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J Biol Chem, Vol. 275, Issue 3, 1601-1607, January 21, 2000


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

Tatiana EfimovaDagger and Richard L. EckertDagger §par **Dagger Dagger

From the Departments of Dagger  Physiology and Biophysics, § Dermatology,  Reproductive Biology, par  Biochemistry, and ** Oncology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-O-Tetradecanoylphorbol-13-acetate (TPA), a keratinocyte-differentiating 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 right-arrow MEKK1 right-arrow MEK3 right-arrow 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 (alpha , beta I, beta II, and gamma ); the novel PKCs (nPKCs) are calcium-independent PKCs (delta , epsilon , eta , theta , and µ); and the atypical PKCs (aPKCs) are calcium- and diacylglycerol-independent (zeta , and lambda ) (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 alpha , delta , epsilon , eta , and zeta  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 isoforms are involved in regulation of the hINV gene.

In the present study we demonstrate that novel PKC isoforms delta , epsilon , and eta , but not the conventional and atypical PKC forms, are involved in regulation of hINV gene expression.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and Reagents-- Keratinocyte serum-free medium (KSFM), gentamicin, trypsin, and Hanks' balanced salt solution were obtained from Life Technologies, Inc. Bisindolylmaleimide (BIS-IM), rottlerin, and Go-6976 were from Calbiochem. Phorbol ester (TPA) and dimethyl sulfoxide were purchased from Sigma. Dispase was obtained from Roche Molecular Biochemicals. The pGL2-Basic plasmid and the chemiluminescent luciferase assay system were purchased from Promega, and chemiluminescence intensity was measured using a Berthold luminometer. PKC isoform-selective (PKCalpha , sc-208; PKCdelta , sc-937; PKCeta , sc-215; PKCepsilon , sc-214) antibodies were from Santa Cruz Biotechnology. The involucrin-specific polyclonal antibody was generated by injecting rabbits with recombinant human involucrin (19).

Plasmids-- We have previously published the structure of the hINV promoter construct pINV-241, which include nucleotides -241/-7 of the hINV promoter, linked to the luciferase reporter gene in pGL2-Basic (7). All positions are defined relative to the hINV gene transcription start site (20).

PKC expression vectors were a generous gift from Dr. S. Ohno (16, 21-25). Dominant-negative PKCdelta (K376R), dndelta (KR), cloned in pLTR was kindly provided by Dr. Weigun Li (26). The c-fos promoter-luciferase reporter plasmid was kindly provided by Dr. Michael Simonson (27).

Tissue Culture, Cell Transfection, and Luciferase Assay-- Normal human foreskin keratinocytes were cultured as described previously. Third passage keratinocytes were transfected in 35-mm diameter dishes when approximately 60% confluent. For transfection experiments, 4 µl of Fugene-6 reagent was added to 96 µl of KSFM and incubated at 25 °C for 5 min. The mixture was then mixed with 2 µg of involucrin promoter reporter plasmid or, for co-transfection experiments, with 1 µg of involucrin reporter plasmid and 1 µg of kinase expression plasmid. The mixture was incubated at 25 °C for 15 min and then added directly to the cells in 2 ml of KSFM. In general, the final DNA concentration in all groups was adjusted to 2 µg of DNA per 4 µl of Fugene-6 reagent per 35-mm dish by addition of empty expression vector. However, in the dose-response experiments, the final DNA concentration was 4 µg per 8 µl of Fugene-6 per 35-mm dish. After 24 h the cells were treated with KSFM in the presence or absence of TPA and/or the indicated inhibitor. After an additional 24 h, the cells were washed with phosphate-buffered saline, dissolved in 250 µl of cell culture lysis reagent (Promega), and harvested by scraping. Luciferase activity was assayed immediately using Promega luciferase assay kit and a Berthold luminometer. All assays were performed in triplicate, and each experiment was repeated a minimum of three times. Luciferase activity was normalized per µg of protein as described previously. A plasmid expressing green fluorescent protein (CLONTECH) was used to monitor transfection efficiency as described (28).

