hINV Promoter Activity Research Articles

Multiple PKCδ Tyrosine Residues Are Required for PKCδ-Dependent Activation of Involucrin Expression—a Key Role of PKCδ-Y311

Protein kinase C-delta (PKCdelta) is a key regulator of human involucrin (hINV) gene expression and is regulated by tyrosine phosphorylation. However, a comprehensive analysis of the requirement for individual PKCdelta tyrosine residues is lacking. We show that multiple tyrosine residues influence the ability of PKCdelta to increase hINV gene expression. Mutation of individual PKCdelta tyrosine residues 52, 64, 155, 187, or 565 does not reduce the ability of PKCdelta to increase hINV promoter activity; however, simultaneous mutation of these five tyrosines markedly reduces activity. Moreover, restoration of any one of these residues results in nearly full activity restoration. It is significant that phosphorylation of PKCdelta-Y(311) is reduced in the five-tyrosine mutant and that mutation of Y(311) results in reduced PKCdelta activity comparable to that observed in the five-tyrosine mutant. Restoration of any one of the tyrosine residues in the five-tyrosine mutant restores Y(311) phosphorylation and biological activity. In addition, reduced phosphorylation of endogenous PKCdelta-Y(311) is associated with reduced biological activity. These findings indicate that PKCdelta activity requires Y(311) and a second tyrosine residue; however, any one of the several tyrosine residues can serve in the secondary role.

Curcumin Suppresses AP1 Transcription Factor-dependent Differentiation and Activates Apoptosis in Human Epidermal Keratinocytes

The diet-derived cancer preventive agent, curcumin, inhibits skin cancer cell proliferation and tumor formation. However, its effect on normal human keratinocyte differentiation, proliferation, and apoptosis has not been adequately studied. Involucrin (hINV) is a marker of keratinocyte differentiation and a useful model for the study of chemopreventive agent action. We show that curcumin suppresses the differentiation agent-dependent activation of hINV gene expression and that an AP1 transcription factor DNA binding site in the hINV gene is required for this regulation. A protein kinase C, Ras, MEKK1, MEK3 signaling cascade controls hINV expression by regulating AP1 factor level. Curcumin treatment inhibits the novel protein kinase C-, Ras-, and MEKK1-dependent activation of hINV promoter activity and reduces the differentiation agent-dependent increase in AP1 factor level and DNA binding. This reduction requires proteasome function. In addition, curcumin treatment reduces cell number, which is associated with a reduced cyclin and cdk1 levels. Curcumin treatment also suppresses the Bcl-xL level, leading to reduced mitochondrial membrane potential and increased cleavage of procaspases and poly(ADP-ribose) polymerase. These studies provide important insights regarding the mechanism whereby curcumin acts as a chemopreventive agent in normal human epidermis.

Apigenin Inhibition of Involucrin Gene Expression Is Associated with a Specific Reduction in Phosphorylation of Protein Kinase Cδ Tyr311

Apigenin is a plant-derived flavanoid that has significant promise as a skin cancer chemopreventive agent. In the present study, we examine the mechanism whereby apigenin regulates normal human keratinocyte differentiation. Expression of involucrin (hINV), a marker of keratinocyte differentiation, is increased by differentiating agents via a protein kinase Cdelta (PKCdelta), Ras, MEKK1, MEK3 cascade that increases AP1 factor level and AP1 factor binding to DNA elements in the hINV promoter. We show that apigenin inhibits this response. Apigenin suppresses the 12-O-tetradeconylphorbol-13-acetate-dependent increase in AP1 factor expression and binding to the hINV promoter and the increase in hINV promoter activity. Apigenin also inhibits the increase in promoter activity observed following overexpression of PKCdelta, constitutively active Ras, or MEKK1. The suppression of PKCdelta activity is associated with reduced phosphorylation of PKCdelta-Y311. The physiological importance of this phosphorylation event was confirmed by showing that the PKCdelta phosphorylation-defective mutant, PKCdelta-Y311F, is less able to increase hINV promoter activity. Activation of hINV promoter activity by the green tea polyphenol, (-)-epigellocathecin-3-gallate, is also inhibited by apigenin, suggesting that the two chemopreventive agents can produce opposing actions in keratinocytes. Additional studies show that the apigenin-dependent suppression of differentiation is associated with reduced cell proliferation but that there is no evidence of apoptosis.

