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-de-pendent 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 and radioactivity autoradiography. Keratinocytes seeded at 10,000 9.5-cm 2 dishes in KSFM and to 48 treated by addition of fresh KSFM in the presence of increasing concentrations of EGCG. After 4 days, were har- vested with balanced salt solution containing 0.025% trypsin

Immunological Detection of Proteins-For immunoblot analysis, equal quantities of protein were electrophoresed on denaturing and reducing polyacrylamide gels and transferred to nitrocellulose. The membranes were blocked and then incubated with the primary antibody followed by an appropriate secondary antibody. Secondary antibody binding was visualized using a chemiluminescent detection system (Amersham Biosciences, Inc.).
Plasmids and Adenoviruses-The hINV reporter plasmids contains various lengths of hINV upstream promoter sequence fused to the luciferase reporter gene in pGL2-basic. These plasmids have been described previously (17,18). Plasmids encoding dominant-negative ERK1 (K71R) and dominant-negative ERK2 (K52R), each cloned in pCEP4, were kindly provided by Dr. Melanie Cobb (19). Dominantnegative Ha-Ras (S17N), cloned in pSR␣, and dominant-negative Raf-1 (K375W), cloned in pRSV, were kindly provided by Dr. Michael Karin (20). Dominant-negative MEK4 (S220A, T224L), cloned in pEECMV, was obtained from Dr. Dennis Templeton (21)(22)(23). Dominant-negative MEKK1 (K432M), cloned in pSR␣, was from Dr. Michael Karin (20). Dominant-negative MKK7 (MKK7KL), cloned in pSR␣, was provided by E. Nishida (24). Dominant-negative MKK3 (MKK3 Ala), cloned in pRSV, dominant-negative p38 MAPK (p38 AGF), cloned in pCMV5, and dominant-negative JNK1 (JNK1 APF), cloned in pcDNA3, were generously provided by Dr. Roger Davis (25)(26)(27). Empty adenovirus was constructed as described previously (28). Adenoviruses encoding wild-FIG. 1. EGCG regulates hINV promoter activity. A, keratinocytes were grown in KSFM until 70% confluent and then were transfected with 2 g of pINV-2473, a plasmid that encodes the full-length hINV promoter (18). The transfected cells were then maintained for 24 h in the absence of treatment (Control) or in the presence of 20 g/ml EGCG or 50 ng/ml TPA. The cells were then harvested for extract preparation and measurement of luciferase activity. B, keratinocytes were transfected with 2 g of pINV-2473 and then treated for 24 h in the presence of different concentrations of EGCG (0, 5, 10, 15, and 20 g/ml). Cell extracts were then prepared and assayed for luciferase activity (17,18). The error bars represent the standard error of the mean. These promoter experiments were repeated a minimum of three times with similar results. C, normal human keratinocytes were treated for 24 h with KSFM or KSFM containing 20 g/ml EGCG or 50 ng/ml TPA. After 24 h, total cell extracts were prepared and assayed for hINV protein level by immunoblot. Equivalent quantities of protein extract were electrophoresed on 8% polyacrylamide gel, transferred to nitrocellulose, and incubated with rabbit anti-hINV-specific antibody (16,66). Identical changes in hINV protein level were observed in each of three experiments. hINV mRNA levels also increased in response to EGCG treatment (data not shown). ␤-Actin levels were monitored as a control to assure appropriate loading.

FIG. 2. hINV promoter sites that mediate the EGCG response.
A, cultured human epidermal keratinocytes were transfected with 2 g of the indicated hINV promoter construct (pINV-241, pINV-128, pINV-110, and pINV-41) (18). The cells were then treated for 24 h in the absence (open bars) or presence (solid bars) of 20 g/ml EGCG. After 24 h, the cells were harvested to assay the level of promoter activity (17). B, cultured human keratinocytes were transfected with the indicated hINV promoter-luciferase reporter constructs. pINV-241 is the native construct. In constructs pINV-241(AP1-1m) and pINV-241 (EBS-2m), the AP1-1 and EBS-2 sites, respectively, are mutated. After a 24-h treatment with 20 g/ml EGCG, the cells were harvested and assayed for hINV promoter-dependent luciferase activity. This experiment is representative of four separate experiments. The bars represent the standard error of the mean. type, flag-tagged p38␣, -␤, -␦, and -␥ were kindly provided by Dr. Y. Wang (29,30) and were used as described previously (28). Adenovirus infection was performed at a multiplicity of infection of 15 in the presence of Polybrene. These conditions are optimized to infect 100% of the cells (28).
