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Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway*

Open AccessPublished:December 07, 2001DOI:https://doi.org/10.1074/jbc.M107897200
      In this study, we investigated the effect of tea polyphenols, (−)-epigallocatechin-3-gallate or theaflavins, on UVB-induced phosphatidylinositol 3-kinase (PI3K) activation in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with these polyphenols inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70 S6 kinase (p70 S6-K), PI3K downstream effectors, were also attenuated by the polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with a specific mitogen-activated protein/extracellular signal-regulated protein kinases (Erks) kinase 1 inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190, blocked UVB-induced activation of both Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling, the Erk pathway is mediated by PI3K. Moreover, pretreatment with rapamycin, an inhibitor of p70 S6-K, inhibited UVB-induced activation of p70 S6-K, but UVB-induced activation of Akt did not change. Interestingly, UVB-induced p70 S6-K activation was directly blocked by the addition of (−)-epigallocatechin-3-gallate or theaflavins, whereas these polyphenols showed only a weak inhibition on UVB-induced Akt activation. Because PI3K is an important factor in carcinogenesis, the inhibitory effect of these polyphenols on activation of PI3K and its downstream effects may further explain the anti-tumor promotion action of these tea constituents.
      MAP
      mitogen-activated protein
      aPKC
      atypical protein kinase C
      EGCG
      (−)-epigallocatechine-3-gallate
      DTT
      dithiothreitol
      EGF
      epidermal growth factor
      Erks
      extracellular signal-regulated protein kinases
      FBS
      fetal bovine serum
      JNKs
      c-Jun N-terminal kinases
      MEM
      minimal essential medium
      mTOR
      mammalian target of rapamycin
      p70 S6-K
      ribosomal p70 S6 protein kinase
      PDK
      phosphatidylinositol 3,4,5-triphosphate-dependent protein kinase
      PI-3
      4,5-P3, phosphatidylinositol 3,4,5-triphosphate
      PI3K
      phosphatidylinositol 3-kinase
      PKB
      protein kinase B
      PKC
      protein kinase C
      PH
      pleckstrin homology
      MOPS
      4-morpholinepropanesulfonic acid
      Nonmelanoma skin cancer is the most frequently diagnosed malignancy in the United States, and the most crucial risk factor in development of nonmelanoma skin cancer is UV radiation from sunlight (
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      • Tong T.
      • Bolden S.
      • Wingo P.A.
      ). Although the UV portion of sunlight is divided into three components based on wavelength, the UV light that reaches the surface of the earth is comprised of UVA (320–400 nm) and UVB (280–320 nm). UVB is a major cause of skin cancer (
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      ). In animal models, UV radiation acts as both a tumor initiator and tumor promoter (
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      ,
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      • Wulf H.C.
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      ). The initiating effects of UV radiation include chromosomal alterations and mutations that result in DNA damage (
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      ). The mitogen-activated protein (MAP)1 kinase family (e.g. extracellular signal-regulated protein kinases (Erks), c-Jun N-terminal kinases (JNKs), and p38 kinase) at least partially mediates the activation of transcription factors (
      • Kyriakis J.M.
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      ,
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      ). In addition, UV radiation triggers activation of tyrosine receptors, such as the epidermal growth factor (EGF) receptor and the insulin receptor, Src family tyrosine kinases, and the small GTP-binding protein, Ras, and MAP kinases (
      • Rosette C.
      • Karin M.
      ,
      • Huang C.
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      • Bowden G.T.
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      • Rubie E.A.
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      ,
      • Smith M.L.
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      ). On the other hand, UV radiation also causes activation of the phosphatidylinositol 3-kinase (PI3K) pathway as well as the activation of tyrosine receptors and Ras (
      • Coffer P.J.
      • Burgering B.M.
      • Peppelenbosch M.P.
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      ,
      • Kabuyama Y.
      • Hamaya M.
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      ). A family of PI3K enzymes phosphorylates the number 3 position of the inositol ring of some different phosphoinositides (
      • Whitman M.
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      • Keeler M.
      • Keller T.
      • Cantley L.
      ). Among the phosphoinositides, phosphatidylinositol (
      • Hall E.J.
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      • Fry M.
      • Geard C.
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      • Mitchell J.
      • Oleinick N.
      • Rubin J.
      • Tu A.
      • Ullrich R.
      • Walden C.
      • Ward J.
      ,
      • Ananthaswamy H.N.
