Cytoprotective effect of phlorofucofuroeckol-A on UVB induced oxidative stress in mouse epidermal JB6 cells

Abstract

피부는 태양광선에 노출되어 있어 자외선에 의한 표적기관이 될 수 있다. 태양광선이 포함하는 여러 가지 전자기파 중에 지표에 도달하여 피부에 생물학적인 영향을 가장 많이 주는 광선은 자외선이다. 특히 UV는 일광화상, 피부노화, 피부암 등의 생물학적 변화를 일으키는 가장 중요한 역할을 하는 파장으로 알려져 있다. UVB 조사에 의해 발생되는 reactive oxygen species (ROS)는 지질과 단백질 그리고 DNA를 산화시키는 원인으로 다양한 피부 질병과 관련이 있다. 본 연구에서는 UVB 조사에 의하여 JB6 세포에 산화적 스트레스를 유도한 후 이에 관련된 mitogen-activated protein kinase (MAPK)의 활성화에 대한 phlorofucofuroeckol-A (PFF-A)의 세포보호효과를 분석하였다. PFF-A를 JB6 세포에 처리한 결과 UVB 조사에 의해 생성된 free radical을 효과적으로 제거하였다. PFF-A를 JB6 세포에 처리한 결과 UVB조사에 의해 활성화된 MAPK family인 ERK1/2, JNK, 그리고 p38 단백질의 인산화를 억제시켰다. 그리고 UVB 조사 전 PFF-A의 처리는 항산화 또는 해독화 전사인자인 NF-E2 related factor-2 (Nrf2)를 활성화시켜 catalase, superoxide dismutase 및 heme oxygenase-1 (HO-1) 전사를 활성화시켰다. 따라서 PFF-A 처리에 의하여 항산화 효소의 발현을 조절하는 전사인자의 활성화로 인하여 항산화 효소의 발현을 증가시킴을 알 수 있었다.