Tissue Culture, Cell Transfection, and Luciferase Assay-Normal human foreskin keratinocytes were cultured as described previously (18). Third passage keratinocytes, growing in 9.5-cm 2 area dishes, were transfected when ϳ70% confluent. For transfection, 4 l of Fugene was added to 96 l of KSFM and incubated at 25°C for 5 min. The mixture was then mixed with 2 g of involucrin promoter reporter plasmid or, for co-transfection experiments, with 1 g of involucrin reporter plasmid and 1 g of a second plasmid. The mixture was incubated at 25°C for 15 min and then added directly to the cultures in 2 ml of KSFM. The final DNA concentration in all groups was adjusted to 2 g of DNA/4 l of Fugene-6 reagent/9.5-cm 2 dish by addition of empty expression vector. After 24 h, the cells were incubated with KSFM in the presence or absence of EGCG. After an additional 24 h, the cells were washed with phosphate-buffered saline, dissolved in 250 l 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 microgram of protein as described previously (17). Transfection efficiency was normalized using a green fluorescent protein-encoding expression vector.
Preparation of Nuclear Extracts-Cultured keratinocytes, grown in KSFM until 80% confluent, were treated with or without 20 g/ml EGCG for 24 h prior to preparation of total cell/nuclear extracts as described previously (17). Nuclear extracts were prepared according to method of Schreiber et al. (31) in the presence of proteinase inhibitors including leupeptin (10 g/ml), aprotinin (10 g/ml), p-benzamide (100 M), and pepstatin (2 g/ml). The protein content was determined using Bradford reagent (Bio-Rad).
Gel Mobility Shift and Supershift Assays-Binding of transcription factors to the hINV promoter AP1-1 site was detected using gel mobility shift assay. Five micrograms of nuclear extract was incubated for 10 min at room temperature in a total volume of 20 l containing 40 mM HEPES (pH 7.6), 10% glycerol, 200 mM KCl, 10 mM dithiothreitol, 2 g/ml poly(dI-dC), and 100,000 cpm of radioactive double-stranded DNA oligonucleotide. The nucleotide, 5Ј-TGTGGTGAGTCAG-GAAGGGGTT (AP1 site in bold), was end-labeled using polynucleotide kinase and [␥-32 P]ATP. For competition studies, radioinert DNA competitor was added to the DNA binding reaction at 10-or 100-fold molar excess. For supershift assays, AP1 factor-specific antibodies (4 g) were added to the reaction mixture after the initial 10 min incubation, and the incubation was continued for an additional 30 min at 25°C. In control reactions, the AP1 antibody was replaced with a nonspecific antibody. Protein-DNA complexes were resolved on a 6% nondenaturing polyacrylamide gel using 0.25ϫ TBE running buffer. The gel was then dried and the radioactivity detected by autoradiography.
Cell Proliferation Studies-Keratinocytes were seeded at 10,000 cells/cm 2 in 9.5-cm 2 dishes in KSFM and allowed to grow for 48 h. The cells were then treated by addition of fresh KSFM in the presence of increasing concentrations of EGCG. After 4 days, the cells were harvested with Hanks' balanced salt solution containing 0.025% trypsin and 1 mM EDTA, fixed in isotonic buffer containing 4% formaldehyde, and counted using a Coulter counter. Cornified envelopes were counted as previously described (32,33).

RESULTS
EGCG Increases hINV Promoter Activity-To determine whether EGCG regulates AP1 factor-dependent events in keratinocytes, we examined its ability to regulate gene expression. Involucrin is a well studied, AP1-responsive marker of keratinocyte differentiation (18,34). Normal keratinocytes were transfected with pINV-2473, a plasmid that encodes the full-length hINV promoter (18), and then treated for 24 h with 20 g/ml EGCG or 50 ng/ml TPA. As shown in Fig. 1A, EGCG increases hINV promoter activity nearly as efficiently as TPA. As shown in Fig. 1B, the EGCG-associated increase is concentration-dependent with a 7-fold increase at 20 g of EGCG/ml. To assure that this response is physiologically relevant, we measured the effect of EGCG treatment on accumulation of endogenous hINV protein. As shown in Fig. 1C, EGCG in-creases endogenous hINV protein levels. Treatment with TPA, shown as a positive control, produces a similar level of increase.
