Elsevier

Journal of Ethnopharmacology

Volume 113, Issue 2, 5 September 2007, Pages 240-247
Journal of Ethnopharmacology

The anti-inflammatory activity of Phellinus linteus (Berk. & M.A. Curt.) is mediated through the PKCδ/Nrf2/ARE signaling to up-regulation of heme oxygenase-1

https://doi.org/10.1016/j.jep.2007.05.032Get rights and content

Abstract

It has been reported that heme oxygenase-1 (HO-1) mediates the anti-inflammatory activity of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. This continuing work aimed to elucidate the signaling pathway to the up-regulation of HO-1 by PL. In RAW264.7 macrophage cells, PL was able to enhance phosphorylation of protein kinase Cδ (PKCδ), but not PKCα/βII, in a time-dependent manner. PL-induced HO-1 expression was dramatically released by GF109203X, a general inhibitor of PKC, and rottlerin, a specific PKCδ inhibitor but not by Gö6976, a selective inhibitor for PKCα/β. Additionally, PL treatment resulted in a marked increase in antioxidant response element (ARE)-driven transcriptional activity, which was dependent on PKCδ but not PKCα. An increase by PL treatment in the ARE-driven transcriptional activity was further enhanced by Nrf2, whereas it was diminished by Keap1. Furthermore, pretreatment of rottlerin and overexpression of PKCδ (K376R), a kinase-inactive form of PKCδ, partly blocked the suppression by PL of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and iNOS promoter activity, which were elevated in the lypopolysaccharide (LPS)-activated macrophages. Similarly, expression of matrix metalloproteinase-9 (MMP-9) and its promoter activity were suppressed by PL, which were dependent upon PKCδ. The present findings indicate that Phellinus linteus gives rise to an anti-inflammatory activity though the PKCδ/Nrf2/ARE signaling to the up-regulation of HO-1 in an in vitro inflammation model.

Introduction

Phellinus linteus Berk. et Curt., an orange color mushroom in the family of Hymenochaetaceae, has been used as a traditional medicine in oriental countries for the treatment of various diseases such as gastroenteric disorders, inflammation, tumors, and lymphatic diseases (Kim et al., 2004b). Its pharmacological activities, especially antitumor and anti-inflammatory activities, have been documented. Its protein-bound polysaccharide shows a direct antitumor effect through apoptosis and cell cycle blockade in human colon cancer cells (Li et al., 2004). Acidic polysaccharide isolated from Phellinus linteus enhances NO production, cell-mediated immunity, and antitumoral activity of peritoneal macrophages, which subsequently supports the antitumor action of Phellinus linteus (Kim et al., 2003, Kim et al., 2004a). Phellinus linteus can also inhibit metastasis of melanoma cells in mice via regulation of urokinase type plasminogen activator associated with tumor cell induced platelet aggregation (Lee et al., 2005). The antitumor activity of Phellinus linteus has also been assessed by antioxidant and anti-angiogenic activities of the ethanolic extract of Phellinus linteus, which additionally possesses strong anti-inflammatory and anti-nociceptive activities (Song et al., 2003, Kim et al., 2004b). Among the subfractions of the ethanolic extract, the n-butanol (n-BuOH) subfraction (PL) is most effective in anti-inflammation and anti-angiogenesis (Kim et al., 2004b).

Heme oxygenases (HOs) catalyze the breakdown of heme into carbon monoxide, biliverdin, and iron using molecular oxygen and reducing equivalents from NADPH:cytochrome P450 reductase. Heme oxygenase-1 (HO-1), localized in the non-neural tissues, is inducible in response to oxidative stress, nitrosative stress, thiol-reactive substances, and cytokines (Ryter and Choi, 2005). Induction of HO-1 is modulated by various natural and synthetic compounds in tissue culture models. Hemin-induced HO-1 expression is attenuated by flavonoids, such as apigenin (Abate et al., 2005), while quercetin, one of other flavonoids, induces HO-1 expression leading to the prevention of oxidative stress-induced apoptosis in RAW264.7 macrophages (Chow et al., 2005). Taurine induces expression of HO-1 in both non-activated and LPS-activated J774.2 macrophages (Olszanecki and Marcinkiewicz, 2004). Nicotine also enhances up-regulation of HO-1 in gingival tissues, which is dependent on the intracellular glutathione (GSH) concentration (Chang et al., 2005). Recently, PL has also been shown to induce HO-1 in RAW264.7 macrophages (Kim et al., 2006).