Immunoblot Analysis-- Cultured keratinocytes, grown in KSFM, were treated with or without 50 ng/ml TPA and/or indicated pharmacological agent (concentrations indicated in each experiment) for 24 h prior to preparation of total cell extracts. Equal quantities of protein were electrophoresed on denaturing polyacrylamide gels and transferred to nitrocellulose. The membranes were blocked and then incubated with the appropriate primary antibody followed by a goat anti-rabbit IgG secondary antibody. Secondary antibody binding was visualized using a chemiluminescent detection system (Amersham Pharmacia Biotech).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKC Isoforms That Regulate hINV Promoter Activity-- Fig. 1A shows that treatment of keratinocytes with phorbol ester increases human involucrin protein levels. This TPA-dependent increase in endogenous gene expression can be inhibited by BIS-IM, an agent that inhibits all PKC isoforms. To identify the specific PKC isoforms responsible for this regulation, we co-transfected keratinocytes with pINV-241 involucrin promoter-luciferase reporter construct (6, 7, 9) and expression plasmids encoding specific wild type PKC isoforms. The results, see Fig. 1B, indicate that novel PKC (nPKC) isoforms delta , epsilon , and eta  increase hINV promoter activity as efficiently as TPA treatment (>10-fold). In contrast, the conventional PKC (cPKC) isoforms alpha , beta I, and gamma , and the atypical isoform, PKCzeta , produce minimal changes. As shown in Fig. 1C, the novel isoform-dependent increase is also observed for the endogenous gene, suggesting that the regulation is physiologically relevant.


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  		Fig. 1.   Novel PKC isoforms activate hINV gene expression. A, keratinocytes were treated for 24 h with 50 ng/ml TPA and/or 3 µM BIS-IM. Total cell extracts were then prepared and assayed for hINV protein level by immunoblot. B, cultured human epidermal keratinocytes were transfected with the 1 µg of pINV-241 reporter plasmid and PKC-encoding plasmids (1 µg) or empty expression vector (EV). After 24 h, the cultures were treated with (+) or without (-) 50 ng/ml TPA. At 48 h after transfection, the cells were harvested, and lysates were assayed for luciferase activity. C, keratinocytes were transfected with 4 µg of PKCdelta , -epsilon , or -eta . At 24 h post-transfection, the cells were harvested for preparation of total cell extracts. Equal quantities of extract was electrophoresed on an 8% denaturing/reducing polyacrylamide gel, transferred to nitrocellulose, and incubated with hINV-specific antibody (19).

Failure of the cPKC isoforms to regulate hINV promoter activity in keratinocytes could result from a failure of the enzymes to be expressed or because they are expressed in an inactive form. To address these concerns, we transfected keratinocytes with PKCalpha , -delta , -epsilon , and -eta , and we monitored for presence of the corresponding protein by immunoblot. As shown in Fig. 2A, each PKC isoform is expressed at a comparable level. Although not evident from the exposure shown here, PKCalpha , -delta , -epsilon , and -eta are also expressed in non-transfected keratinocytes. To ensure that the transfected cPKC isoforms are active, we transfected the c-fos promoter (27), which responds to phorbol ester-sensitive PKC isoforms (29-32), with each cPKC isoform. Fig. 2B shows that PKCalpha , -beta 1, and -gamma strongly increase c-fos promoter activity, confirming activity of these enzymes in keratinocytes. Thus, the lack of hINV promoter activation by the cPKC isoforms is not due to low expression or lack of activity of the expressed enzymes.


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  		Fig. 2.   Transfected PKC isoforms are expressed in keratinocytes. A, cultured keratinocytes were transfected with expression plasmids encoding indicated PKC isozymes (+) or empty expression vector (-). After 48 h, total cell extracts were prepared. Equal quantities of protein were electrophoresed, transferred to nitrocellulose, and immunoblotted with PKC isozyme-specific antibodies. The arrows indicate migration of the each respective PKC isoform. The molecular masses are indicated in kilodaltons. B, keratinocytes were transfected with c-fos promoter reporter plasmid in the presence of expression plasmids encoding the conventional alpha , beta 1, and gamma  PKC isozymes or empty expression vector (EV). After 48 h, total cell extracts were prepared and assayed for luciferase activity.