Localization of hINV Promoter Site That Mediates EGCG Response-To determine whether the EGCG-associated response is linked to AP1 sites within the hINV promoter, we tested the response of a series of hINV promoter 5Ј deletion constructs (18,35). Fig. 2A shows that constructs pINV-241 and pINV-128 respond to EGCG with a 3-4-fold increase in promoter activity. Plasmids pINV-110 and pINV-41, in contrast, were not responsive, suggesting that the relevant EGCG-response element is located within the segment spanning nucleotides Ϫ128 to Ϫ110. Previous studies show that the Ϫ128/Ϫ110 region of the hINV promoter contains functional Ets factor binding sties (EBS-2), and an activator protein 1 (AP1-1) transcription factor binding site (18,34). We therefore tested whether the EGCG response is mediated via these elements. Fig. 2B shows that the mutation of EBS-2 site does not influence the EGCG-associated increase in promoter activity. However, in contrast, mutation of the AP1-1 site reduces overall activity and eliminates the EGCG-associated regulation. Thus, the AP1-1 site appears to be required for EGCGdependent gene activation.
EGCG Increases AP1 Factor Level and DNA Binding-To explore the mechanism responsible for this regulation, we monitored the effects of EGCG treatment on AP1 factor expression. As shown in Fig. 3, EGCG treatment increases nuclear Fra-1, Fra-2, c-Fos, FosB, c-Jun, JunB, and JunD levels. To determine whether EGCG enhances AP1 factor DNA binding, we performed gel mobility shift assays using an hINV AP1-1 site-encoding oligonucleotide, 5Ј-TGTGGTGAGTCAGGAAGGGGTT (AP1 binding motif in bold). Fig. 4A reveals that EGCG markedly increases transcription factor binding to the hINV AP1-1 site. Moreover, this binding is specific, as addition of a 10-or 100-fold molar excess of radioinert oligonucleotide eliminates binding. In contrast, no inhibition of binding to the 32 P-labeled AP1-1 oligonucleotide was observed in the presence of a 100-fold molar excess of oligonucleotide encoding a mutated AP1-1 binding site (data not shown). These results show that AP1 DNA binding is increased by EGCG. To identify the factors that interact with the AP1-1 site, nuclear extracts were incubated with 32 P-labeled AP1-1 in the presence or absence of antibodies reactive with specific Jun and Fos family members.
FIG. 3. EGCG increases AP1 factor levels. Keratinocytes were treated with or without 20 g/ml EGCG for 24 h followed by preparation of nuclear extracts. Equivalent quantities of protein extract were electrophoresed on a denaturing and reducing 10% acrylamide gel and transferred to nitrocellulose for detection using appropriate rabbit polyclonal AP1 factor-specific antibodies. Binding of the primary antibody was detected by incubation with an appropriate anti-rabbit IgG secondary antibody, and binding was visualized using a chemiluminescent detection system. Changes in level were measured using laser densitometry. Similar results were obtained in each of four experiments.
As shown in Fig. 4B, only antibodies specific for Fra-1 and JunD produced supershifted bands (indicated by asterisks). No supershift was observed in extracts incubated with a nonspecific IgG, or antibodies specific for c-Fos, FosB, Fra-2, c-Jun, or JunB, suggesting that Fra-1 and JunD are the AP1 factors associated with the complex.
Tyrosine Kinase Activity Is Required for EGCG-dependent Regulation of hINV Gene Expression-To investigate the mech-anism whereby EGCG regulates AP1 factor level and hINV gene expression, we examined the signal transduction pathways associated with the EGCG response. Genistein is an efficient inhibitor of tyrosine kinases, including the dual specificity MEK. Fig. 5 shows that genistein causes a concentrationdependent reduction in hINV promoter activity, suggesting a role of MAPK pathways in this regulation.
EGCG Regulation of hINV Gene Expression Requires Ras and MEKK1 Activity-As Ras is an important regulator of MAPK cascade activity in keratinocytes (17), we evaluated the role of Ras in the EGCG-dependent regulation. As shown in Fig. 6A, expression of a dominant-negative form of Ras, dnRas, results in the complete suppression of the EGCG-dependent increase in hINV promoter activity. We next assessed the role of Raf-1 and MEKK1, kinases located immediately downstream of Ras. As shown in Fig. 6B, dominant-negative MEKK1 (dnMEKK1) efficiently inhibits EGCG action. In contrast, dominant-negative Raf-1 (dnRaf-1) does not alter the response. The immunoblot results (Fig. 6C) confirm that the expression vectors do produce dnRas, dnMEKK1, and dnRaf-1 in keratinocytes.