      • Pierceall W.E.
      ) diphosphate is believed to be the preferred substrate in vivo generating the second messenger phosphatidylinositol (
      • Ananthaswamy H.N.
      • Loughlin S.M.
      • Cox P.
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      • Ullrich S.E.
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      ,
      • Hall E.J.
      • Astor M.
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      • Fry M.
      • Geard C.
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      • Mitchell J.
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      • Rubin J.
      • Tu A.
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      ,
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      ) triphosphate (
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      • Graziani A.
      • Kapeller R.
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      ,
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ). 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 (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Keely P.J.
      • Westwick J.K.
      • Whitehead I.P.
      • Der C.J.
      • Parise L.V.
      ). In addition, accumulating evidence suggests the importance of PI3K signaling in carcinogenesis (
      • Stambolic V.
      • Mak T.W.
      • Woodgett J.R.
      ,
      • Krasilnikov M.A.
      ). Initially PI3K was a subject of interest because of its known ability to form complexes with some viral oncoproteins (
      • Sugimoto Y.
      • Whitman M.
      • Cantley L.C.
      • Erikson R.L.
      ,
      • Macara I.G.
      • Marinetti G.V.
      • Balduzzi P.C.
      ) and also because of its involvement in the viral transformation process (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ). Later, expression of a retroviral oncogene, v-p3k, which codes for the catalytic subunit (p110α) of PI3K, was shown to induce oncogenic transformation in chicken cells (
      • Chang H.W.
      • Aoki M.
      • Fruman D.
      • Auger K.R.
      • Bellacosa A.
      • Tsichlis P.N.
      • Cantley L.C.
      • Roberts T.M.
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      ). The oncogenic transformed phenotype was observed in mammalian fibroblasts transfected with the constitutively active p110α (
      • Klippel A.
      • Escobedo M.A.
      • Wachowicz M.S.
      • Apell G.
      • Brown T.W.
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      • Kavanaugh W.M.
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      ). In fact, alterations or amplifications of PI3K have been detected in a number of human malignances (
      • Shayesteh L.
      • Lu Y.
      • Kuo W.L.
      • Baldocchi R.
      • Godfrey T.
      • Collins C.
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      ,
      • Phillips W.A.
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      • Thomas R.J.
      • Mitchell C.A.
      ). Our previous studies also demonstrated that PI3K is necessary for 12-O-tetradecanoylphorbol-13-acetate- or EGF-induced cell transformation in mouse epidermal JB6 cells (
      • Huang C.
      • Schmid P.C.
      • Ma W.Y.
      • Schmid H.H.
      • Dong Z.
      ,
      • Huang C.
      • Ma W.Y.
      • Dong Z.
      ). Those findings suggested that PI3K activation by UV exposure is a critical factor in UV-induced carcinogenesis. The serine/threonine proto-oncogene Akt (also known as protein kinase B or PKB) was first identified as the cellular homolog of the transforming oncogene product v-Akt (
      • Bellacosa A.
      • Testa J.R.
      • Staal S.P.
      • Tsichlis P.N.
      ). It plays an important role in the regulation of a wide spectrum of cellular signaling events including apoptosis, glycogen metabolism, protein synthesis, and cell cycle regulation (
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      • Woodgett J.R.
      ,
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      ). Akt is activated by growth factors and oncogenes through PI3K-dependent signaling (
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ,
      • Hemmings B.A.
      ). Akt has been shown to be overexpressed in ovarian, prostate, breast, and pancreatic cancers and is associated with a poor prognosis and increased tumorigenicity (
      • Stambolic V.
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      • Woodgett J.R.
      ,
      • Cheng J.Q.
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      • Nevi G.
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      ). Alteration and down-regulation of the tumor suppressor gene PTEN/MMAC1, which directly antagonizes PI3K, are frequently observed in a number of cancers (
      • Stambolic V.
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      ,
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      ). The mutations are thought to be associated with Akt activation as well as an increased level of phosphatidylinositol 3,4,5-triphosphate (
      • Dahia P.L.
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      ). An accumulation of evidence suggests that ribosomal p70 S6 kinase (p70 S6-K) is also a crucial effector of PI3K in response to growth factors (
      • Duronio V.
      • Scheid M.P.
      • Ettinger S.
      ). Activation of p70 S6-K regulates a variety of cellular functions involved in the mitogenic response including protein synthesis, translation of specific mRNA species, cell cycle regulation, and cell motility (
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      ,
      • Volarevic S.