Concentration-dependent Regulation of hINV Promoter Activity by Individual PKC Isoforms-- PKC-dependent responses can be concentration-dependent, and so we studied the effects of various concentrations of PKC expression plasmid on promoter activity. PKCalpha did not regulate promoter activity at any concentration examined (Fig. 3). Although not evident in this figure, because the responses are minimal, cPKCbeta I and cPKCgamma slightly stimulated promoter activity at high plasmid concentrations, and promoter activity was increased a modest 2-fold by 0.25 µg of PKCzeta plasmid with no further increase at higher plasmid concentrations. In contrast to these minimal responses, the nPKC isoforms, -delta , -epsilon , and -eta , produced similar dose-response curves and a 12-35-fold increase in promoter activity.


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  		Fig. 3.   Concentration dependence of PKC-dependent activation of hINV gene expression. Cultured keratinocytes were transfected with 2 µg of pINV-241 and 0-2 µg of each PKC isoform. The total concentration of plasmid in each transfection was maintained constant at 4 µg by addition of empty expression vector. After 24 h extracts were prepared and assayed for luciferase activity.

Rottlerin, but Not Go-6976, Suppresses hINV Promoter Activity-- To examine this regulation further, we used isoform-specific PKC inhibitors. Rottlerin specifically inactivates PKCdelta (33), and Go-6976, a staurosporine-related compound, inactivates cPKC isozymes (34). pINV-241 reporter plasmid-transfected cells were treated with increasing concentrations of each inhibitor. Fig. 4A shows that rottlerin inhibits hINV promoter activity at concentrations that selectively inhibit PKCdelta (33). In contrast, the cPKC inhibitor, Go-6976, which is normally active in the nanomolar range (34), did not inhibit promoter activity even at micromolar concentrations (Fig. 4B).


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  		Fig. 4.   Pharmacological evidence for novel PKC regulation of hINV promoter activity. Cultured human epidermal keratinocytes were transfected with pINV-241 according to the procedure outlined under "Materials and Methods" and then treated for 24 h with culture medium containing the indicated concentration of rottlerin (A) or Go-6976 (B). At 24 h, the cells were harvested, and extracts were assayed for luciferase activity.

Dominant-negative PKCdelta Suppresses PKCdelta -, -epsilon -, and -eta -dependent Promoter Activation-- The previous experiment suggests that PKCdelta may be the primary PKC controlling hINV gene expression. However, additional nPKC isoforms may also have a role. To confirm a role for PKCdelta and to study the role of the other nPKC isoforms, we used a dominant-negative form of PKCdelta , dndelta (KR), in which the ATP-binding site is mutated (26). We show, in Fig. 5A, that dndelta (KR) completely inhibits TPA-dependent promoter activation. Thus, ligand-dependent activation of the promoter is inhibited by dndelta (KR). In Fig. 5B we show that dndelta (KR) completely inhibits PKCdelta -dependent promoter activation. However, it is interesting that dndelta (KR) also inhibits wild type PKCeta - and PKCepsilon -dependent promoter activation, although less efficiently compared with the dndelta (KR)-dependent inhibition of PKCdelta -driven activity. This result suggest caution in assigning the sole regulatory role to PKCdelta .


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  		Fig. 5.   Dominant-negative PKCdelta inhibits novel PKC-dependent promoter activation. A, keratinocytes were transfected with 2 µg of pINV-241 in the presence (+) or absence of (-) dndelta (KR) and treated with 50 ng/ml TPA. After 24 h, extracts were assayed for promoter activity. B, keratinocytes were transfected with 2 µg of pINV-241 and 1 µg of PKCdelta , -eta , or -epsilon in the presence (+) or absence (-) of 1 µg of dominant-negative PKCdelta (dndelta (KR)). At 24 h after transfection extracts were prepared and assayed for luciferase activity.

Activation by PKC Isoforms in the Presence of TPA-- The results presented above indicate that nPKC isoforms activate basal hINV promoter activity. To examine the effects of TPA on PKC isoform-dependent activation, keratinocytes were co-transfected with pINV-241 reporter vector and PKC expression plasmid and treated with TPA. As shown in Fig. 6, none of the classical PKC isoforms (alpha , beta 1, and gamma ) or the atypical isoform (zeta ) altered the TPA-dependent response. However, PKCepsilon and PKCeta produced a dramatic superinduction of hINV promoter activity in the presence of TPA (100-fold activation). Unexpectedly, and in contrast to the PKCdelta -dependent activation observed in the absence of TPA (Fig. 3), PKCdelta suppressed the TPA-dependent activation to TPA-nonstimulated levels.


