involucrin and epigallocatechin-gallate

Persistent environmental insult can convert a normal cell into a cancer cell. However, various natural chemopreventive agents called antioxidants can retard this progression. We have recently explored the effects of several chemopreventive agents, including green tea polyphenol and curcumin, on normal human keratinocyte function. Our findings suggest that a bioactive polyphenol from green tea, (-)-epigallocatechin-3-gallate (EGCG), acts to increase involucrin gene expression, suggesting that EGCG treatment enhances normal human keratinocyte differentiation. Mechanistic studies indicate that EGCG alters mitogen-activated protein kinase cascade function to activate involucrin gene transcription via a Ras, MEKK1, MEK3, ERK1/2-p38delta cascade that targets AP1 and CAATT enhancer binding protein transcription factors. These findings suggest that EGCG may inhibit disease progression by promoting keratinocyte differentiation. Parallel studies indicate that not all antioxidants produce a similar response. Curcumin, an antioxidant derived from the turmeric, antagonizes the EGCG-dependent response by interfering in this signaling pathway. These studies suggest that different antioxidant may produce antagonistic effects in tissues.

Topics: Antioxidants; Catechin; Cell Differentiation; Chemoprevention; Curcumin; Flavonoids; Gene Expression; Humans; Keratinocytes; p38 Mitogen-Activated Protein Kinases; Phenols; Polyphenols; Protein Precursors; Signal Transduction; Tea

Previous studies suggest that the novel protein kinase C (PKC) isoforms initiate a mitogen-activated protein kinase (MAPK) signaling cascade that regulates keratinocyte differentiation. However, assigning these functions has relied on treatment with pharmacologic inhibitors and/or manipulating kinase function using overexpression of wild-type or dominant-negative kinases. As these methods are not highly specific, an obligatory regulatory role for individual kinases has not been assigned. In this study, we use small interfering RNA knockdown to study the role of individual PKC isoforms as regulators of keratinocyte differentiation induced by the potent differentiating stimulus, 12-O-tetradecanoylphorbol-13-acetate (TPA). PKC-delta knockdown reduces TPA-activated involucrin promoter activity, nuclear activator protein-1 factor accumulation and binding to DNA, and cell morphology change. Knockdown of PKC downstream targets, including MEKK-1, MEK-6, MEK-3, or p38-delta, indicates that these kinases are required for these responses. Additional studies indicate that knockdown of PKC-eta inhibits TPA-dependent involucrin promoter activation. In contrast, knockdown of PKC-alpha (a classical PKC isoform) or PKC-epsilon (a novel isoform) does not inhibit these TPA-dependent responses. Further studies indicate that PKC-delta is required for calcium and green tea polyphenol-dependent regulation of end responses. These findings are informative as they suggest an essential role for selected PKC and MAPK cascade enzymes in mediating a range of end responses to a range of differentiation stimuli in keratinocytes.

Topics: Anticarcinogenic Agents; Calcium; Carcinogens; Catechin; Cell Differentiation; Cells, Cultured; Epidermal Cells; Humans; Keratinocytes; MAP Kinase Kinase 3; MAP Kinase Kinase 6; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 13; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C-delta; Protein Precursors; RNA, Small Interfering; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