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      ,
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      ). The p70 S6-K is constitutively activated in small cell lung cancer cells, and anchorage-independent proliferation in these cells is mediated through an Akt and p70 S6-K-dependent pathway (
      • Moore S.M.
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      • Haslett C.
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      ). Kl-Ras-mediated transformation is inhibited by blocking both the MAP kinase and p70 S6-K pathways (
      • Fukazawa H.
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      ). Activation of p70 S6-K has been shown to be essential for the UVB-induced increase in MMP-1 and MMP-3 proteins in human dermal fibroblasts (
      • Brenneisen P.
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      ).
      Tea and tea polyphenol preparations have been shown to inhibit carcinogenesis at several organ sites in rodent models (
      • Yang C.S.
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      ,
      • Katiyar S.K.
      • Mukhtar H.
      ). The polyphenols from green tea and black tea, (−)-epigallocatechin-3-gallate (EGCG) and theaflavins, respectively, are generally considered to be the most effective components for inhibition of carcinogenesis (
      • Huang M.T.
      • Ho C.T.
      • Wang Z.Y.
      • Ferraro T.
      • Finnegan-Olive T.
      • Lou Y.R.
      • Mitchell J.M.
      • Laskin J.D.
      • Newmark H.
      • Yang C.S.
      • Conney A.H.
      ,
      • Dong Z.
      • Ma W.
      • Huang C.
      • Yang C.S.
      ,
      • Ahmad N.
      • Gupta S.
      • Mukhtar H.
      ). Tea polyphenols also have been shown to inhibit UV-induced carcinogenesis in animal models (
      • Huang M.T.
      • Ho C.T.
      • Wang Z.Y.
      • Ferraro T.
      • Finnegan-Olive T.
      • Lou Y.R.
      • Mitchell J.M.
      • Laskin J.D.
      • Newmark H.
      • Yang C.S.
      • Conney A.H.
      ,
      • Wang Z.Y.
      • Huang M.T.
      • Ferraro T.
      • Wong C.Q.
      • Lou Y.R.
      • Reuhl K.
      • Iatropoulos M.
      • Yang C.S.
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      ,
      • Gensler H.L.
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      • Wachter G.A.
      • Dorr R.
      • Dvorakova K.
      • Alberts D.S.
      ,
      • Record I.R.
      • Dreosti I.E.
      ). The most widely recognized biological properties of tea polyphenols are their antioxidant activity in scavenging reactive oxygen or nitrogen species (
      • Wiseman S.A.
      • Balentine D.A.
      • Frei B.
      ). The molecular mechanism of their anti-tumor promotion effects might be related to their ability to block signal transduction pathways leading to carcinogenesis (
      • Dong Z.
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      • Huang C.
      • Yang C.S.
      ,
      • Ahmad N.
      • Gupta S.
      • Mukhtar H.
      ,
      • Mukhtar H.
      • Ahmad N.
      ). Recently, we have shown that tea polyphenols inhibit UVB-induced AP-1 activation in mouse epidermal JB6 cells (
      • Nomura M.
      • Ma W.Y.
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      • Yang C.S.
      • Bowden G.T.
      • Miyamoto K.
      • Dong Z.
      ). Thus, the inhibitory effect of the tea polyphenols in UVB-induced carcinogenesis may result from the blocking of these signal transduction pathways. In the present study, we investigated the effects of tea polyphenols on UVB-induced activation of the PI3K and its downstream effectors, which are critical factors in carcinogenesis.

      DISCUSSION

      Green tea is widely used as a beverage in China, Japan, and other Asian countries, whereas black tea is more popular in Western countries (
      • Yang C.S.
      • Wang Z.Y.
      ,
      • Katiyar S.K.
      • Mukhtar H.
      ,
      • Trevisanato S.I.
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      ). In recent years many animal studies and several epidemiological studies have reported the anti-carcinogenic effects of tea (
      • Yang C.S.
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      ,
      • Katiyar S.K.
      • Mukhtar H.
      ,
      • Huang M.T.
      • Ho C.T.
      • Wang Z.Y.
      • Ferraro T.
      • Finnegan-Olive T.
      • Lou Y.R.
      • Mitchell J.M.
      • Laskin J.D.
      • Newmark H.
      • Yang C.S.
      • Conney A.H.
      ,
      • Wang Z.Y.