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  		Fig. 6.   Synergistic activation of promoter activity by TPA and novel PKCs. Cultured human epidermal keratinocytes were transfected with the 1 µg of pINV-241 reporter plasmid and PKC-encoding plasmids (1 µg) or empty expression vector (EV). At 24 h, the indicated groups (+) were treated with 50 ng/ml TPA for 24 h. At 48 h post-transfection, cell extracts were prepared for assay of luciferase activity.

To investigate further the nPKCdelta effect, we transfected keratinocytes with a fixed amount of hINV reporter plasmid (2 µg) and increasing concentrations of nPKCdelta expression plasmid (0.25-2 µg), and we treated with TPA (Fig. 7). PKCdelta , at 0.25 µg expression plasmids per dish, produced a strong promoter activation. A comparable increase was observed at lower PKCdelta concentrations (0.06 µg of PKCdelta per dish, not shown) indicating that very small concentrations of this isoform can activate transcription. Interestingly, a smaller increase is observed at intermediate plasmid concentrations, and inhibition is observed at high (2 µg) plasmid levels. In contrast, nPKCepsilon and nPKCeta (Fig. 7) markedly enhance the TPA-induced hINV promoter activity at all concentrations tested.


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  		Fig. 7.   Concentration dependence of PKC-dependent activation in the presence of TPA. Keratinocytes were transfected with pINV-241 and increasing concentrations of PKCdelta , -eta , or -epsilon . After 24 h, the cells were treated in the presence () or absence (open circle ) or TPA. At 48 h, the cells were extracts were assayed for luciferase activity.

One mechanism whereby PKC is inactivated is via degradation (10, 35). We therefore determined whether PKCdelta , epsilon  and eta  levels change in response to TPA treatment. The results, shown in Fig. 8, indicate that PKCepsilon is markedly decreased by TPA treatment; PKCdelta is decreased by 50%,; and PKCeta is slightly increased. These results suggest that PKC level is not correlated with ability to drive TPA/PKC-dependent hINV promoter activation.


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  		Fig. 8.   Regulation of PKC isoform level by TPA. Keratinocytes were treated with 50 ng/ml TPA for 24 h followed by preparation of nuclear extracts. Equivalent quantities of extract, layered based on protein concentration, were electrophoresed on an 8% acrylamide gel and transferred to nitrocellulose for detection using PKCepsilon -, -delta -, and -eta -specific antibodies. Binding of the primary antibody was detected by incubation with an appropriate secondary antibody, and binding was visualized using ECL technology.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Involucrin Promoter Activity and Endogenous hINV Gene Expression Are Regulated by Novel PKC Isoforms-- We have previously presented evidence indirectly implicating PKC in the signal transduction pathway leading to hINV promoter activation (7, 9). Evidence includes the finding that TPA increases hINV mRNA levels and promoter activity (7), and BIS-IM, a general PKC inhibitor, blocks these responses (9). The present studies were designed, in part, to identify which PKC isoform(s) are involved in this regulation. Keratinocytes express classical, novel, and atypical PKC isoforms, including cPKCalpha , nPKCdelta , nPKCepsilon , nPKCeta , and aPKCzeta (14-18, 36). All of these forms, with the exception of PKCzeta , can be activated by TPA (25). Because of their distinct pattern of expression, primary sequence, differing response to stimuli, and differing cofactor dependence, each PKC is expected to have a different function (10, 13). Therefore, it is important to determine which isoforms regulate keratinocyte target gene expression and which pathways convey the regulatory signal. To address these issues, we expressed wild type PKC isoforms in normal human keratinocytes and monitored the effects on basal hINV promoter activity. These experiments identify the novel PKC isoforms delta , epsilon , and eta  as potent inducers of promoter activity. In contrast, atypical PKCzeta , and the conventional PKC isoforms alpha , beta 1, and gamma , failed to regulate activity. The lack of activation by PKCalpha , -beta 1, and -gamma was not due to inadequate expression of these kinases, as each was detected at high level by immunoblot. Moreover, activity of these kinases was confirmed by demonstrating PKCalpha , -beta 1, and -gamma -dependent regulation of the c-fos promoter. c-fos is known to be regulated by TPA-dependent PKC isoforms (29-32). A regulatory role for the novel PKC isoforms is further supported by the finding that Go-6976, an inhibitor of conventional but not novel PKC isoforms (34), does not inhibit promoter activity.

