Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 3-O-β-d-glucopyanosylspinasterol via blocking NF-κB and STAT1 signaling pathways in TNF-α and IFN-γ-induced HaCaT keratinocytes

doi:10.1016/j.bbrc.2012.08.087

Biochemical and Biophysical Research Communications

Volume 427, Issue 2, 19 October 2012, Pages 236-241

https://doi.org/10.1016/j.bbrc.2012.08.087 Get rights and content

Abstract

A phytosterol derivative, 3-O-β-d-glucopyanosylspinasterol (spinasterol-Glc) isolated from leaves of Stewartia koreana was reported to inhibit LPS-induced cytokine production in macrophage cells. Thymus and activation regulated chemokine (TARC/CCL17) is produced in response to pro-inflammatory cytokines in keratinocytes, which is implicated in the development of inflammatory skin diseases. In present study, we investigated the effect of spinasterol-Glc on production of TARC/CCL17 induced by TNF-α and IFN-γ in human HaCaT keratinocytes. Spinasterol-Glc inhibited the mRNA and protein expression of TARC/CCL17 induced by TNF-α/IFN-γ in a dose-dependent manner. Inhibitors of c-Raf-1, p38 MAPK, and JAK2, suppressed the TNF-α/IFN-γ-induced production of TARC/CCL17, and phosphorylation of these signaling molecules were attenuated by spinasterol-Glc. The compound also inhibited phosphorylation of IKKα/β and IκB-α, and reduced translocation of NF-κB to the nucleus. We demonstrated that spinasterol-Glc suppressed the NF-κB-driven and the GAS-driven expression of luciferase reporter gene induced by TNF-α and IFN-γ. In addition, spinasterol-Glc inhibited the DNA binding of NF-κB and STAT1 to its cognate binding site. These results suggest that spinasterol-Glc has effective inhibitory effects on production of TARC/CCL17 in keratinocytes via inhibition of NF-κB as well as STAT activation, and could be utilized for development of a potential therapeutic agent against skin inflammatory diseases.

Highlights

► Spinasterol-Glc suppressed expression of CCL17 induced by TNF-α and IFN-γ in HaCaT cells. ► Activation of c-Raf-1, p38 MAPK, JAK2, and IKK was attenuated by spinasterol-Glc in HaCaT cells. ► Spinasterol-Glc inhibits TNF-α/IFN-γ-induced production of CCL17 via blocking NF-κB and STAT1 activation.

Introduction

Keratinocytes play an important function in the pathogenesis of inflammatory skin diseases such as atopic dermatitis (AD). AD is commonly characterized by infiltration of Th2-type lymphocytes into skin lesions and is known to be associated with high levels of chemokines like TARC/CCL17 [1], [2], [3], [4]. TARC/CCL17 belongs to a CC chemokine family, attracts CCR4 + Th2 type T cells, and is therefore thought to be related to the development of Th2-mediated inflammatory diseases. It is constitutively expressed in the thymus and keratinocytes, and also produced in other various cell types including endothelial cells and dendritic cells [5], [6], [7]. Exposure of keratinocytes to TNF-α and/or IFN-γ induced an abnormal expression of cytokines and chemokines including TARC/CCL17, leading to infiltration of T cells or leukocytes into the site of inflammatory lesion in the skin [1]. It has been reported that TNF-α and IFN-γ stimulate production of TARC/CCL17 synergistically not only in primary human keratinocyte but also in human keratinocyte cell line, HaCaT cells [7], [8]. Thus, the downregulation of TARC/CCL17 production in keratinocytes can be effective for treatment of skin diseases.

A variety of herbs and plants have been traditionally used in oriental folk medicine for the treatment of inflammatory diseases. Plant extracts and natural compounds are widely recognized as potential sources of therapeutic agents for prevention and treatment of diseases including inflammatory skin diseases [9], [10], [11]. It has been reported that the extracts from Stewartia koreana leaves induced angiogenesis, extracellular matrix remodeling in a mouse model, and stimulate wound healing on punched skin of the back of mice [12]. A phytosterol derivative, 3-O-β-d-glucopyanosylspinasterol (spinasterol-Glc), was isolated from the extracts of S. koreana and was identified as an active compound with strong anti-inflammatory activities [13], [14], [15].