      • Huang M.T.
      • Ferraro T.
      • Wong C.Q.
      • Lou Y.R.
      • Reuhl K.
      • Iatropoulos M.
      • Yang C.S.
      • Conney A.H.
      ,
      • Gensler H.L.
      • Timmermann B.N.
      • Valcic S.
      • Wachter G.A.
      • Dorr R.
      • Dvorakova K.
      • Alberts D.S.
      ,
      • Record I.R.
      • Dreosti I.E.
      ,
      • Trevisanato S.I.
      • Kim Y.I.
      ). The polyphenols from green tea and black tea, EGCG and theaflavins, respectively, are generally considered to be the most effective components for inhibition of carcinogenesis (
      • Huang M.T.
      • Ho C.T.
      • Wang Z.Y.
      • Ferraro T.
      • Finnegan-Olive T.
      • Lou Y.R.
      • Mitchell J.M.
      • Laskin J.D.
      • Newmark H.
      • Yang C.S.
      • Conney A.H.
      ,
      • Dong Z.
      • Ma W.
      • Huang C.
      • Yang C.S.
      ,
      • Ahmad N.
      • Gupta S.
      • Mukhtar H.
      ,
      • Trevisanato S.I.
      • Kim Y.I.
      ). Although several mechanisms explaining the anti-carcinogenic effects of tea have been presented (
      • Wiseman S.A.
      • Balentine D.A.
      • Frei B.
      ,
      • Trevisanato S.I.
      • Kim Y.I.
      ,
      • Chen C., Yu, R.
      • Owuor E.D.
      • Kong A.N.
      ,
      • Fu Y.C.
      • Jin X.P.
      • Wei S.M.
      ), we and others suggested that tea components target specific cell signaling pathways responsible for regulating cell growth, survival, and cell transformation (
      • Dong Z.
      • Ma W.
      • Huang C.
      • Yang C.S.
      ,
      • Ahmad N.
      • Gupta S.
      • Mukhtar H.
      ,
      • Mukhtar H.
      • Ahmad N.
      ,
      • Nomura M.
      • Ma W.Y.
      • Huang C.
      • Yang C.S.
      • Bowden G.T.
      • Miyamoto K.
      • Dong Z.
      ). PI3K is an important regulatory protein that is involved in different signaling pathways and in the control of cell growth, survival, and malignant transformation (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Keely P.J.
      • Westwick J.K.
      • Whitehead I.P.
      • Der C.J.
      • Parise L.V.
      ,
      • Stambolic V.
      • Mak T.W.
      • Woodgett J.R.
      ,
      • Krasilnikov M.A.
      ). PI3K forms complexes with some viral or cellular oncoproteins (
      • Sugimoto Y.
      • Whitman M.
      • Cantley L.C.
      • Erikson R.L.
      ,
      • Macara I.G.
      • Marinetti G.V.
      • Balduzzi P.C.
      ), which have transforming activities, and by itself also has oncogenic activity (
      • Chang H.W.
      • Aoki M.
      • Fruman D.
      • Auger K.R.
      • Bellacosa A.
      • Tsichlis P.N.
      • Cantley L.C.
      • Roberts T.M.
      • Vogt P.K.
      ). The transforming effect of PI3K is suggested to be related to the activation of its downstream effector kinases, including Akt and p70 S6-K (
      • Stambolic V.
      • Mak T.W.
      • Woodgett J.R.
      ,
      • Bellacosa A.
      • Testa J.R.
      • Staal S.P.
      • Tsichlis P.N.
      ,
      • Cheng J.Q.
      • Ruggeri B.
      • Klein W.M.
      • Sonoda G.
      • Altomare D.A.
      • Watson D.K.
      • Testa J.R.
      ,
      • Cheng J.Q.
      • Godwin A.K.
      • Bellacosa A.
      • Taguchi T.
      • Franke T.F.
      • Hamilton T.C.
      • Tsichlis P.N.
      • Testa J.R.
      ,
      • Bellacosa A.
      • de Feo D.
      • Godwin A.K.
      • Bell D.W.
      • Cheng J.Q.
      • Altomare D.A.
      • Wan M.
      • Dubeau L.
      • Scambia G.
      • Masciullo V.
      • Ferrandina G.
      • Panici P.B.
      • Mancuso S.
      • Nevi G.
      • Testa J.R.
      ,
      • Moore S.M.