The Role of PKCdelta -- An important role for PKCdelta is suggested by the finding that promoter activity is inhibited by concentrations of rottlerin (33) that selectively inhibit PKCdelta . However, our results also support a role for PKCepsilon and PKCeta in two ways. First, transfection of keratinocytes with PKCepsilon or -eta activates hINV promoter activity and expression of the endogenous hINV gene. Second, dominant-negative PKCdelta inhibits PKCepsilon - and PKCeta -dependent activity. Dominant-negative mutants have been extensively utilized to map signal transduction cascades. These proteins function to inhibit the activity of the endogenous wild type enzymes by a variety of mechanisms. For PKCs, dominant-negative mutants have been constructed by mutating threonine phosphorylation sites in the activation loop of the kinase domain (37) or by mutating the site that binds ATP, a necessary cofactor for enzyme activity (26, 29, 38-41). We have used a form of PKCdelta in which a conserved lysine at the ATP-binding site is converted to arginine to inactivate the enzyme (26). Our experiments show that dominant-negative PKCdelta inhibits PKCdelta -dependent promoter activation, a result that is consistent with a role for PKCdelta in regulating hINV gene expression. However, albeit to a lesser extent, dnPKCdelta also inhibits PKCeta - and PKCepsilon -dependent promoter activation. There are several possible mechanisms whereby dnPKCdelta could inhibit PKCeta - and PKCepsilon -dependent responses. First, dnPKCdelta may titrate a kinase that is required for activation of all novel PKCs and, thereby, inhibit activity of all nPKC isoforms (i.e. dominant-negative PKCdelta may not specifically inhibit of PKCdelta in our system). Second, dnPKCdelta may interfere with chaperone "docking" proteins that may regulate the function of multiple PKC isoforms. Experiments that suggest these possibilities have been noted using activation-loop mutants of PKC (10, 37). The dnPKCdelta used in the present studies is an ATP-binding site mutant (26). ATP-binding site mutants may be more specific inhibitors of the corresponding wild type PKC isoform than activation-loop mutants; however, this has not been rigorously tested. Third, PKCdelta , -epsilon , and -eta may indirectly regulate the level/activity of each other by regulating expression of the corresponding genes. This interesting possibility is not unprecedented, as a recent study in mouse lymphoma cells shows that PKCalpha increases PKCdelta protein level by regulating PKCdelta mRNA level (42). Fourth, PKCdelta , -epsilon , and -eta may share a common substrate(s). Fifth, overexpression of individual PKC isoforms could lead to non-selective activation of downstream targets of other PKC isoforms. Thus, although our present studies strongly point to a role for PKCdelta , it is likely that PKCepsilon and -eta also play an important role.

Function of PKCs in the Presence of the PKC Activator, Phorbol Ester-- Diacylglycerol is a ligand that directly activates PKC isoforms (11). TPA is a stable diacylglycerol analog that mimics the effects of diacylglycerol and strongly activates PKC (43-45) and is a potent inducer of keratinocyte differentiation (46). Treating cultured keratinocytes with TPA increases cell differentiation, and this change is correlated with an increase in hINV mRNA and protein (7, 9, 47, 48). To study the effects of TPA-dependent activation of individual PKC isoforms on hINV promoter activity, we transfected cells with PKC expression constructs and then treated with TPA. The results indicate a synergistic activation (>= 100-fold, Fig. 6) of promoter activity when PKCepsilon - or -eta -treated cells are incubated with TPA. This increase depends directly on the concentration of PKCepsilon or -eta expression plasmid transfected. In contrast, no potentiation was observed for PKCalpha , -beta 1, -gamma , or -zeta . PKCdelta , however, caused synergistic promoter activation at moderate plasmid concentrations and inhibition at higher plasmid concentrations. It is not clear why the response to delta  is biphasic; however, this result suggests that the PKC-dependent regulation is complex. One possible explanation is that PKCdelta levels are reduced by TPA treatment. However, immunoblot results suggest that PKCdelta levels are decreased by only 30-50% in response to TPA treatment. In contrast, PKCepsilon levels are reduced substantially, and PKCeta levels are relatively unchanged. These results suggest that TPA-dependent regulation of PKC level does not explain the difference in activity. There are other possible explanations. For example, PKCdelta undergoes tyrosine phosphorylation in response to various stimuli, including epidermal growth factor, platelet-derived growth factor, transforming growth factor-alpha , carbachol, extracellular ATP or UTP, and hydrogen peroxide (35). Moreover, tyrosine kinases of the Src family phosphorylate PKCdelta in vitro, although the functional significance of this phosphorylation has not been clearly established (45). Several reports in keratinocytes suggest that phosphorylation of PKCdelta on tyrosine residues in the regulatory domain diminishes activity (49-51), although studies in other systems report increased PKC activity following tyrosine phosphorylation (52-54)). Thus, high level overexpression of PKCdelta may result in tyrosine phosphorylation-dependent inactivation of PKCdelta . However, although PKCdelta could be inactivated by phosphorylation, it is difficult to understand the unique plasmid concentration dependence of the inhibition. As noted above, PKCdelta is expressed at high levels in keratinocytes. It is possible that very high plasmid concentrations inhibit promoter activity by saturating the system with PKCdelta which "squelches" the response.