MEK3 and p38 Mediate the EGCG-dependent Regulation-MEKK1 is known to activate, in a stimulus-and cell typespecific manner, several MAPK modules (36). MEK4, MEK7, and MEK3 are direct downstream targets of MEKK1 (17,28,37). We therefore evaluated whether MEK4, MEK7, or MEK3 activity is required for the EGCG-dependent regulation. As shown in Fig. 7A, dominant-negative forms of MEK4 or MEK7 do not inhibit hINV promoter activation. In contrast, expression of dnMEK3 completely inhibits promoter activation, suggesting that MEK3 activity is required for EGCG-dependent regulation. Fig. 7B shows that the vector-encoded kinase mutants are expressed in keratinocytes.
To assess the ability of individual MAPKs to function as mediators of the EGCG/MEK3 signal, we measured the ability of various dnMAPKs to inhibit the EGCG-related response. Dominant-negative ERK2 slightly reduces the response, whereas dominant-negative ERK1 potentiates the EGCG-associated regulation (Fig. 8A). In addition, dominant-negative JNK causes a slight reduction in the EGCG-dependent re- FIG. 4. Gel mobility shift analysis of binding to AP1-1 element. A, nuclear extracts were prepared from near-confluent keratinocyte cultures following a 24-h treatment in the absence (Ϫ) or presence (ϩ) of 20 g/ml EGCG. Nuclear extracts (N. Ext.) were then incubated with 32 P-labeled double-stranded oligomer, 5Ј-TGTGGTGAGTCAG-GAAGGGGTT ( 32 P-AP1-1), containing the hINV AP1-1-binding site (bold). Specific binding was demonstrated by including a 10-and 100fold molar excess of homologous competitor oligonucleotide (AP1-1) during the binding reaction. Complexes were separated by electrophoresis on a nondenaturing gel and visualized by autoradiography. AP1 indicates the migration position of the AP1/oligonucleotide complex, and FP indicates migration of the free probe. B, interaction of specific Jun/Fos proteins with the AP1-1 binding site. Nuclear extracts, prepared and incubated with 32 P-labeled double-stranded hINV AP1-1 site oligomer, followed by incubation with an appropriate AP1 factorspecific antibody (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, or Fra-2) or a nonspecific antibody (IgG). Complexes were separated by electrophoresis on a nondenaturing 8% acrylamide gel and visualized by autoradiography. FP indicates free probe, and the bar indicates AP1 binding. Lane C indicates the migration of probe in the absence of added nuclear extract. AP1 indicates migration of the AP1 factor/oligonucleotide complex, and the asterisks indicate the positions of the supershifted bands in the JunD and Fra-1 lanes. 5. Genistein inhibits the EGCG-dependent increase in hINV promoter activity. Keratinocytes were grown in KSFM until 70% confluent and then were transfected 2 g of pINV-241 (18). The transfected cells were maintained for 24 h in the presence or absence of 20 g/ml EGCG and increasing concentrations of genistein. Genistein was administered 30 min prior to addition of EGCG. The cells were then harvested for extract preparation and measurement of luciferase activity (17,18). The error bars represent the standard error of the mean. These promoter experiments were repeated a minimum of three times with similar results. sponse (Fig. 8B). However, the most striking response is evoked by dnp38, which completely inhibits the response to EGCG (Fig. 8B). Fig. 8C shows that the vector-encoded dominantnegative kinases are expressed in keratinocytes. Based on these results, we suggest that EGCG regulates hINV gene expression via the pathway presented in Fig. 8D.

FIG.
p38␦ MAPK Mediates the EGCG-dependent Regulation-The above results argue that the EGCG-dependent signal is transmitted via activation of p38 MAPK. To further test this hypothesis, we performed p38 kinase activity assays. We also assayed for EGCG-dependent activation of ERK1/2 and JNK1/2. As shown in Fig. 9A, a transient increase in ERK1/2 activity, as measured by ELK1 phosphorylation, is observed at 15-30 min after EGCG treatment. Total p38 activity, as measured based on ATF2 phosphorylation, is increased at 15 min and remains elevated at 4 h. JNK activity is not regulated. Immunological analysis, shown in Fig. 9B, indicates that these kinase activity changes are not caused by changes in JNK1/2, ERK1/2, or p38 levels. To determine whether the ERK activity observed in Fig.  9A is preferentially caused by activation of ERK1 or ERK2, we measured the level of phosphorylated ERK1/2. Fig. 9C shows that EGCG treatment is not associated with preferential activation of ERK1 or ERK2. To determine which p38 isoform is activated by EGCG, we expressed FLAG-tagged forms of each isoform in keratinocytes and then treated with EGCG. At various times after EGCG addition, the FLAG-p38 kinases were precipitated and assayed for ability to phosphorylated ATF2. Fig. 9D shows that p38␦ is the only form activated by EGCG. We next confirmed that the endogenous p38␦ isoform is activated. As shown in Fig. 9E, p38␦ levels are not altered by EGCG treatment (p38␦), but activity (P-ATF2) increases within 15 min of EGCG treatment and remains elevated at 4 h.