Well-established epidemiological evidences indicate that ultraviolet (UV) radiation of the sunlight is the major environmental carcinogen responsible for the development of skin cancer (Armstrong et al., 2001). UV radiation has proved to be a potent carcinogen as it can induce skin cancer in the absence of any other potential tumor promoter. The process of skin cancer induction can be divided into three overlapping stages: initiation, promotion, and progression of tumors (Digiovanni, 1992). Initiation of skin cancer by UV involves the induction of an irreversible DNA damage and formation of photoproducts in critical genes. If this damage remains unrepaired, it may result in the mutation of proto-oncogenes and tumor suppressor genes. Chronic exposure to UV light and clonal expansion of the initiated cells leads to tumor promotion and the development of a benign tumor. It has been documented that UV radiation-induced skin tumor promotion is closely associated with alterations in the induced signal transduction pathways, including the activation of mitogen-activated protein kinases (MAPKs) that lead eventually to the transcription of specific set of genes as part of a general UV response (Bode et al., 2003). Progression of skin cancer entails the transformation of the benign tumors into malignant skin cancers (Madronich et al., 1998). UV irradiation is potent inducer of reactive oxygen species, which have been implicated in cutaneous aging as well as in skin cancer and various cutaneous inflammatory disorders (Devary et al., 1992; Bender et al., 1997). UV irradiation have been reported to upregulate expression of genes such as c-fos and c-jun (Karin, 1995; Su et al., 1996). Transcription of many genes is mediated by the sequential activation of cytoplasmic protein kinases, and the mitogen-activated protein kinase (MAPK) plays a major role in triggering and coordinating these gene response (Cobb et al., 1995). MAPKs, a group of serine/threonine-specific, proline-directed protein kinases are known to modulate transcription factor activities. Three structurally related but biochemically and functionally distinct MAPK signal transduction pathways have been identified and include the extracellular signal regulated kinases (ERK), c-jun N-terminal kinases (JNK), and p38 (Guyton et al., 1996). Transient activation of ERK is responsible for proliferation and differentiation (Chen et al., 1996) and has also been shown to be involved in tumor promotion processes especially stimulated by the oxidant state (Ip et al., 1998). Stimulation of JNK and p38 can mediate differentiation, inflammatory response, and cell death (Robinson et al., 1997; Wang et al., 1998; Wilmer et al., 1997). There is evidence that antioxidants can attenuate MAPK activation (Kensler et al., 1997; Chen et al., 2004). UV radiation cause activation of the phosphatidylinositol 3-kinase (PI3K) pathway as well as the activation of tyrosine receptors and Ras (Kabuyama et al., 1998; Whitman et al., 1998). A family of PI3K enzymes phosphorylates the number 3 position of the inositol ring of some different phosphoinositides (Cantley et al., 1991). Among the phosphoinositides, phosphatidylinositol diphosphate is believed to be the preferred substrate in vivo generating the second messenger phosphatidylinositol (3,4,5) triphosphate (Stephens et al., 1991; Carpenter et al., 1996). PI3K plays a central role in a broad range of biological effects such as cell growth, apoptosis, intercellular vesicle trafficking/secretion, regulation of actin, cell migration, and integrin function (Keely et al., 1997; Stambolic et al., 1999). In addition, accumulating evidence suggests the importance of PI3K signaling in carcinogenesis (Krasilnikov et al., 2000; Sugimoto et al., 1984). Initially PI3K was a subject of interest because of its known ability to form complexes with some viral oncoproteins (Macara et al., 1984; Klippel et al., 1998) and also because of its involvement in the viral transformation process (Shayesteh et al., 1998). The oncogenic transformed phenotype was observed in mammalian fibroblasts transfected with the constitutively active 110a (Phillips et al., 1998). In fact, alterations or amplification of PI3K have been detected in a number of human malignances (Marte et al., 1997; Dudek et al., 1997). UV irradiation induces the activation of Akt. In a wide range of cellular systems, Akt has been shown to control intracellular pathways responsible for preventing cell death in response to a variety of extracellular stimuli (Kulik et al., 1997; Medema et al., 2000; Muise-Helmericks et al., 1998). Furthermore, several reports has shown that Akt is not only a "cell survival" kinase but it may play an important role in protein synthesis, glycogenesis, and regulation of cell cycle progression (Bellacosa et al., 1991; Cerutti, 1994). In contrast, identification of the gene encoding Akt as a transforming oncogene that causes thymic lymphomas in mice suggests a role for Akt in tumorigenesis (Darr et al., 1994). Nrf2, NF-E2-related factor 2 is sequestered in the cytoplasm as an inactive complex with its cytosolic repressor Kelch-like ECH associated protein 1 (Keap1). Dissociation of Nrf2 from the inhibitory protein Keap1 is a prerequisite for nuclear translocation and subsequent DNA binding of Nrf2. After forming a heterodimer with small Maf protein inside the nucleus, the active Nrf2 binds to cis-acting ARE or EpRE, also alternatively known as Maf recognition element (Lee et al., 2005a). Besides the dissociation of the Nrf2-Keap1 complex that is facilitated by covalent modification or oxidation of critical cysteine residues contained in Keap1 may facilitate the dissociation of Keap1-Nrf2 complex or increase the stability of Nrf2. Cysteine residues present in Keap1 serve as a molecular sensor for recognizing the altered intracellular redox-status triggered by electrophiles or ROS (Lee et al., 2005b). Besides the direct oxidation or covalent modification of thiol groups contained in Keap1, the Nrf2-Keap1 signaling can be modulated directly by post-transcriptional modification of Nrf2. Phosphorylation of Nrf2 on its serine and threonine residues by several kinases such as protein kinase C (PKC), phosphoinositol 3-kinase (PI3K), and mitogen activated protein kinases (MAPKs) has been demonstated to activate Nrf2 (Owuor et al., 2002). Upon nuclear translocation, Nrf2 not only binds to the specific consensus cis-element called ARE or EpRE present in the promoter region of genes encoding many antioxidant enzymes but also to other trans-acting factors such as small Maf-F/G/K as well as the coactivators of ARE including cAMP response element binding protein(CREB)-binding protein (CBP)/p300 that can coordinately regulate the ARE-driven antioxidant gene transcription. AREs have been found in the 5'-flanking region of many genes involved with cytoprotection from oxidative stress, such as catalase, superoxide dismutase (SOD), heme oxygenase 1 (HO-1), and glutathion (Jaiswal, 2004; Juan et al., 2005). HO-1 is the inducible form of three isozymes of heme oxygenase, a microsomal enzyme which catalyzes the rate-limiting step in heme catabolism (Tyrrell, 1991). Three isoforms of HO that are the products of separate genes have been identified in mammals (Maines et al., 1997; Tenhunen et al., 1969; Yoshidat et al., 1978). HO-1 is transcriptionally upregulated as a sensitive anti-inflammatory protein by various types of oxidative stress, such as oxidized LDL (Maines et al., 1974.), UV radiation (McCoubrey et al., 1994), thiol scavengers (Shibahara et al., 1993), and hypoxia (McCoubrey et al., 1997; Wang et al., 1998), as well as substrate heme (Keyse et al., 1998) in the cardiovascular system. A common feature of these HO-1 inducers is their ability to regulate the intracellular redox state. HO-1 is also transcriptionally activated through several regulatory mechanisms. Studies on the promoter region of HO-1 have revealed transcriptionally responsive elements, including activator protein I, activator protein II, nuclear factor-kB, interleukin-6-responsive elements, and an antioxidant response element (ARE) (Maines et al., 1997; Lee et al., 1997; Morita et al., 1995). HO-2 is constitutively expressed in many organs throughout the body, although it is particularly high in the brain and testes, but is unresponsive to any of the inducers of HO-1 (Shibahara et al., 1979). The third isoform, HO-3, is nearly devoid of catalytic activity (Alam et al., 1992), However, Hayashi and colleagues (Hayashi et al., 2004) recently reported that HO-3 is a pseudo gene derived from HO-2 transcripts. Moreover, HO-1 is a sensitive marker for oxidative stress induced by the substate heme itself, as well as a wide variety of cellular stressors including reactive oxygen species such as hydrogen peroxide, hydroxyl radical, nitric oxide, and 1O2 (Maines et al., 1986; Applegate et al., 1991). In dermal fibroblasts, photochemically generated 1O2 seems to be the main effector species for UVA-mediated HO-1 up-regulation (Basu-Modak et al., 1993). Ecklonia stolonifera OKAMURA (Fig. 1) is a perennial brown alga, belonging to the family Laminariaceae. It is abundant in the Southern coast of Korea and Japan. It is popular as a health food and is occasionally used as a gynecopathy in Japan (Sugiura et al., 2006). Several compounds from Ecklonia species exhibits radical scavenging activity (Kang et al., 2003), anti-plasmin inhibiting activity (Fukuyama et al., 1990), anti-mutagenic activity (Han et al., 2000), tyrosinase inhibitory activity (Park et al., 2003). Phlorotinnins which is oligomeric polyphenol of phloroglucinol units are responsible for the biological activities of Ecklonia. Mouse keratinocyte JB6 cells are well stuited to study tumor promotion, as these cells are sensitive to tumor promoter-mediated cell transformation and promotion (Suzukawa et al., 2002). In this study, the cytoprotective effect of PFF-A on the UVB-induced oxidative stress was evaluated on the activation of Nrf2 transcription factor and the activations of its up-stream proteins and expression of its down-stream proteins.