PKC Isoforms and Keratinocyte Function-- Our results suggest that PKCdelta , -epsilon , and -eta regulate hINV gene expression. Takahashi et al. (55) showed that PKCgamma increases basal hINV promoter activity by 2-3-fold in the absence of TPA, whereas PKCalpha and -eta increase activity by 2-fold in TPA-treated cells. These studies differ from ours in that we do not observe PKCalpha -dependent regulation. Moreover, the magnitude of our responses are much larger. We attribute the different findings to the fact that we use normal human keratinocytes, whereas Takahashi et al. (55) used SV40-immortalized keratinocytes. Signal transduction is known to be altered in immortalized cell lines, and a significant amount of circumstantial evidence indicates that the involucrin gene is not always appropriately regulated in immortalized keratinocyte cell lines. For example, in cell lines, the level of hINV gene expression and the response to stimuli is significantly reduced compared with normal cells.2 However, the studies in both cell types support a role for PKCeta as a regulator of hINV gene expression.

In epidermis, involucrin is expressed in the late spinous and granular layers but not in the basal layer (1, 3, 5). Type I transglutaminase is another marker of keratinocyte differentiation that displays a similar spatial and temporal pattern of expression (56, 57). Thus, it is useful to compare mechanisms that regulate expression of the involucrin and transglutaminase type 1 (TG1) genes. Recent studies indicate that overexpression of the eta  and delta  PKC isoforms in human keratinocytes causes an increase in TG1-encoding mRNA. This is correlated with growth inhibition and morphological changes (58). In contrast, the alpha  and zeta  PKC isoforms do not regulate TG1 expression. Regulation in response to PKCepsilon was not studied. In addition, it has been reported that expression of exogenous PKCeta in a rat keratinocyte cell line efficiently induces TG1 transcription, but PKCalpha , PKCbeta II, PKCgamma , and PKCzeta did not regulate activity (59). Yuspa and co-workers (60) have shown that TPA blocks the calcium-dependent increase in K1 and K10 (spinous layer markers) and simultaneously increases filaggrin and loricrin expression (granular layer markers). This TPA-dependent response is blocked by bryostatin, a PKC inactivating agent or cycloheximide, a protein synthesis inhibitory agent. This suggests that PKC regulates genes in the transition from spinous to granular layers (60). These results are consistent with ours, as involucrin is predominantly a granular cell marker (2). Moreover, PKCeta is known to be localized in the epidermal granular layer (61). Thus, our studies suggest a role for novel PKCs as regulators of hINV gene expression.

    ACKNOWLEDGEMENT

The Skin Diseases Research Center of Northeast Ohio was supported by National Institutes of Health Grant AR39750.

    FOOTNOTES

* This work was supported by grants from the National Institutes of Health (to R. L. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Physiology/Biophysics, Rm E532, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106-4970. Tel.: 216-368-5530; Fax: 216-368-5586.

2 R. L. Eckert, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: hINV, human involucrin; TPA, 12-O-tetradecanoylphorbol-13-acetate; AP1, activator protein 1; PKC, protein kinase C; nPKC, novel PKCs; aPKCs, atypical PKCs; cPKC, conventional/classical PKCs; TG1, transglutaminase type 1; dn, dominant-negative; BIS-IM, bisindolylmaleimide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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