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
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.
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2021, Journal of the Taiwan Institute of Chemical EngineersCitation Excerpt :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.