      • Rintoul R.C.
      • Walker T.R.
      • Chilvers E.R.
      • Haslett C.
      • Sethi T.
      ,
      • Fukazawa H.
      • Uehara Y.
      ,
      • Brenneisen P.
      • Wenk J.
      • Wlaschek M.
      • Krieg T.
      • Scharffetter-Kochanek K.
      ). We previously demonstrated that PI3K is necessary for 12-O-tetradecanoylphorbol-13-acetate- or EGF-induced cell transformation and AP-1 activation (
      • Huang C.
      • Schmid P.C.
      • Ma W.Y.
      • Schmid H.H.
      • Dong Z.
      ,
      • Huang C.
      • Ma W.Y.
      • Dong Z.
      ). We also recently found that rapamycin, which blocks p70 S6-K activation, inhibits EGF-induced cell transformation independently of AP-1 activation in mouse epidermal JB6 cells.
      M. Nomura, A. Kaji, Z. He, W.-Y. Ma, K.-I. Miyamoto, and Z. Dong, manuscript in preparation.
      UVB is a crucial risk factor for skin cancer (
      • Ananthaswamy H.N.
      • Loughlin S.M.
      • Cox P.
      • Evan R.L.
      • Ullrich S.E.
      • Kripke M.L.
      ,
      • Hall E.J.
      • Astor M.
      • Bedford J.
      • Borek C.
      • Curtis S.B.
      • Fry M.
      • Geard C.
      • Hei T.
      • Mitchell J.
      • Oleinick N.
      • Rubin J.
      • Tu A.
      • Ullrich R.
      • Walden C.
      • Ward J.
      ,
      • Ananthaswamy H.N.
      • Pierceall W.E.
      ,
      • Staberg B.
      • Wulf H.C.
      • Klemp P.
      • Poulsen T.
      • Brodthagen H.
      ) and also induces PI3K activation (
      • Coffer P.J.
      • Burgering B.M.
      • Peppelenbosch M.P.
      • Bos J.L.
      • Kruijer W.
      ,
      • Kabuyama Y.
      • Hamaya M.
      • Homma Y.
      ). Thus, PI3K and its downstream effectors are suggested to be important regulatory proteins in the tumor promotion effects of UVB.
      In this study, we found that EGCG and theaflavins inhibit UVB-induced activation of PI3K and its downstream effectors, Akt and p70 S6-K. Furthermore, we demonstrated that the Erks and p38 kinase pathways are critical for UVB-induced activation of Akt and p70 S6-K. Fig.8 shows a schematic of the proposed signaling model for Akt and p70 S6-K induced by UVB. Generally, Akt is believed to be regulated by a pathway mediated by PI3K and PDK-1 (
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ,
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ,
      • Hemmings B.A.
      ,
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ). In contrast, we recently demonstrated that UVB-induced Akt activation is mediated through Erks and p38 kinase-dependent mitogen- and stress-activated protein kinase-1 rather than the PI3K/PDK-1 pathway (
      • Nomura M.
      • Kaji A.
      • Ma W.
      • Zhong S.
      • Liu G.
      • Bowden G.T.
      • Miyamoto K.
      • Dong Z.
      ). That study (
      • Nomura M.
      • Kaji A.
      • Ma W.
      • Zhong S.
      • Liu G.
      • Bowden G.T.
      • Miyamoto K.
      • Dong Z.
      ) and the present study also suggested that PI3K is located upstream of Erks, because pretreatment of cells with a PI3K inhibitor, LY294002, or the expression of a dominant negative mutant of the PI3K p85 subunit inhibited UVB-induced phosphorylation of Erks, but not JNKs or p38 kinase.
      Figure thumbnail gr8
      Figure 8Effect of tea polyphenols on PI3K-dependent pathway induced by UVB irradiation.Exposure to UVB results in activation of the PI3K pathway. EGCG and theaflavins inhibit UVB-induced activation and phosphorylation of PI3K and its downstream effectors, Akt (Thr308/Ser473) and p70 S6-K (Thr389 and Thr421/Ser424). The tea polyphenols did not affect phosphorylation of Akt (Thr308) by PDK-1. UVB-induced p70 S6-K activation is also blocked by EGCG or theaflavins. The Erk kinase pathway, critical for UVB-induced activities of Akt and p70 S6-K, is also inhibited by tea polyphenols. ↑, activation; ⊥, inhibition; tea bags, tea polyphenols.