The mechanisms of action by which spinasterol-Glc exhibits anti-inflammatory activities in different cell types still remain unclear. In the present study, we investigated the effects of spinastol-Glc on production of TARC/CCL17 induced by TNF-α/IFN-γ and the mechanism of its action by which it inhibits the TNF-α/IFN-γ-induced production of TARC/CCL17 in HaCaT keratinocyte cells. We showed that spinasterol-Glc suppressed expression of TARC/CCL17 mRNA and protein induced by TNF-α/IFN-γ. We found that spinasterol-Glc inhibited phosphorylation of Raf1, p38 MAP kinase, JAK2 and STAT1. Furthermore, we demonstrated that spinasterol-Glc inhibited TNF-α/IFN-γ-induced activation of NF-κB and STAT1 and the binding of the factors to the DNA elements contained in TARC/CCL17 promoter.

Section snippets

Cell culture and reagents

The human keratinocyte HaCaT cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and antibiotics (100 unit/ml penicillin, 100 μg/ml streptomycin) at 37 °C in a humidified incubator containing 5% CO₂ and 95% air. Dulbecco’s modified Eagle’s medium (DMEM), FBS, antibiotics, and trypsin–EDTA were obtained from Invitrogen (Carlsbad, CA). Signal inhibitors, BAY11-7082, c-Raf Inhibitor(1-(3-(1,4-dihydroimidazo[4,5-c]pyrazol-5-yl)-4-methylphenyl)-3-(3-(4-methyl-1H

Spinasterol-Glc inhibited TNF-α/IFN-γ-induced expression of TARC/CCL17 in HaCaT keratinocyte cells

To investigate the effects of spinasterol-Glc on TARC/CCL17 production in TNF-α/IFN-γ-stimulated keratinocyte cells, HaCaT cells were pretreated with various concentrations of spinasterol-Glc for 1 h and then stimulated with TNF-α/IFN-γ for 18 h. TARC/CCL17 production increased markedly up to 186 pg/ml in response to stimulation with TNF-α/IFN-γ, comparing to that of basal level (18 pg/ml). Spinasterol-Glc profoundly inhibited the TNF-α/IFN-γ-induced production of TARC/CCL17 in a dose-dependent

Discussion

We have previously demonstrated that spinasterol-Glc suppressed LPS-induced expression of iNOS and cytokine genes such as TNF-α, IL-1β and IL-6 in RAW264.7 murine macrophage cells via inhibiting nuclear translocation and binding of NF-κB to the DNA elements of its target genes [15]. Concomitant with the decreased NF-κB binding, spinasterol-Glc inhibited the activation of ERK1/2, JNK, and p38 MAP kinases, which contribute to the regulation of LPS-stimulated cytokine production in macrophage

Acknowledgement

This research was supported by the grant from the Next Generation BioGreen 21 program [PJ007985], Rural Development Administration, Republic of Korea.

References (25)

B. Homey et al.
Cytokines and chemokines orchestrate atopic skin inflammation
J. Aller. Clin. Immunol.
(2006)
T. Kakinuma et al.
Thymus and activation-regulated chemokine in atopic dermatitis: serum thymus and activation-regulated chemokine level is closely related with disease activity
Aller. Clin. Immunol.
(2001)
Y. Shimada et al.
Both Th2 and Th1 chemokines (TARC/CCL17, MDC/CCL22, and Mig/CXCL9) are elevated in sera from patients with atopic dermatitis
J. Dermatol. Sci.
(2004)
C. Vestergaard et al.
A Th2chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin
J. Invest. Dermatol.
(2000)
T. Imai et al.
Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein-Barr virus vector
J. Biol. Chem.
(1996)
H. Saeki et al.
Thymus and activation regulated chemokine (TARC)/CCL17 and skin diseases
J. Dermatol. Sci.
(2006)
M. Komine et al.
Mechanism of thymus- and activation-regulated chemokine (TARC)/CCL17 production and its modulation by roxithromycin
J. Invest. Dermatol.
(2005)
S.M. Ju et al.
Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-beta-d-glucose via blockade of NF-kappaB and STAT1 activation in the HaCaT cells
Biochem. Biophys. Res. Commun.
(2009)
E.H. Han et al.
Psidium guajava extract inhibits thymus and activation-regulated chemokine (TARC/CCL17) production in human keratinocytes by inducing heme oxygenase-1 and blocking NF-κB and STAT1 activation
Environ. Toxicol. Pharmacol.
(2011)
D.J. Kwon et al.
Casuarinin suppresses TARC/CCL17 and MDC/CCL22 production via blockade of NF-κB and STAT1 activation in HaCaT cells
Biochem. Biophys. Res. Commun.
(2012)

C.K. Park et al.

