title: PKC-delta and -eta, MEKK-1, MEK-6, MEK-3, and p38-delta are essential mediators of the response of normal human epidermal keratinocytes to differentiating agents.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Gautam
authors.initials: G
authors.lastname: Adhikary
authors.forename: Yap Ching
authors.initials: YC
authors.lastname: Chew
authors.forename: E Albert
authors.initials: EA
authors.lastname: Reece
authors.forename: Richard L
authors.initials: RL
authors.lastname: Eckert
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D000078404:Epidermal Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D048371:MAP Kinase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D048669:MAP Kinase Kinase 6 / Q000235:genetics / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D048292:Mitogen-Activated Protein Kinase 13 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics; D034741:RNA, Small Interfering; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism
chemical_list: D016588:Anticarcinogenic Agents; D002273:Carcinogens; D011498:Protein Precursors; D034741:RNA, Small Interfering; D018808:Transcription Factor AP-1; C032253:involucrin; D002392:Catechin; C045651:epigallocatechin gallate; D048292:Mitogen-Activated Protein Kinase 13; C081975:protein kinase C eta; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; C482530:MAP2K3 protein, human; C482566:MAP2K6 protein, human; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium
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