An Involucrin Promoter AP1 Transcription Factor Binding Site Is Required for Expression of Involucrin in the Corneal Epithelium In Vivo

Cell division of corneal limbal stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. The mechanisms that regulate changes in gene expression during this process are not well understood. In the present study, the involucrin gene was used as a model to study this regulation. Regulation of human involucrin gene expression and promoter activity was assessed using in vivo transgenic mouse models and cultured primary human corneal epithelial cells. Human involucrin (hINV) is a structural protein that is selectively expressed in differentiating corneal epithelial cells. The results reveal that an activator protein one (AP1) DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-5, results in a loss of hINV gene expression in the corneal epithelium in vivo and in cultured corneal epithelial cells. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1 and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased AP1 factor (Fra-1 and JunB) binding to the hINV gene AP1-5 response element. These findings point to an essential role for AP1 transcription factors, acting through a distal regulatory region AP1-5 element, in the regulation of involucrin gene expression during corneal epithelial cell differentiation.

Regulation of involucrin expression in normal human corneal epithelial cells: a role for activator protein one.

Understanding the mechanisms that regulate gene expression in the human cornea is an important goal. In the present study, the involucrin gene was used as a model to study this regulation. Human involucrin (hINV) is a structural protein that is selectively expressed in surface epithelia, including corneal epithelial cells. Regulation of involucrin gene expression was monitored in cultures of normal human primary corneal epithelial cells. The studies revealed that an activator protein (AP)-1 DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-1, results in a loss of hINV promoter activity. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1, Fra-2, and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased nuclear AP1 factor level and binding to the hINV gene AP1-1 response element. Expression of the endogenous hINV gene is also increased by TPA treatment. These findings point to an important role for AP1 transcription factors in the regulation of human corneal epithelial cell involucrin gene expression.

Novel Protein Kinase C Isoforms Regulate Human Keratinocyte Differentiation by Activating a p38δ Mitogen-activated Protein Kinase Cascade That Targets CCAAT/Enhancer-binding Protein α

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 p38 alpha, p38 beta, and p38 delta are expressed in keratinocytes, only a single isoform, p38 delta, 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 alpha transcription factor expression and DNA binding to the hINV promoter and increased hINV gene expression. Expression of PKC delta, PKC epsilon, or PKC eta causes a 10-fold increase in hINV promoter activity, whereas C/EBP alpha 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 delta and inhibit ERK1/2 and ultimately increase C/EBP alpha 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. PKCalpha and PKCdelta are two PKC isoforms that are expressed at high levels in keratinocytes. In the present study, we examine the effect of PKCdelta and PKCalpha on calcium-dependent keratinocyte differentiation as measured by effects on involucrin (hINV) gene expression. Our studies indicate that calcium increases hINV promoter activity and endogenous hINV gene expression. This response requires PKCdelta, as evidenced by the observation that treatment with dominant-negative PKCdelta inhibits calcium-dependent hINV promoter activity, whereas wild type PKCdelta increases activity. PKCalpha, in contrast, inhibits calcium-dependent hINV promoter activation, a finding that is consistent with the ability of dominant-negative PKCalpha and the PKCalpha inhibitor, Go6976, to increase hINV 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 PKCdelta-dependent regulation; moreover, both calcium and PKCdelta 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 PKCalpha or delta levels or cause a marked redistribution to membranes. However, tyrosine phosphorylation of PKCdelta is markedly increased following calcium treatment. These findings suggest that PKCalpha and PKCdelta are required for, and modulate, calcium-dependent keratinocyte differentiation in opposing directions.

Green Tea Polyphenol Stimulates a Ras, MEKK1, MEK3, and p38 Cascade to Increase Activator Protein 1 Factor-dependent Involucrin Gene Expression in Normal Human Keratinocytes

(-)-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.

MEK6 Regulates Human Involucrin Gene Expression via a p38α- and p38δ-dependent Mechanism

A signaling cascade that includes protein kinase C (PKC), Ras, and MEKK1 regulates involucrin (hINV) gene expression in epidermal keratinocytes (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). Because signal transfer downstream of MEKK1 may involve several MAPK kinases (MEKs), it is important to evaluate the regulatory role of each MEK isoform. In the present study we evaluate the role of MEK6 in transmitting this signal. Constitutively active MEK6 (caMEK6) increases hINV promoter activity and increases endogenous hINV levels. The caMEK6-dependent increase in gene expression is inhibited by the p38 MAPK inhibitor, SB203580, and is associated with a marked increase in p38alpha MAPK activity; JNK and ERK kinases are not activated. In addition, hINV gene expression is inhibited by dominant-negative p38alpha and increased when caMEK6 and p38alpha are co-expressed. caMEK6 also activates p38delta, but p38delta inhibits the caMEK6-dependent activation. These results suggest that MEK6 increases hINV gene expression by regulating the balance between activation of p38alpha, which increases gene expression, and p38delta, which decreases gene expression.

