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 p38α MAPK activity; JNK and ERK kinases are not activated. In addition, hINV gene expression is inhibited by dominant-negative p38α and increased when caMEK6 and p38α are co-expressed. caMEK6 also activates p38δ, but p38δ inhibits the caMEK6-dependent activation. These results suggest that MEK6 increases hINV gene expression by regulating the balance between activation of p38α, which increases gene expression, and p38δ, which decreases gene expression.

Involucrin is a component of the keratinocyte cornified envelope that is expressed in the suprabasal layers of stratifying squamous epithelia and serves as a marker of keratinocyte differentiation (1). Differentiation-dependent hINV 1 gene activation involves a signaling cascade that includes the novel protein kinase C, Ras, and MEKK1 (2,3). The MEKK1-associated signal is transmitted via several MAPK kinases (MEKs) (2,3). These in turn activate p38 kinases, which regulate hINV gene expression (2,3). Several lines of evidence support this mechanism. For example, dominant-negative forms of protein kinase C, Ras, MEKK1, and p38 inhibit basal and differentiation-dependent hINV gene expression in cultured keratinocytes (2,3).
Although MEKs are structurally related (4), they can differentially regulate downstream responses. For example, MEK6 is reported to activate all four p38 isoforms (5), whereas MEK3 activates p38␣, ␦, and ␥, but not p38␤ (5). Although MEK3 and MEK7 have been implicated as regulators of hINV gene expression (2,3), the role of the other MEKs has not been examined. Because of the central role of MEK and p38 kinases in regulation of hINV gene expression, it is important to determine how each p38 isoform is regulated by each MEK and how these events influence hINV gene expression. In the present study, we examined the role of MEK6 in conjunction with the p38 isoforms as regulators of hINV gene expression. We demonstrate that MEK6 regulates p38 MAPK activity and that hINV gene expression is influenced by the balance between MEK6dependent p38␣ and p38␦ activation. These results suggest that the selective activation of specific p38 isoforms is one mechanism whereby the MAPK cascade influences differentiation-dependent gene expression in keratinocytes.

MATERIALS AND METHODS
Tissue Culture, Cell Transfection, and hINV Promoter Assay-Normal human foreskin keratinocytes were cultured as described previously (6). Third passage keratinocytes, grown in 35-mm dishes, were utilized when 50% confluent. Cells were transfected with 2 g of pINV-2473, a plasmid that encodes the full-length hINV promoter fused to the firefly luciferase gene (7), in the presence of 4 l of FuGENE6 transfection reagent (8). At 24 h posttransfection, adenoviruses encoding specific signal transduction kinases were added at 5-15 pfu/cell in the presence of 2.5 g/ml of Polybrene®. In some cases the cells were treated 24 h later with a pharmacological agent and incubated for an additional 24 h. To detect promoter activity, the cells were washed with phosphate buffered saline, dissolved in 140 l of cell lysis reagent (Promega), and harvested by scraping. Luciferase activity was assayed immediately in triplicate as previously described and normalized per g of protein as previously described (2,3,6). We used a green fluorescent protein-encoding expression plasmid to normalize transfection efficiency (8,9). Each experiment was repeated a minimum of three times. ERK1/2, p38, and JNK Kinase Activity Assays-p38, ERK1/2, and JNK activity were assayed using a non-radioactive method. Cell lysates were prepared in 20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 1 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Kinase activity was monitored in buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM ␤-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , and 10 mM MgCl 2 . For total p38 activity assessment, an agarose-conjugated monoclonal antibody (New England Biolabs number 9219) that binds to the phosphorylated form (Thr 180 / Tyr 182 ) of all p38 isoforms was used to immunoprecipitate active p38 kinase. The precipitate was assayed for p38 activity as measured by ability to phosphorylate ATF-2. ATF-2 phosphorylation was measured by immunoblot using a rabbit anti-phospho-ATF-2 (New England Biolabs number 9221S). ERK1/2 activity was monitored by immunoprecipitation using an agarose-conjugated monoclonal antibody (New England Biolabs number 9109) that binds phospho-ERK1/2. Activity of the precipitated enzyme was assayed based on ability to phosphorylate ELK-1. Phospho-ELK-1 was detected using a rabbit anti-phospho-ELK1 (New England Biolabs number 9181S). To measure JNK/SAPK * This work was supported by the National Institutes of Health (R. L. E.) and utilized the facilities of the Skin Diseases Research Center of Northeast Ohio. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Western Blot Analysis-Cells were rinsed with phosphate-buffered saline, lysed in Laemmli buffer (11), and an equivalent amount of protein (10 g) was electrophoresed on 8% acrylamide gels. The blots were incubated with the primary antibody, washed, and exposed to horseradish peroxidase-conjugated secondary antibody. Specific antibody binding was visualized using chemiluminescence detection reagents.