      In addition, we previously showed that overexpression of a dominant negative mutant of PKCλ/τ (
      • Huang C.
      • Ma W.
      • Bowden G.T.
      • Dong Z.
      ) or pretreatment with antisense oligonucleotides of PKCζ specifically inhibited UVB-induced phosphorylation of Erks (
      • Huang C.
      • Li J.
      • Chen N.
      • Ma W.
      • Bowden G.T.
      • Dong Z.
      ). PKCζ is regulated in vitroby PI-3,4,5-P3 (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ). PKCλ, as well as PKCζ, is activated in vivo through a pathway involving PI3K (
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takayanagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S.
      • Mizuno K.
      • Hirai S.
      • Kazlauskas A.
      • Ohno S.
      ,
      • Standaert M.L.
      • Galloway L.
      • Karnam P.
      • Bandyopadhyay G.
      • Moscat J.
      • Farese R.V.
      ). Atypical PKCs (aPKCs), such as the PKCλ and PKCζ, have also been shown to be activated by PDK-1 (
      • Chou M.M.
      • Hou W.
      • Johnson J.
      • Graham L.K.
      • Lee M.H.
      • Chen C.S.
      • Newton A.C.
      • Schaffhausen B.S.
      • Toker A.
      ,
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      ), and the maximal phosphorylation and activation of aPKCs by PDK-1 require PI-3,4,5-P3. These findings suggested that in UVB signaling Erk activation is mediated by pathways involving PI3K/aPKCs or PI3K/PDK-1/aPKCs. On the other hand, PDK-1 is also suggested to serve as a Thr229 kinase of p70 S6-K because the sequence surrounding the activation loop including Thr229 of p70 S6-K is highly homologous to that of Akt, which is activated by PDK-1. Pullen et al. (
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ) reported that PDK-1 activates a p70 S6-K mutant with acidic residues at Thr389 and four phosphorylation sites in the C terminus and that the catalytically inactive PDK-1 blocked insulin-induced p70 S6-K activation. However, the activation of p70 S6-K by PDK-1 was found to be independent of PI-3,4,5-P3 or pretreatment with wortmannin or rapamycin (
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ). Romanelli et al. (
      • Romanelli A.
      • Martin K.A.
      • Toker A.
      • Blenis J.
      ) showed that expression of a dominant negative mutant of PKCλ antagonized p70 S6-K activation by EGF and PDK-1. They also demonstrated that p70 S6-K forms complexesin vivo with PDK-1 and PKCλ. In addition, the interaction of the N-terminal region of p70 S6-K with the kinase domain of PKCλ and the requirement for interaction with the regulatory domain of PKCλ of the C-terminal region of p70 S6-K have been reported by Akimoto et al. (
      • Akimoto K.
      • Nakaya M.
      • Yamanaka T.
      • Tanaka J.
      • Matsuda S.
      • Weng Q.P.
      • Avruch J.
      • Ohno S.
      ).
      We found that UVB irradiation causes translocation of PKCλ to cell membrane (
      • Huang C.
      • Li J.
      • Chen N.
      • Ma W.
      • Bowden G.T.
      • Dong Z.
      ). Therefore, PDK-1 may be able to colocalize with aPKCs at the membrane. In this study, we showed that U0126, an inhibitor of MAP kinase/Erk kinase, repressed UVB-induced p70 S6-K phosphorylation at Thr389 and Thr421/Ser424. Thus, the activation of p70 S6-K by PI3K is suggested to be mediated through interaction with aPKCs or phosphorylation at Thr389 and Thr421/Ser424 by PDK-1/aPKCs/Erk pathways. Furthermore, although whether Akt phosphorylates mTOR is not clear (
      • Conus N.M.
      • Hemmings B.A.
      • Pearson R.B.
      ,
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.
      • Hemmings B.A.
      • Thomas G.
      ), several studies have reported that phosphorylation and activation of mTOR is mediated by the PI-3/Akt pathway (
      • Scott P.H.
      • Brunn G.J.
      • Kohn A.D.
      • Roth R.A.
      • Lawrence Jr., J.C.
      ,
      • Aoki M.
      • Blazek E.
      • Vogt P.K.
      ,
      • Sekulic A.
      • Hudson C.C.
      • Homme J.L.
      • Yin P.
      • Otterness D.M.
      • Karnitz L.M.
      • Abraham R.T.
