Tatiana Efimova

Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.

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.

A Regulatory Role for p38δ MAPK in Keratinocyte Differentiation EVIDENCE FOR p38δ-ERK1/2 COMPLEX FORMATION

p38 MAPK isoforms are important in the regulation of a variety of cellular processes. Among the four described p38 isoforms, p38α, β, and δ are expressed in keratinocytes (Dashti, S. R., Efimova, T., and Eckert, R. L. (2001) J. Biol. Chem. 276, 8059–8063). However, very little is known about how individual p38 isoforms regulate keratinocyte function. In the present study, we use okadaic acid (OA) as a tool to study the role of p38 MAPKs as regulators of keratinocyte differentiation. We demonstrate that OA activates p38δ but not other p38 isoforms. p38δ activation is increased as early as 0.5 h after OA addition, and activity is maximal at 8 and 24 h. ERK1 and ERK2 activity are reduced on an identical time course. We show that p38δ forms a complex with ERK1/2, and overexpression of p38δ inhibits ERK1/2 activity without reducing ERK1/2 level. Thus, p38δ may directly suppress ERK1/2 activity. Additional studies show that p38δ is expressed in the epidermis, suggesting a role for p38δ in regulating differentiation. To evaluate its function, we show that increased p38δ activity is associated with increased levels of AP1 and CAATT enhancer binding protein factors, increased binding of these factors to the involucrin (hINV) promoter, and increased expression. Moreover, these responses are maintained in the presence of SB203580, an agent that inhibits p38α and β, further suggesting a central role for the p38δ isoform. Dominant-negative p38 also inhibits these responses. These unique observations suggest that p38δ is the major p38 isoform driving suprabasal hINV gene expression and that p38δ directly regulates ERK1/2 activity via formation of a p38δ-ERK1/2 complex.

Novel protein kinase C isoforms regulate human keratinocyte differentiation by activating a p38 delta mitogen-activated protein kinase cascade that targets CCAAT/enhancer-binding protein alpha.

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 mitogen-activated 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 p38α, p38β, and p38δ are expressed in keratinocytes, only a single isoform, p38δ, 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/EBPα transcription factor expression and DNA binding to the hINV promoter and increased hINV gene expression. Expression of PKCδ, PKCε, or PKCη causes a 10-fold increase in hINV promoter activity, whereas C/EBPα 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 p38δ and inhibit ERK1/2 and ultimately increase C/EBPα binding to the hINV promoter and hINV gene expression.

Calcium-dependent Involucrin Expression Is Inversely Regulated by Protein Kinase C (PKC)α and PKCδ

Calcium is an important physiologic regulator of keratinocyte function that may regulate keratinocyte differentiation via modulation of protein kinase C (PKC) activity. PKCα and PKCδ are two PKC isoforms that are expressed at high levels in keratinocytes. In the present study, we examine the effect of PKCδ and PKCα on calcium-dependent keratinocyte differentiation as measured by effects on involucrin (hINV) gene expression. Our studies indicate that calcium increaseshINV promoter activity and endogenous hINV gene expression. This response requires PKCδ, as evidenced by the observation that treatment with dominant-negative PKCδ inhibits calcium-dependent hINV promoter activity, whereas wild type PKCδ increases activity. PKCα, in contrast, inhibits calcium-dependent hINV promoter activation, a finding that is consistent with the ability of dominant-negative PKCα and the PKCα inhibitor, Go6976, to increasehINV gene expression. The calcium-dependent regulatory response is mediated by an AP1 transcription factor-binding site located within the hINV promoter distal regulatory region that is also required for PKCδ-dependent regulation; moreover, both calcium and PKCδ produce similar, but not identical, changes in AP1 factor expression. A key question is whether calcium directly influences PKC isoform function. Our studies show that calcium does not regulate PKCα or δ levels or cause a marked redistribution to membranes. However, tyrosine phosphorylation of PKCδ is markedly increased following calcium treatment. These findings suggest that PKCα and PKCδ are required for, and modulate, calcium-dependent keratinocyte differentiation in opposing directions.