The physiological relevance of the HO-1 induction and increased HO-1 activity has been demonstrated in several pathological states such as atherosclerosis and inflammation, wherein it confers cytoprotection (Yet et al., 2003, Kapturczak et al., 2006). In support of this in vivo evidence, results obtained from cell culture studies have shown that induction of HO-1 is also involved in the inhibitory mechanism of some anti-inflammatory flavonoids on LPS-induced inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in mouse macrophages (Lin et al., 2005). Opposite regulation of iNOS and HO-1 is similarly observed in response to cytokine exposure and oxidative stress, and carbon monoxide, a key product of HO, suppress inflammation via a mitogen-activated protein kinase (MAPK) pathway (Otterbein, 2002).

Protein kinase C (PKC) is a multigene family of related serine/threonine protein kinases that play vital roles in cellular signaling and various biological phenomena, and can phosphorylate key substrates such as receptors, ion channels, cytoskeletal elements, regulatory factors and enzymes (Graves and Krebs, 1999). Based on the structure and physiological characteristics, PKC isoforms are classified into three subgroups; Ca2+-dependent classical cPKC isoforms-α, -βI, -βII, and -γ which are activated by diacylglycerol and phosphatidylserine, Ca2+-independent novel nPKC isoforms-δ, -ɛ, -η, -μ, and -θ which are activated by 1,2′-diacylglycerol and phosphatidylserine, and the atypical aPKC isoforms-λ/I and -ζ which are activated by phosphatidylserine (Suzuki et al., 2006, Zhou et al., 2006). Among them, PKCδ is known to play a crucial role in growth regulation, tissue remodeling and regulation of immune system (Perletti and Terrian, 2006). The tyrosine-phosphorylated form of PKCδ accumulates in the soluble fraction of cells exposed to oxidative stress, displays lipid-independent kinase activity, and is uniquely positioned to phosphorylate target substrates throughout the cells (Steinberg, 2004). Oxidative stress can activate PKCδ by translocation, tyrosine phosphorylation, or proteolysis (Kanthasamy et al., 2003). During proteolysis, caspase-3 cleaves the native PKCδ (72–74 kDa) into 41-kDa catalytically active and 38-kDa regulatory fragments to persistently activate the kinase. The proteolytic activation of PKCδ plays a pivotal role in promoting apoptosis in various cell types (Kanthasamy et al., 2003). Since PKCδ can be recruited both as a backup kinase for gatekeeper tumor suppression and as an activator of the Ras/Raf/MEK/MAP kinase signaling pathway in cell proliferation, it acts as both a tumor suppressor and a positive regulator of cell cycle progression (Jackson and Foster, 2004). The current study demonstrates the signaling pathway leading to up-regulation of HO-1 by PL in the LPS-activated RAW264.7 macrophages.

Section snippets

Cell culture and DNA constructs

RAW264.7, a mouse macrophage cell line, obtained from American Type Culture Collection (Manassas, VA), was cultured in Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The mammalian cells were maintained at 37 °C in a humidified air/CO2 (19:1) atmosphere. Kinase-inactive forms of PKC constructs (pHACE-PKCα (K368R) and pHACE-PKCδ (K376R)) were gifts from Dr. Young-Geun Kwon (Yonsei

Participation of PKCδ in PL-induced HO-1 expression in the macrophages

In the previous work, we clearly showed that, in RAW264.7 macrophages, HO-1 plays a crucial role in anti-inflammatory activity elicited by PL. To identify signaling pathway leading to PL-induced HO-1 expression in the RAW264.7 cells, the PL's capability to phosphorylate PKC isoforms was tested. As shown in Fig. 1, PL treatment was able to enhance phosphorylation of PKCδ, with the level of phosphorylation reaching a maximum at 10–30 min and then declining to the basal level. However, PL could not

Discussion

In the previous work, PL was shown to exhibit its anti-inflammatory activity through the induction of HO-1 in the RAW264.7 macrophage cells in concentration- and time-dependent manner (Kim et al., 2006). However, the signaling pathway of PL to HO-1 induction was not previously suggested. PL was markedly able to increase phosphorylation of PKCδ but not PKCα/βII in the non-stimulated RAW264.7 macrophage cells (Fig. 1). Using general and selective PKC inhibitors, the specific induction of PKCδ by

Acknowledgements

This study was supported by Kangwon BIO-NURI. We are very grateful to Pro. Seung-Tak Lee of Yonsei University for kindly donating plasmids used in this study. Special thanks to Mr. Sang-Ro Han, Samsung Folk Medicine Co., Chuncheon, Korea for kindly donating the mushroom.