Antioxidants are important candidate agents for the prevention of disease. However, the possibility that different antioxidants may produce opposing effects in tissues has not been adequately explored. We have reported previously that (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol antioxidant, stimulates expression of the keratinocyte differentiation marker, involucrin (hINV), via a Ras, MEKK1, MEK3, p38delta signaling cascade (Balasubramanian, S., Efimova, T., and Eckert, R. L. (2002) J. Biol. Chem. 277, 1828-1836). We now show that EGCG activation of this pathway results in increased CCAAT/enhancer-binding protein (C/EBPalpha and C/EBPbeta) factor level and increased complex formation at the hINV promoter C/EBP DNA binding site. This binding is associated with increased promoter activity. Mutation of the hINV promoter C/EBP binding site eliminates the regulation as does expression of GADD153, a dominant-negative C/EBP factor. In contrast, a second antioxidant, curcumin, inhibits the EGCG-dependent promoter activation. This is associated with inhibition of the EGCG-dependent increase in C/EBP factor level and C/EBP factor binding to the hINV promoter. Curcumin also inhibits the EGCG-dependent increase in endogenous hINV levels. The curcumin-dependent suppression of C/EBP factor level is inhibited by treatment with the proteasome inhibitor MG132, suggesting that the proteasome function is required for curcumin action. We conclude that curcumin and EGCG produce opposing effects on involucrin gene expression via regulation of C/EBP factor function. The observation that two antioxidants can produce opposite effects is an important consideration in the context of therapeutic antioxidant use.

Topics: Antioxidants; Binding Sites; Catechin; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Curcumin; Cysteine Endopeptidases; DNA; Enzyme Activation; Flavonoids; Humans; Keratinocytes; MAP Kinase Kinase 3; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 13; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Mutation; Phenols; Polyphenols; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Protein Precursors; Protein-Tyrosine Kinases; Tea; Transcription Factor CHOP; Transcription Factors

Cancer begins with a normal cell that, due to persistent environmental insult, is transformed, via a series of progressively more insidious steps, into a cancer cell. A major goal of chemopreventive therapy is to alter the normal cell response to the environmental agent with the goal of inhibiting disease progression. (-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive green tea antioxidant that possesses remarkable cancer chemopreventive properties. We have recently explored the hypothesis that EGCG prevents cancer by promoting keratinocyte differentiation. Based on our findings, we argue that EGCG acts to enhance the differentiation of normal keratinocytes. This is a potentially important finding, as it represents a novel mechanism of disease inhibition by EGCG--cancer preventive "differentiation therapy". However, not all antioxidant chemopreventive agents work by this mechanism. Curcumin, for example, inhibits the differentiation-promoting activity of EGCG. This report discusses the mechanism of EGCG and curcumin action in regulating expression of involucrin, a marker of keratinocyte differentiation.

Topics: Anticarcinogenic Agents; Catechin; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Curcumin; Drug Interactions; Humans; Keratinocytes; Mitogen-Activated Protein Kinase 13; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Precursors; Tea

(-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea that efficiently reduces epidermal cancer cell proliferation. This inhibition is associated with a reduction in activator protein 1 (AP1) transcription factor level and activity. However, its effects on AP1 function in normal epidermal cells have not been extensively explored. Our present studies show that EGCG regulates normal keratinocyte function. To understand the mechanism of action, we examined the effects of EGCG on AP1 factor activity, MAPK signal transduction, and expression of the AP1 factor-regulated human involucrin (hINV) gene. EGCG increases hINV promoter activity in a concentration-dependent manner that requires the presence of an intact hINV promoter AP1 factor binding site. This response appears to be physiologic, as endogenous hINV gene expression is also increased. Fra-1, Fra-2, FosB, JunB, JunD, c-Jun, and c-Fos levels are increased by EGCG treatment, as is AP1 factor binding to hINV promoter AP1 site. Gel mobility shift studies show that this complex contains Fra-1 and JunD. Signal transduction analysis indicates that the EGCG response requires Ras, MEKK1, MEK3, and p38 kinases. Kinase assays and inhibitor studies suggest that p38delta is the p38 isoform responsible for the regulation. These changes are also associated with a cessation of cell proliferation and enhanced cornified envelope formation. These studies show that in normal human keratinocytes EGCG markedly increases, via a MAPK signaling mechanism, AP1 factor-associated responses.

Topics: Catechin; Cells, Cultured; Gene Expression Regulation; Humans; Keratinocytes; MAP Kinase Kinase 3; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Protein Precursors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; ras Proteins; Tea; Transcription Factor AP-1