Detection of MEK6 mRNA-Poly(A ϩ ) RNA was isolated from cultured keratinocytes using the Oligotex Direct mRNA System (Qiagen). For real time RT-PCR, the RNA amplification kit (SYBR Green I, Roche Molecular Biochemicals) used 20 ng of mRNA and 0.5 M of each MEK6 primer (5Ј-AGC GGA TCC GAG CCA CAG TAA ATA-3Ј and 5Ј-CCC GAA ACA GTG CGC CAT AAA AG-3Ј) in a total reaction volume of 20 l. The denaturation, annealing, and elongation steps were 95°C for 1 s, 55°C for 10 s, and 72°C for 20 s, respectively. The primers were designed using the DNASTAR computer program based on the published MEK6 sequence (17) to amplify a 115-nucleotide segment of MEK6. Control reactions to evaluate primer specificity include no template or 0.1 ng of plasmid encoding MEK3, MEK6, or MEK7.

MEK6
Regulates hINV Promoter Activity-To evaluate the role of MEK6 as a regulator of hINV gene expression, keratinocytes were transfected with the pINV-2473 reporter plasmid and then infected with empty adenovirus (EV) or adenovirus encoding constitutively active MEK6. As shown in Fig. 1A, mock-infected and EV-infected cells display basal hINV promoter activity. In contrast, caMEK6 increases hINV promoter activity by 2.7-fold. To determine whether MEK6 similarly regulates endogenous hINV expression, cells were infected with MEK6-encoding virus, and endogenous hINV protein levels were assayed by immunoblot (2). Basal involucrin protein levels were detectable in mock-infected and EV-infected cells (Fig. 1B). In contrast, caMEK6 produced a 3-to 4-fold increase in endogenous involucrin level. To confirm expression of the exogenously delivered protein, we measured the level of adenovirus-expressed MEK6 proteins by immunoblot. As shown in Fig. 1C, caMEK6 is clearly expressed.
SB203580 Inhibits MEK6-dependent hINV Gene Expression-To determine whether MEK6 acts via p38, we tested the ability of SB203580, an inhibitor of p38␣ and ␤, to block the caMEK6-dependent increase in hINV protein levels. SB203580 at a concentration of 1 M inhibits p38␣ and ␤ but not p38␥ or FIG. 1. MEK6 regulates hINV gene expression. A, cells were transfected with the full-length hINV promoter luciferase reporter construct, pINV-2473, as described under "Materials and Methods." At 24 h posttransfection, the cells were infected with the indicated recombinant adenoviruses at a multiplicity of infection of 15 pfu/cell in keratinocyte serum-free media containing 2.5 g/ml Polybrene®. After 48 h, the cells were harvested and assayed for luciferase activity/g protein. B, cells were infected with empty adenovirus (EV) or adenovirus encoding caMEK6 at a multiplicity of infection of 15 pfu/cell in keratinocyte serum-free medium plus 2.5 g/ml Polybrene®. At 48 h, the cells were lysed, and extracts were prepared for immunoblot using rabbit anti-hINV, diluted 1:4000 (12,42). Binding of the primary antibody was visualized using peroxidase-conjugated donkey anti-rabbit IgG (Amersham Pharmacia Biotech NA934, diluted 1:10,000). C, to confirm expression of vector-produced MEK protein, cells were mock infected or infected with adenovirus expressing caMEK6. Cells were lysed, and equal amounts of protein were electrophoresed for immunodetection with goat anti-MEK6 (Santa Cruz sc-6073, diluted 1:1000). Binding of the MEK6 primary antibody was detected using peroxidase-conjugated donkey anti-goat IgG (Santa Cruz sc-2020, diluted 1:10,000). ␤-Actin level was monitored to confirm appropriate protein loading in B and C.
