Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614 -12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.

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(؊)-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 factorregulated 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 p38␦ 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.The polyphenol constituents of green tea inhibit carcinogenesis in a variety of tissues (1-4); however, their mechanism of action is not well understood. (Ϫ)-Epigallocatechin-3-gallate (EGCG) 1 is the major polyphenol isolated from green tea. AP1 transcription factors and AP1 factor-associated signal transduction are important targets of EGCG action (5-7). AP1 proteins consist of homodimers of Jun proteins (c-Jun, JunB, and JunD) and heterodimers of Jun and Fos (c-Fos, FosB, Fra-1, and Fra-2) factors (8). These proteins regulate transcription, differentiation, and cell proliferation by binding to specific recognition motifs in target genes (9 -13). EGCG produces specific changes in AP1 factor function in immortalized and transformed keratinocytes. These changes include an EGCGdependent reduction in phorbol ester-dependent mitogen-activated protein kinase (MAPK) activity (6), and reduced AP1 factor level and activity (7,14). EGCG also inhibits ultraviolet light-associated activation of c-Fos gene expression and the accumulation of c-Fos protein (7). Based on these studies, it has been suggested that the cancer-preventive role of EGCG may be due, in part, to its ability to reduce AP1 factor-related responses (6,7,14). However, very little information is available regarding how EGCG effects AP1 factor-regulated responses in normal keratinocytes. Our present studies show that EGCG increases AP1 factor levels in normal keratinocytes, and increases human involucrin gene expressio...

The epidermis is a dynamic renewing structure that provides life-sustaining protection from the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a carefully choreographed program of differentiation. Alteration of these events results in a variety of debilitating and life-threatening diseases. Understanding how this process is regulated is an important current goal in biology. In this review, we summarize the literature regarding regulation of involucrin, an important marker gene that serves as a model for understanding the mechanisms that regulate the differentiation process. Current knowledge describing the role of transcription factors and signaling cascades in regulating involucrin gene expression are presented. These studies describe a signaling cascade that includes the novel protein kinase C isoforms, Ras, MEKK1, MEK3, and a p38delta-extracellular signal regulated kinase 1/2 complex. This cascade regulates activator protein one, Sp1, and CCATT/enhancer-binding protein transcription factor DNA binding to two discrete involucrin promoter regions, the distal- and proximal-regulatory regions, to regulate involucrin gene expression.

Human involucrin (hINV) mRNA level and promoter activity increase when keratinocytes are treated with the differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). This response is mediated via a p38 mitogen-activated protein kinase-dependent pathway that targets activator protein 1 (Efimova, T., LaCelle, P. T., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395). In the present study we examine the role of various PKC isoforms in this regulation. Transfection of expression plasmids encoding the novel PKC isoforms ␦, ⑀, and increase hINV promoter activity. In contrast, neither conventional PKC isoforms (␣, ␤, and ␥) nor the atypical isoform () regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKC␦-selective inhibitor, rottlerin, but not by Go-6976, an inhibitor of conventional PKC isoforms, and novel PKC isoform-dependent promoter activation is inhibited by dominant-negative PKC␦. This regulation appears to be physiologically important, as transfection of keratinocytes with PKC␦, -⑀, or -increases expression of the endogenous hINV gene. Synergistic promoter activation (>100-fold) is observed when PKC⑀-or --transfected cells are treated with TPA. In contrast, the PKC␦-dependent response is more complex as either activation or inhibition is observed, depending upon PKC␦ concentration. Human involucrin (hINV)1 is a marker of keratinocyte differentiation that is exclusively expressed in differentiated, suprabasal keratinocytes, both in vivo and in vitro (1-5). 12-OTetradecanoylphorbol-13-acetate (TPA), a keratinocytedifferentiating agent, is extensively used to induce keratinocyte differentiation. We have previously shown that TPA treatment of human keratinocytes increases hINV mRNA level and promoter activity. This increase is mediated via a Ras 3 MEKK1 3 MEK3 3 p38 signaling cascade. One target of this cascade is activator protein 1 (AP1) that binds an AP1-binding site, AP1-1, in the hINV proximal regulatory region (6 -9). A key question to be resolved is the identity of the kinase(s) that initiate this cascade and mediate the effects of TPA in normal human keratinocytes. The various isoforms of PKC are key candidates for this regulatory role.The protein kinase C (PKC) family consists of at least 11 distinct serine/threonine protein kinases that are classified into three groups. The conventional/classical PKCs (cPKCs) are calcium-, phospholipid-, and diacylglycerol-dependent (␣, ␤I, ␤II, and ␥); the novel PKCs (nPKCs) are calcium-independent PKCs (␦, ⑀, , , and ); and the atypical PKCs (aPKCs) are calcium-and diacylglycerol-independent (, and ) (10 -12). The differences in cofactor requirements, tissue distribution, subcellular localization, and substrate specificity suggest distinct biological functions for each PKC isozyme (10, 12, 13). Epidermal keratinocytes express ␣, ␦, ⑀, , and isoforms (14 -18). As involucrin is a model for the study of gene expression in stratifying epithelia, it is important to identify which of these PKC i...