Regulation of Human Involucrin Promoter Activity by Novel Protein Kinase C Isoforms

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 δ, ε, and η increase hINV promoter activity. In contrast, neither conventional PKC isoforms (α, β, and γ) nor the atypical isoform (ζ) regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKCδ-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 PKCδ. This regulation appears to be physiologically important, as transfection of keratinocytes with PKCδ, -ε, or -η increases expression of the endogenous hINV gene. Synergistic promoter activation (≥100-fold) is observed when PKCε- or -η-transfected cells are treated with TPA. In contrast, the PKCδ-dependent response is more complex as either activation or inhibition is observed, depending upon PKCδ concentration.

Protein Kinase Cδ Regulates Keratinocyte Death and Survival by Regulating Activity and Subcellular Localization of a p38δ-Extracellular Signal-Regulated Kinase 1/2 Complex

ABSTRACT Protein kinase Cδ (PKCδ) is an important regulator of apoptosis in epidermal keratinocytes. However, little information is available regarding the downstream kinases that mediate PKCδ-dependent keratinocyte death. This study implicates p38δ mitogen-activated protein kinase (MAPK) as a downstream carrier of the PKCδ-dependent death signal. We show that coexpression of PKCδ with p38δ produces profound apoptosis-like morphological changes. These morphological changes are associated with increased sub-G1 cell population, cytochrome c release, loss of mitochondrial membrane potential, caspase activation, and PARP cleavage. This death response is specific for the combination of PKCδ and p38δ and is not produced by replacing PKCδ with PKCα or p38δ with p38α. A constitutively active form of MEK6, an upstream activator of p38δ, can also produce cell death when coupled with p38δ. In addition, concurrent p38δ activation and extracellular signal-regulated kinase 1/2 (ERK1/2) inactivation are required for apoptosis. Regarding this inverse regulation, we describe a p38δ-ERK1/2 complex that may coordinate these changes in activity. We further show that this p38δ-ERK1/2 complex relocates into the nucleus in response to PKCδ expression. This regulation appears to be physiological, since H2O2, a known inducer of keratinocyte apoptosis, promotes identical PKCδ and p38δ-ERK1/2 activity changes, leading to similar morphological changes.

MEK7-dependent Activation of p38 MAP Kinase in Keratinocytes

Previous studies suggest that a PKC/Ras/MEKK1 cascade regulates involucrin (hINV) gene expression in human epidermal keratinocytes. MEK7, which is expressed in epidermis, has been identified as a member of this cascade (Efimova, T., LaCelle, P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387–24395 and Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601–1607). However, the kinase that functions downstream of MEK7 has not been identified. Our present studies show that MEK7 expression in keratinocytes markedly activates p38α and modestly activates JNK. Activation of p38 MAPK by MEK7 is a novel finding, as previous reports have assigned MEK7 as a JNK regulator. We also demonstrate that this regulation is physiologically important, as the p38α- and JNK-dependent activities regulate hINV promoter activity and expression of the endogenous hINVgene.

CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of keratinocyte differentiation and of involucrin gene expression. In the present study we show that a CCAAT/enhancer-binding protein (C/EBP) site in the proximal regulatory region is required for the phorbol ester response. Mutation of the C/EBP site results in the loss of basal and TPA-responsive activity. Gel mobility supershift analysis shows that C/EBPα binding to this site is increased by TPA treatment. Moreover, cotransfection of the human involucrin reporter plasmid with C/EBPα increases promoter activity to an extent comparable with TPA treatment. Mutation of the C/EBP-binding site eliminates these responses. Transfection experiments using GADD153 to create C/EBP-null conditions confirm that C/EBP factors are absolutely required for promoter activity and TPA responsiveness. C/EBPβ and C/EBPδ inhibit both TPA- and C/EBPα-dependent promoter activation, indicating functional differences among C/EBP family members. These results suggest that C/EBP transcription factor activity is necessary for basal promoter activity and TPA response of the involucrin gene.

Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein alpha (C/EBPalpha) DNA binding.

Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50 ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10 nM and maximal at 100 nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPalpha-dependent promoter activation via a mechanism that involves inhibition of C/EBPalpha binding to DNA without changing C/EBPalpha protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.