1 (HO-1) expression in the keratinocytes. In this study, we examined the signaling pathways that lead to the up-regulation of HO-1 expression by celastrol. In HaCaT cells, celastrol-induced HO-1 expression was dependent on ROS generation. ERK and p38 MAPK were major MAPK pathways responsible for celastrol-induced HO-1 expression. Celastrol induced Nrf2 activation. Nrf2 knockdown using small interfering RNA (siRNA) inhibited celastrol-induced HO-1 expression. Treatment with celastrol resulted in a marked increase in antioxidant response element (ARE)-driven transcriptional activity, which was dependent on ROS generation and activation of ERK and p38 MAPK. Furthermore, Nrf2 siRNA significantly reversed the inhibitory effect of celastrol on IFN-γ-induced expression of ICAM-1 in the keratinocytes. Taken together, our results indicate that celastrol can activate the ROS-ERK/p38-Nrf2-ARE signaling cascades leading to the up-regulation of HO-1 which is partly responsible for its anti-inflammatory activity in the keratinocytes.

Highlights

► In HaCaT cells, celastrol-induced HO-1 expression was dependent on ROS generation. ► ERK and p38 MAPK were major MAPK pathways responsible for celastrol-induced HO-1 expression. ► Celastrol induced Nrf2 activation. ► Celastrol can activate the ROS-ERK/p38-Nrf2-ARE signaling cascades leading to the upregulation of HO-1 in the keratinocytes.

Introduction

Celastrol, a quinone methide triterpenoid derived from Tripterygium wilfordii Hook, has been used as a traditional medicine for the treatment of various diseases such as inflammation [1]. Celastrol has been demonstrated to exert anti-oxidant, anti-tumor, and anti-inflammatory activities in a variety of in vitro and in vivo models [2], [3], [4]. However, the molecular mechanism by which celastrol exerts its biological activities has not been fully elucidated (for review, see [5]). Celastrol has been shown to suppress production of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β by modulating NF-κB signaling pathways in LPS-stimulated cells [6], [7], [8]. Celastrol exhibited its anti-allergic activity by inhibiting FcepsilonRI signaling through binding to ERK in antigen-stimulated mast cells [9]. Recently, we observed that celastrol suppresses IFN-γ-induced ICAM-1 expression and subsequent monocyte adhesiveness by a mechanism involving expression of HO-1 in the keratinocytes [10].

Heme oxygenase-1 (HO-1), an inducible antioxidant enzyme, mediates the degradation of heme into ferrous iron, carbon monoxide (CO) and biliverdin. The products resulting from HO-1 activity have antioxidant and anti-inflammatory effects [11]. In response to oxidative stress, the epidermal keratinocyte, the major cell type in the skin, can express HO-1. Increased HO-1 activity in the skin has been reported to exert cytoprotective and anti-inflammatory effects in several pathological conditions including inflammation [12]. Recently, it was demonstrated that HO-1 expression has a regulatory role in skin inflammation such as atopic dermatitis [13], [14], [15].

Although we reported that celastrol induced HO-1 expression, its signaling pathways involved in the up-regulation of HO-1 expression were not elucidated. In this study, we investigated the signaling cascades that mediate celastrol-induced HO-1 expression in a human keratinocyte cell line HaCaT. We show that celastrol induces HO-1 expression via ROS-ERK/p38-Nrf2-ARE signaling cascades in the HaCaT cells.

Section snippets

Cell culture and reagents

The immortalized human keratinocyte cell line, HaCaT, was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin) at 37 °C in a humidified incubator containing 5% CO2 and 95% air. Human THP-1 monocytic cells were maintained in RPMI 1640 medium supplemented with 2 mM l-glutamine and 10% fetal bovine serum. Nrf2 specific siRNA, control siRNA, primary antibodies against ICAM-1, HO-1, Nrf2, actin,

Generation of ROS by celastrol in HaCaT cells

In the previous study, we demonstrated that HO-1 expression plays a role in the anti-inflammatory responses by celastrol in the keratinocytes [10]. We further studied the signaling pathway leading to HO-1 expression by celastrol. Since the antioxidant enzymes such as HO-1 are known to respond to a variety of oxidative stresses [11], ROS may be involved in celastrol-induced up-regulation of HO-1 expression by human keratinocytes. We investigated ROS generation in the HaCaT cells treated with

Discussion

Infiltration of monocytes/T cells into the inflamed skin is a characteristic feature of skin inflammation. Up-regulation of adhesion molecules such as ICAM-1 in the keratinocytes is an important regulator to initiate interaction between monocytes/T cells and keratinocytes during the processes of skin inflammation [17]. Therefore, suppression of ICAM-1 expression is considered one of strategies for treatment of inflammatory skin diseases. We recently reported that celastrol exerted its

Acknowledgments

This work was supported by the Regional Core Research Program funded by the Korea Ministry of Education, Science and Technology (MEST) (Medical & Bio-material Research Center) and in part by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093812).

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