The novel protein kinase C (nPKC) isoforms are important regulators of human involucrin (hINV) gene expression during keratinocyte differentiation (Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). Although the regulatory mechanism involves mitogenactivated protein kinase (MAPK) activation, the role of individual MAPK isoforms has not been elucidated. We therefore examined the effects of individual nPKCs on MAPK activation. We observe unique changes whereby nPKC expression simultaneously increases p38 activity and decreases ERK1 and ERK2 activity. Although p38␣, p38␤, and p38␦ are expressed in keratinocytes, only a single isoform, p38␦, accounts for the increased p38 activity. Parallel studies indicate that this isoform is also activated by treatment with the keratinocyte regulatory agents, 12-O-tetradecanoylphorbol-13-acetate, calcium, and okadaic acid. These changes in MAPK activity are associated with increased C/EBP␣ transcription factor expression and DNA binding to the hINV promoter and increased hINV gene expression. Expression of PKC␦, PKC⑀, or PKC causes a 10-fold increase in hINV promoter activity, whereas C/EBP␣ expression produces a 25-fold increase. However, simultaneous expression of both proteins causes a synergistic 100-fold increase in promoter activity. These responses are eliminated by the dominant-negative C/EBP isoform, GADD153, and are also inhibited by dominant-negative forms of Ras, MEKK1, MEK3, and p38. These results suggest that the nPKC isoforms produce a unique shift in MAPK activity via a Ras, MEKK1, MEK3 pathway, to increase p38␦ and inhibit ERK1/2 and ultimately increase C/EBP␣ binding to the hINV promoter and hINV gene expression.Protein kinase C (PKC) 1 family members are classified into three major groups. The conventional/classical PKCs (cPKC␣, cPKC␤I, cPKC␤II, and cPKC␥) are calcium-, diacylglycerol-, and phospholipid-dependent kinases; the atypical PKC kinases (aPKC and aPKC ) are calcium-and diacylglycerol-independent (1-3); and the novel PKCs (nPKC␦, nPKC⑀, nPKC , nPKC , and nPKC ), are calcium-independent enzymes (1-3). Each PKC isozyme has unique co-factor requirements, tissue distribution, subcellular localization, and substrate specificity (1, 3, 4). Epidermal keratinocytes express ␣, ␦, ⑀, , and isoforms (5-9). However, the role of each isoform in regulating differentiation is not well understood.In previous studies, we demonstrated that the novel PKC isoforms PKC␦, PKC⑀, and PKC , but not the conventional and atypical PKC forms, activate keratinocyte differentiation as measured by effects on human involucrin (hINV) gene expression (10, 11). Involucrin is a precursor of the keratinocytecornified envelope and a marker of early keratinocyte differentiation (12). Dominant-negative PKC␦ inhibits this response (10). This pathway appears to operate by triggering a cascade that includes Ras, MEKK1, and MEK3. Although this pathway is known to require MAPK activity, whether individual nPKCs activate different MAPKs has not been explored. In the present study...

The epidermis is a dynamic and continually renewing surface that provides and maintains a life-sustaining interface with the environment. The epidermal keratinocyte, the major cell type of the epidermis, undergoes a complex and carefully choreographed program of differentiation. This process requires a balance between keratinocyte proliferation, differentiation, and apoptosis. This overview will concentrate on cascades that regulate the balance between keratinocyte cell proliferation and survival, and apoptosis and cell differentiation, with a particular emphasis on the role of the mitogen-activated protein kinase cascades. A summary of the literature suggests that extracellular regulated kinases function to promote keratinocyte proliferation and survival, whereas p38 mitogen-activated protein kinase functions to promote differentiation and apoptosis.

The p38 family of mitogen-activated protein kinases includes p38 alpha (SAPK2a, CSBP), p38 beta (SAPK2b), p38 delta (SAPK4), and p38 gamma (SAPK3/ERK6). p38 alpha and p38 beta are widely expressed p38 isoforms that are involved in regulation of cell proliferation, differentiation, development, and response to stress. Relatively less is known regarding the function of the p38 delta isoform. In this review, we discuss the role of the p38 alpha, p38 beta, and p38 gamma isoforms and then present recent findings that define a role for p38 delta as a regulator of differentiation-dependent gene expression in keratinocytes.