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 p38alpha 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 p38alpha- and JNK-dependent activities regulate hINV promoter activity and expression of the endogenous hINV gene.

Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein α (C/EBPα) 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/EBPα-dependent promoter activation via a mechanism that involves inhibition of C/EBPα binding to DNA without changing C/EBPα protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.

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

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 delta, epsilon, and eta increase hINV promoter activity. In contrast, neither conventional PKC isoforms (alpha, beta, and gamma) nor the atypical isoform (zeta) regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKCdelta-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 PKCdelta. This regulation appears to be physiologically important, as transfection of keratinocytes with PKCdelta, -epsilon, or -eta increases expression of the endogenous hINV gene. Synergistic promoter activation (>/=100-fold) is observed when PKCepsilon- or -eta-transfected cells are treated with TPA. In contrast, the PKCdelta-dependent response is more complex as either activation or inhibition is observed, depending upon PKCdelta concentration.

Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types

The gene for human involucrin (hINV) is selectively expressed in stratifying epithelial cells lining external body surfaces. Previously, we characterized the hINV promoter 5' distal regulatory region (DRR) located between nt -2473 and -2088 upstream of the transcription start site. This region is required for optimal hINV gene expression. The DRR contains weak and strong activator elements. The strong activator comprises AP1- and Sp1-binding sites that combine to drive high-level promoter expression in human keratinocytes. Here we show that the hINV promoter is expressed in a cell-specific manner in vitro and that the DRR contains elements that are partly responsible for cell-type-specific expression of hINV. hINV promoter activity is barely detectable in 3T3 fibroblasts or HEK-293 human embryonic kidney cells. Reporter plasmids containing the full-length promoter or the isolated DRR can, however, be activated in 3T3 and HEK-293 cells by co-transfection with a plasmid encoding the transcription factor Sp1. Consistently with the lower hINV promoter activity, immunoblotting studies indicate that Sp1 protein levels are lower in 3T3 and HEK-293 cells than in human epidermal keratinocytes. Increased Sp1 protein in transfected 3T3 cells and HEK-293 cells correlates with increased promoter activity. In addition, Sp1 transfection activates the expression of the endogenous gene for hINV in HEK-293 cells. These studies suggest that Sp1 might have a role in cell-specific expression of hINV.

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.

Regulation of Human Involucrin Promoter Activity by POU Domain Proteins

POU domain transcription factors are expressed in the epidermis and are thought to be important regulators of keratinocyte gene expression. In the present article we demonstrate that POU transcription factors suppress transcription of the human involucrin (hINV) promoter. Cotransfection of pINV-2473, a construct containing 2473 base pairs of hINV upstream sequence linked to luciferase, with POU homeodomain transcription factors Oct1, Oct2, Brn4, SCIP, Skn1a or Skn1i, results in a strong suppression of basal promoter activity. The hINV upstream region includes a consensus POU transcription factor binding site, 5'-ATGCAAAT-3', centered around nucleotide -1277. Although this site interacts with POU factors, assays of promoter activity for a series of progressive 5' end truncations demonstrate that this site is not required for POU factor-dependent transcriptional suppression. Suppression is observed with the shortest truncation construct tested, pINV-41, suggesting that this inhibition may be mediated by effects on TATA box proteins. SCIP mutants that lack transactivation or DNA binding domains were shown to suppress transcription, suggesting that the DNA binding and transactivation domains are not required for suppression. Moreover, cotransfection of the pINV-2473 with pKSM13(+)OCT, which contains a single consensus OCT binding site, results in an increase in basal promoter activity, suggesting that endogenous POU factors suppress hINV promoter activity. In addition to inhibiting basal transcription, POU transcription factors also suppress phorbol ester-stimulated hINV promoter activity. These studies suggest that suppression of hINV promoter activity does not require the amino-terminal segment of the POU factor or direct POU factor interaction with DNA and suggest that the suppression may be via indirect interaction with other proteins in the vicinity of the TATA box. Thus, involucrin joins the ranks of a small set of genes that are regulated by POU factors in an octamer binding site-independent manner.