MEK6 Regulates p38 Kinase Activity-To determine whether MEK6 regulates p38 activity in cultured human keratinocytes, cells were infected with caMEK6-encoding virus and incubated for 48 h. The cells were then harvested, and an in vitro kinase assay was performed by selectively immunoprecipitating the activated form of all p38 isoforms using anti-phospho-p38. Kinase activity was then measured based on the ability of the precipitated enzyme to phosphorylate ATF-2. As shown in Fig.  3A, caMEK6 robustly increases endogenous p38 kinase activity in cultured keratinocytes. However, caMEK6 does not regulate ERK or JNK activity. To assure that the observed response is due to a change in activity and not p38 kinase level, we measured endogenous p38 levels in cells infected with constitutively active MEK6. As shown in Fig. 3B, treatment with MEK6 does not change p38 protein level or the level of ERK or JNK.

FIG. 4. Regulation of p38 MAPK isoforms in keratinocytes.
A, to measure the enzymatic activity of individual p38 isoforms in response to caMEK6, keratinocytes were co-infected with empty vector (Control (EV)), or caMEK6 and FLAG-p38␣, ␤, ␥, or ␦. After 48 h, the p38 isoforms were immunoprecipitated using mouse monoclonal anti-FLAG antibody M2 (Sigma number F3165, diluted to 5 g/ml) and 30 l of protein G/A-agarose (Oncogene IP05). p38 activity was monitored based on ability of the precipitated kinase to phosphorylate ATF-2. Phosphorylated ATF-2 was detected by immunoblot as in Fig. 3B. Immunoblot showing that p38␣, ␤, ␥, and ␦ are expressed in comparable levels when delivered by adenovirus vector. Cells were lysed 48 h after infection, and each p38 isoform was detected by immunoblot using peroxidaseconjugated mouse monoclonal anti-FLAG (Sigma A8592, diluted 1:10,000). C, endogenous p38␣ activity was measured by mock infecting keratinocytes, infecting with empty (EV) adenovirus, or infecting with adenovirus encoding caMEK6. After 48 h, the cells were harvested and endogenous p38␣, ␤, and ␦ were immunoprecipitated, respectively, using rabbit anti-p38␣ (Sigma number M0800), mouse anti-p38-␤2 (Amersham Pharmacia Biotech number 33-8700), and goat anti-p38␦ (Santa Cruz sc-7585), each at 5 g/precipitation. p38 kinase activity was monitored based on the ability to phosphorylate ATF-2. Phosphorylated ATF-2 was detected by immunoblot as in Fig. 3. This result was confirmed in three separate experiments.

FIG. 2. SB203580 inhibits MEK6-dependent expression of endogenous hINV gene.