Inhibitory effects of S. koreana on osteoclast differentiation and bone resorption

Int. Immunopharmacol.

(2007)

T.H. Lee et al.

Inhibitory effects of a spinasterol glycoside on lipopolysaccharide-induced production of nitric oxide and proinflammatory cytokines via down-regulating MAP kinase pathways and NF-κB activation in RAW264.7 macrophage cells

Int. Immunopharmacol.

(2012)

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Consequently, the downregulation of keratinocytes inflammatory chemokine production and the inhibition of their interaction with immune cells may be an effective target in the treatment of inflammatory skin diseases (Kwon et al., 2012; Yang et al., 2015). Anti-TNF-α antibodies have been used efficiently for the management of immune-mediated inflammatory diseases including rheumatoid arthritis, Crohn's disease, ulcerative colitis and psoriasis (Jung et al., 2012 #441). Although blockage of TNF-α, by anti-TNF-α agents, such as Infliximab, Etanercept and Adalimumab, which bind TNF-α with high affinity, has been shown to successfully treat psoriasis, they can cause serious side effects (Chan et al., 2008; Ganguly, 2009; Makol and Grover, 2008; Silva et al., 2010).
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Althought all the extracts inhibited the TNF-α-induced IL-8 release, just mERC and EWRC suppressed NF-κB activation, ICAM-1, and MMP-9 secretion. EWRC showed higher inhibition on ICAM-1 and MMP-9 with IC₅₀s of 1.76 ± 0.24 and 1.24 ± 0.33 μg/mL, respectively (mean ± s.d.). On the contrary, mERC significantly decreased VEGF levels whereas EWRC did not show any effect. The HPLC-UV profile of the extracts revealed higher amount of anthocyanins in EWRC in comparison with mERC.
Our results suggest the potential positive effect of R. coriaria fruit extracts, mostly mERC, as preventive agent in the treatment of keratinocyte inflammation through their inhibitory effect on the production of skin pro-inflammatory mediators.
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Atopic dermatitis (AD) is identified by an increase in infiltrations of several inflammatory cells including type 2 helper (Th2) lymphocytes. Th2-related chemokines such as thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), and pro-inflammatory cytokines including interleukin (IL)-1β and IL-6 are considered to play a crucial role in AD. Tumor necrosis factor (TNF)-α- and interferon (IFN)-γ induce the inflammatory condition through production of TARC, MDC, IL-1β and IL-6, and activations of related transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT) in keratinocytes. Apamin, a peptide component of bee venom, has been reported its beneficial activities in various diseases. However, anti-inflammatory effects of apamin on inflammatory condition in keratinocytes have not been explored. Therefore, the present study aimed to demonstrate the anti-inflammatory effect of apamin on TNF-α- and IFN-γ-induced inflammatory condition in keratinocytes.
HaCaT was used as human keratinocytes cell line. Cell Counting Kit-8 was performed to measure a cytotoxicity of apamin. The effects of apamin on TNF-α-/IFN-γ-induced inflammatory condition were determined by real-time PCR and Western blot analysis. Further, NF-κB signaling pathways, STAT1, and STAT3 were analyzed by Western blot and immunofluorescence.
Apamin ameliorated the inflammatory condition through suppression of Th2-related chemokines and pro-inflammatory cytokines. Further, apamin down-regulated the activations of NF-κB signaling pathways and STATs in HaCaT cells.
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Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 3-O-β-d-glucopyanosylspinasterol via blocking NF-κB and STAT1 signaling pathways in TNF-α and IFN-γ-induced HaCaT keratinocytes

Abstract

Highlights

Introduction

Section snippets

Cell culture and reagents

Spinasterol-Glc inhibited TNF-α/IFN-γ-induced expression of TARC/CCL17 in HaCaT keratinocyte cells

Discussion

Acknowledgement

J. Aller. Clin. Immunol.

Aller. Clin. Immunol.

J. Dermatol. Sci.

J. Invest. Dermatol.

J. Biol. Chem.

J. Dermatol. Sci.

J. Invest. Dermatol.

Biochem. Biophys. Res. Commun.

Environ. Toxicol. Pharmacol.

Biochem. Biophys. Res. Commun.

Int. Immunopharmacol.

Int. Immunopharmacol.