      ). The Thr389 residue of p70 S6-K is known to be rapamycin-sensitive, whereas the residues in the C terminus (like Thr421 and Ser424) are considered insensitive (
      • Pearson R.B.
      • Dennis P.B.
      • Han J.W.
      • Williamson N.A.
      • Kozma S.C.
      • Wettenhall R.E.
      • Thomas G.
      ). However, more recent reports have questioned this difference (
      • Weng Q.P.
      • Kozlowski M.
      • Belham C.
      • Zhang A.
      • Comb M.J.
      • Avruch J.
      ,
      • Valentinis B.
      • Navarro M.
      • Zanocco-Marani T.
      • Edmonds P.
      • McCormick J.
      • Morrione A.
      • Sacchi A.
      • Romano G.
      • Reiss K.
      • Baserga R.
      ). We also showed that rapamycin, an inhibitor of mTOR, not only represses phosphorylation of p70 S6-K at Thr389 but blocks phosphorylation at Thr421/Ser424. In addition, SB202190, a specific p38 kinase inhibitor, inhibited UVB-induced p70 S6-K phosphorylation at Thr389 and Thr421/Ser424 and p70 S6-K has also shown to be phosphorylate by MAP kinases (
      • Zhang Y.
      • Dong Z.M.
      • Nomura M.
      • Zhong S.
      • Chen N.
      • Bode A.M.
      • Dong Z.
      ,
      • Mukhopadhyay N.K.
      • Price D.J.
      • Kyriakis J.M.
      • Pelech S.
      • Sanghera J.
      • Avruch J.
      ). These findings suggested that p38 kinase and Erks activate p70 S6-K directly by themselves or indirectly through other kinases including mTOR mediated by the mitogen- and stress-activated protein kinase 1/Akt pathway.
      Our previous study showed that EGCG or theaflavins inhibit UVB-induced Erks phosphorylation (
      • Nomura M.
      • Kaji A.
      • Ma W.
      • Zhong S.
      • Liu G.
      • Bowden G.T.
      • Miyamoto K.
      • Dong Z.
      ), and the present study demonstrated that these polyphenols attenuated UVB-induced PI3K activation. Although the inhibition of Erks phosphorylation may be due to suppressed PI3K activation, the blocking of the Erks-mediated pathway was suggested to be involved in the inhibitory effects of Akt and p70 S6-K by tea polyphenols. On the other hand, EGCG and theaflavins also directly inhibited the activation of p70 S6-K but not Akt activation. Polyphenols are known to bind to proteins, as exemplified by their binding to the proline-rich salivary proteins (
      • Murray N.J.
      • Williamson M.P.
      • Lilley T.H.
      • Haslam E.
      ,
      • Baxter N.J.
      • Lilley T.H.
      • Haslam E.
      • Williamson M.P.
      ,
      • Siebert K.J.
      ). These proteins have the general characteristics of a large size, a loose open structure, and a high proportion of hydrophobic amino acids and proline. Among the regulatory phosphorylation sites that are implicated in the activation of p70 S6-K, Ser404 is surrounded by large bulky hydrophobic amino acids in the −1 and +1 positions, similar to Thr229 and Thr389, and the remaining phosphorylation sites in C-terminal are followed by proline at the +1 position, similar to Ser371 (
      • Grove J.R.
      • Banerjee P.
      • Balasubramanyam A.
      • Coffer P.J.
      • Price D.J.
      • Avruch J.
      • Woodgett J.R.
      ). Therefore, EGCG and theaflavins may modulate the conformation of p70 S6-K by binding to the hydrophobic amino acids described above (Fig. 7 B). In contrast, the binding of these polyphenols to Akt was suggested to be unnecessary or insufficient for inhibition of its activation (Fig.7 B).
      In summary, the present results show that EGCG and theaflavins inhibited the activation of PI3K and also attenuated the activation of Akt and p70 S6-K, downstream effectors of PI3K, by inhibiting PI3K and Erk activation in UVB signaling. Furthermore, these polyphenols directly blocked UVB-induced p70 S6-K activation. Because PI3K and its downstream effectors are considered to play a critical role in carcinogenesis, these results provide insight into the biological actions of tea polyphenols on UV-induced carcinogenesis and on the molecular basis for the development of new chemopreventive agents.

      ACKNOWLEDGEMENTS

      We thank Dr. Ann M. Bode for critical reading and Andria Hansen for secretarial assistance.

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