To determine whether MEK6 acts via p38 to activate hINV gene expression, cells were mock-infected, or infected with 15 pfu/cell of empty adenovirus or adenovirus encoding caMEK6 in keratinocyte serum-free medium containing 2.5 g/ml Polybrene®. Cells were infected with caMEK6-expressing adenovirus in the presence of treatment with 0, 0.1, 1.0, and 10 M SB203580-HCl. The media was replaced every 12 h with fresh keratinocyte-serum free medium containing fresh SB203580HCl. At 48 h after infection, the cells were lysed, and hINV protein level was assayed by immunoblot (12). Identical results were observed in each of three experiments. ␤-actin was monitored as an internal control to confirm appropriate protein loading.
isoform be expressed at a comparable level in keratinocytes. This was confirmed by immunoblot using an anti-FLAG antibody (Fig. 4B). As previously reported, overexpressed p38␣ is active in control cells (21,22). Therefore, to determine whether caMEK6 activates p38␣, endogenous p38␣ was precipitated using an antibody that specifically detects p38␣, and the precipitated p38␣ enzyme was assayed for ability to phosphorylate ATF-2. As shown in Fig. 4C, endogenous p38␣ activity is substantially increased in the presence of caMEK6. Similarly, the endogenous p38␤ and ␦ isoforms were precipitated using specific antibodies and assayed for the ability to phosphorylate ATF-2. Fig. 4C also shows that caMEK6 does not increase endogenous p38␤ kinase activity; however, endogenous p38␦ kinase activity is increased.
p38 Isoforms Differentially Regulate caMEK6-dependent hINV Promoter Activation-If p38␣ is an activator of hINV gene expression, we would expect that dominant-negative p38␣ would inhibit and wild type p38␣ would stimulate the caMEK6dependent promoter activation. As shown in Fig. 5A, caMEK6dependent hINV promoter activity is inhibited by expression of dnp38␣. In addition, caMEK6 and p38␣ each increase promoter activity, and coexpression of these kinases results in a slightly greater increase (Fig. 5B). In contrast, as shown in Fig. 6, expression of wild type p38␦ inhibits caMEK6-dependent promoter activity.
p38␣ and ␦ Differentially Regulate Endogenous hINV Gene Expression-To determine whether p38␣ regulates endogenous hINV gene expression, cells were infected with empty adenovirus or adenovirus encoding p38␣ or caMEK6. After 48 h, cells were lysed and assayed for hINV protein expression. Fig. 7A shows that p38␣ and caMEK6 increase endogenous hINV protein levels. We next measured the ability of p38␦ to regulate both the caMEK6-and the TPA-dependent increase in endog- FIG. 6. p38␦ inhibits hINV promoter activity. Keratinocytes were transfected with 2 g/dish of the pINV-2473 hINV promoter construct. After 24 h, the cells were infected with either p38␦-encoding or empty (control) adenovirus at 10 pfu/cell. After an additional 24 h, the cells were harvested and assayed for hINV promoter activity. The experiment was repeated three times with essentially identical results.
FIG . 5. p38␣-dependent regulation of hINV promoter activity. A, keratinocytes were transfected with the full-length hINV promoter construct fused to the firefly luciferase gene and either pCMV-p38(AGF) (dnp38␣) (17) or empty pCMV plasmid (empty vector). After 24 h, cells were infected with empty adenovirus (EV) or adenovirus encoding caMEK6 at 10 pfu/cell and incubated for an additional 48 h. The cells were then harvested and assayed for luciferase activity, which was normalized per g of protein as previously described (43). B, keratinocytes were transfected with pINV-2473 as in A and after 24 h were infected with empty adenovirus (EV) or adenoviruses encoding p38␣ and/or caMEK6 at 5 pfu/cell. The total virus concentration/dish was maintained at 10 pfu/cell. After 48 h, the cells were harvested and assayed for luciferase activity. This experiment is representative of three separate determinations. enous hINV gene expression. TPA is a keratinocyte differentiating agent that is known to increase hINV levels (2,6). Treatment with TPA, caMEK6, or TPA/caMEK6 increases hINV protein level (Fig. 7B), and expression of p38␦ inhibits the increase. It is important to note that in one-half of the experiments co-treatement with caMEK6 and TPA results in enhanced levels of hINV compared with each individual treatment (not shown). In each case, the presence of p38␦ inhibits the increase.