References (46)

  • A. Abate et al.

    Apigenin decreases hemin-mediated heme oxygenase-1 induction

    Free Radical Biology and Medicine

    (2005)
  • L. Carpenter et al.

    Protein kinase Cδ activation by interleukin-1β stabilizes inducible nitric-oxide synthase mRNA in pancreatic β-cells

    Journal of Biological Chemistry

    (2001)
  • Y.C. Chang et al.

    The up-regulation of heme oxygenase-1 expression in human gingival fibroblast stimulated with nicotine

    Journal of Periodontal Research

    (2005)
  • J.-M. Chow et al.

    Quercetin, but not rutin and quercitrin, prevention of H2O2-induced apoptosis via antioxidant activity and heme oxygenase 1 gene expression

    Biochemical Pharmacology

    (2005)
  • S. Dhakshinamoorthy et al.

    Functional characterization and role of INrf2 in antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase gene

    Oncogene

    (2001)
  • D. Gao et al.

    Resveratrol reduces the elevated level of MMP-9 induced by cerebral ischemia-reperfusion in mice

    Life Sciences

    (2006)
  • J.D. Graves et al.

    Protein phosphorylation and signal transduction

    Clinical Pharmacology and Therapeutics

    (1999)
  • S.S. Greenberg et al.

    Role of PKC and tyrosine in ethanol-mediated inhibition of LPS-inducible nitric oxide synthase

    Alcohol

    (1998)
  • T. Grimm et al.

    Inhibition of NF-κB activation and MMP-9 secretion by plasma of human volunteers after ingestion of marine pine bark extract (Pycnogenol)

    Journal of Inflammation

    (2006)
  • M. Gschwendt et al.

    Rottlerin, a novel protein kinase inhibitor

    Biochemical and Biophysical Research Communications

    (1994)
  • T. Ishii et al.

    Oxidative stress-inducible proteins in macrophages

    Free Radical Research

    (1999)
  • K. Itoh et al.

    Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain

    Genes and Development

    (1999)
  • D.N. Jackson et al.

    The enigmatic protein kinase Cδ: complex roles in cell proliferation and survival

    The FASEB Journal

    (2004)
  • A.G. Kanthasamy et al.

    Role of proteolytic activation of protein kinase Cδ in oxidative stress-induced apoptosis

    Antioxidants and Redox Signaling

    (2003)
  • M.H. Kapturczak et al.

    Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1-deficient mouse

    American Journal of Pathology

    (2006)
  • Y.S. Keum et al.

    Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element-mediated heme oxygenase-1 in human hepatoma HepG2 cells

    Cancer Research

    (2006)
  • B.-C. Kim et al.

    Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts

    Journal of Biological Chemistry

    (1999)
  • G.Y. Kim et al.

    Acidic polysaccharide isolated from Phellinus linteus induces nitric oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C

    Biochemical and Biophysical Research Communications

    (2003)
  • G.Y. Kim et al.

    Acidic polysaccharide isolated from Phellinus linteus enhances through the up-regulation of nitric oxide and tumor necrosis factor-alpha from peritoneal macrophages

    Journal of Ethnopharmacology

    (2004)
  • S.-H. Kim et al.

    Anti-inflammatory and related pharmacological activities of the n-BuOH subfraction of mushroom Phellinus linteus

    Journal of Ethnopharmacology

    (2004)
  • B.-C. Kim et al.

    Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom Phellinus linteus in LPS-stimulated RAW264.7 macrophages

    Journal of Ethnopharmacology

    (2006)
  • R.C. Lantz et al.

    The effect of turmeric extracts on inflammatory mediator production

    Phytomedicine

    (2005)
  • H.-J. Lee et al.

    Cambodian Phellinus linteus inhibits experimental metastasis of melanoma cells in mice via regulation of urokinase type plasminogen activator

    Biological and Pharmaceutical Bulletin

    (2005)
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      Phellinus linteus is a medicinal mushroom that exhibits various pharmacological effects, including antitumor [1,2], anticancer [3,4], immunomodulatory [3], antidiabetic [3,5], hypoglycemic [6], anti-inflammatory [3,7], and antioxidative effects [3,8]. Hispidin is a yellow-colored polyphenol pigment and a secondary metabolite produced by P. linteus and possesses antioxidant [9,10], anticancer [11,12], anti-inflammatory [13,14], anti-viral [15], anti-obesity [16], and anti-dementia [17] properties. However, the fruiting body of P. linteus is difficult to obtain in the wild and the number of mushrooms available in the wild is insufficient to meet the medical demand.

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