MEK6 Is Expressed in Keratinocytes-For the present studies to be physiologically relevant, it is important to show that MEK6 is expressed in keratinocytes. To test this, we used real time RT-PCR to measure MEK6 mRNA levels. As shown in Fig. 8A, nonspecific background signal was detected in RT-PCR reactions programmed without template (blank) or in reactions programmed with 0.1 ng of MEK3-or MEK7-encoding plasmid (background signal was detected at 33-34 PCR cycles). In contrast, a signal was detected at cycle 16 when the reaction was programmed with 0.1 ng of MEK6-encoding plasmid. Thus, the primers are specific for MEK6 and do not detect related MEK family members. The reaction programmed with 20 ng of keratinocyte mRNA generated an RT-PCR curve that was detected at FIG. 7. p38␣ and ␦ differentially regulate endogenous hINV gene expression. A, keratinocytes were infected with 12.5 pfu/cell of empty adenovirus (EV), p38␣-encoding, or caMEK6-encoding adenovirus. After 48 h, the cells were harvested and hINV protein level was assayed by immunoblot as in Fig. 2. Involucrin sometimes runs as a doublet on gel electrophoresis. B, keratinocytes were infected adenoviruses encoding p38␦ and/or caMEK6 at 5 pfu/cell. Total viral load was maintained at 15 pfu/cell by addition of empty adenovirus (EV). After 48 h, the cells were treated with 50 ng/ml TPA for 24 h. The cells were then harvested and extracts were prepared for detection of hINV by immunoblot. Fluorescence intensity is directly correlated with accumulation of PCR product. B, to detect endogenous MEK6 protein expression, a total cell extract was prepared, and 100 g of extract was electrophoresed on a 6% polyacrylamide gel. An extract, prepared from cells infected with MEK6encoding adenovirus, was electrophoresed in parallel as a positive control for antibody activity (expressed). MEK6 was detected by immunoblot using rabbit anti-MEK6 (Chemicon International, AB3185) diluted 1:700. Binding of the primary antibody was visualized using peroxidaseconjugated donkey anti-rabbit IgG (Amersham Pharmacia Biotech NA934, diluted 1:10,000), and the signal was visualized by chemiluminescence. PCR cycle 26, indicating that the MEK6 mRNA is expressed. In addition, low level MEK6 protein expression was confirmed by immunoblot using a MEK6-specific antibody (Fig. 8B). DISCUSSION The MAP kinases are ubiquitously expressed enzymes that transfer signals from the cell surface to the nucleus (23). These enzymes are organized as part of three-kinase modules that include a MAPK kinase kinase (MEKK), a MAPK kinase (MEK), and a MAPK (24). Four major mitogen-activated protein kinases (MAPK) have been identified including ERK1/2, ERK5, p38 MAPK, and the c-jun NH 2 -terminal kinase (JNK) (24 -27). MEKdependent dual phosphorylation of the regulatory loop of the MAPK results in kinase activation (24). MEKs are known to differentially activate specific MAPKs. For example, MEK1/2 activates ERK1/2, MEK5 activates ERK5/BMK1, MEK4 activates p38 and JNK, MEK7 activates JNK and p38, and MEK3 and MEK6 activate p38 (23, 24, 28 -30). Thus, an understanding of signal transduction requires an evaluation of how each MEK regulates MAPK activity in each particular cell type.
Involucrin is a marker of keratinocyte differentiation that is specifically expressed only in differentiated keratinocytes. Involucrin expression is restricted to the suprabasal layers in stratifying squamous surface epithelia (31,32). Involucrin expression in cultured cells is increased by agents that enhance keratinocyte differentiation such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (1,31,33) and calcium (2,6). Previous studies have shown that the MAPK cascades play a central role in maintaining both basal and regulated expression (2,3,8,9,34). Studies using dominant-negative kinases and pharmacological agents indicate that p38 kinase activity is required for activation of hINV gene expression. This hINV gene regulatory cascade includes the novel protein kinase C isoforms, Ras, and MEKK1 (2, 3). MEKK1, in turn, targets several MEKs, including MEK1, MEK3, and MEK7 but not MEK4 (3,22). Downstream targets of this pathway include the C/EBP and AP1 transcription factors (6 -8). These transcriptional regulators, in turn, bind to specific elements in the hINV promoter proximal and distal regulatory regions to regulate transcription (2, 3, 6, 9, 34, and 35).
Although MEK3 and MEK7 are known to function as regulators of hINV gene expression (2,3,22), the role of MEK6 has not been evaluated. MEK6 is a potential regulator, as it has been shown to regulate p38 MAPK in other systems (36 -38). Our results showed that MEK6 is expressed in keratinocytes and that caMEK6 increases hINV promoter activity and expression of endogenous hINV.
p38 MAPK is a downstream mediator of the MEK6-dependent response, as endogenous p38 MAPK activity is dramatically increased in caMEK6-expressing keratinocytes, and this correlates with increased involucrin expression. This is in contrast to JNK and ERK activity, which are not caMEK6-responsive. The p38 MAP kinase family consists of four isoforms, p38␣, ␤, ␥, and ␦. Although these isoforms share 50 to 60% homology, they demonstrate differential substrate specificity and activation by MEKs (5, 20, 27, 39 -41). The p38-dependent signal that activates hINV gene expression appears to be mediated by p38␣. Thus, caMEK6 increases p38␣ enzyme activity, and the MEK6-dependent increase in gene expression is inhibited by an agent, SB203580, which inhibits p38␣. SB203580 also inhibits p38␤ at the concentrations used in this study, however, it appears unlikely that p38␤ mediates the response, as it is not activated by MEK6 in our system. p38␥ is also a candidate mediator of the MEK6-associated response, as it is also activated by caMEK6. However, our recent report using quantitative real time RT-PCR to measure p38 isoform mRNA levels, showed that p38␥ is not expressed in cultured keratino-cytes (22). p38␦ activity is also increased by caMEK6. However, we observed that the MEK6-dependent increase in hINV gene expression is reduced by p38␦, suggesting that this isoform is an inhibitor of expression. In addition, p38␦ inhibits the TPAassociated increase in hINV gene expression. As TPA is known to enhance hINV gene expression via a nPKC, Ras, MEKK1, MEK, p38 cascade (2,3), this result further suggests the potential physiological importance of p38␦ as an inhibitor of hINV gene expression. In addition, dominant-negative p38␣ strongly inhibits MEK6-mediated activation of hINV promoter activity. Thus, the most likely scenario is that the MEK6 stimulus is carried downstream by p38␣. An interesting observation is the high level of p38␣ activity observed in p38␣ overexpressing cells in the absence of upstream stimulus (Fig. 4). We would have expected, based on the other results presented in the manuscript, that this activity would be low. However, increased p38␣ activity has been previously reported in cells expressing high levels of p38␣. It has been suggested that this is due to the tendency of this p38 isoform to become selectively activated by low levels of MEK6 (21,22). This effect is not observed for the other p38 MAPKs (21). Thus, when p38␣ is present at high levels, mass action may drive its activation. Our previous report shows that p38␣ is more abundant in keratinocytes than p38␦ (22). In fact, the tendency of p38␣ to become active at lower MEK6 concentrations may explain why the net effect of MEK6 activation is to increase hINV gene expression, despite the fact that it can also activate a MAPK, p38␦, which functions to decrease hINV gene expression.
We propose that the effect of MEK6 on hINV gene expression is determined by the balance between MEK6-dependent activation of p38␣ and ␦ and that in the conditions used in the present experiments, the p38␣-dependent activation of gene expression is the predominant response.