EGCG Inhibits Keratinocyte Proliferation-The observation that EGCG increases AP1 expression and hINV gene expression suggests that EGCG may enhance keratinocyte differentiation. Because differentiation is associated with reduced cell proliferation (38, 39), we measured the effect of EGCG on keratinocyte proliferation. Subconfluent dishes of keratinocytes were treated with increasing concentrations of EGCG for 4 days and then harvested and counted. The open bars in each panel show that number of cells present on the first day of treatment. As shown in Fig. 10A, keratinocyte FIG. 6. Ras and MEKK1 activity are required for EGCG-dependent regulation of hINV gene expression. Keratinocytes were grown in KSFM until 70% confluent and then were transfected with 1 g of pINV-241 in the presence of 1 g of empty expression vector (EV) or dominant-negative Ras-encoding expression plasmid (A), or empty expression vector or expression vectors encoding dominantnegative Raf-1 or dominant-negative MEKK1 (B). The transfected cells were maintained for 24 h in the presence or absence of 20 g/ml EGCG and assayed for luciferase activity. This experiment is representative of four separate experiments. C, keratinocytes were transfected with 1 g of empty expression plasmid (Ϫ) or expression plasmids encoding dnRas, dnMEK1, or dnRaf-1 (ϩ). At 24 h, extracts were prepared and each protein was detected by immunoblot using antibodies specific for Ras (Santa Cruz, sc-520, diluted 1:1000), MEKK1 (Santa Cruz, sc-437, diluted 1:500), or Raf-1 (Santa Cruz, sc-7198, diluted 1:500). It is important to note that longer exposure times revealed the presence of endogenous Ras, MEKK1, and Raf-1 (data not shown). ␤-Actin levels were measured to assure that each lane contained an equivalent amount of protein.

FIG. 7. EGCG-dependent regulation of hINV gene expression requires MEK3 activity.
A, keratinocytes were grown in KSFM until 70% confluent and then were transfected 1 g of pINV-241 in the presence of 1 g of empty expression vector (EV) or vector encoding dominant-negative MEK4, MEK7, or MEK3. The transfected cells were maintained for 24 h in the presence or absence of 20 g/ml EGCG, and then extracts were prepared for assay of luciferase activity. Identical results were observed in each of three experiments. The error bars represent the mean Ϯ S.E. B, to confirm expression of each kinase, keratinocytes were transfected with 1 g of empty expression plasmid (Ϫ) or expression plasmids encoding dnMEK4, dnME7, dnMEK3 (ϩ). After 24 h, the cells were harvested and total extracts was prepared and assayed for dnMEK4, dnMEK7, and dnMEK3 expression using specific rabbit polyclonal antibodies from Santa Cruz diluted 1:1000 (MEK4, sc-837; MEK7, sc7104; MEK3, sc-960). In empty vector-transfected cells (Ϫ), endogenous MEK7, MEK4, and MEK3 was detected at longer exposure times (data not shown). ␤-Actin levels were measured to assure that each lane contained an equivalent amount of protein.
cell number is markedly reduced by EGCG concentrations greater than 10 g/ml. To determine whether EGCG nonspecifically causes toxic cell death, confluent cultures of normal keratinocytes treated for 4 days with 0 -30 g/ml EGCG. As shown in Fig. 10B, EGCG treatment did not cause the nonspecific loss of cells.
EGCG Increases Cornified Envelope Formation-The process of terminal keratinocyte differentiation is characterized by the formation of cornified envelopes (40 -43). Fig. 11 compares the effect of EGCG on viable cell number and cornified envelope formation. Cells were treated for 4 days with 0, 10, or 20 g/ml EGCG. The open bars in Fig. 11 show the number of cells per dish present on the first day of treatment, and the cross-hatched bars show the counts after 4 days of EGCG treatment. In the absence of EGCG treatment, the number of cells doubled over 4 days (Fig. 11A). This increase was partially inhibited by 10 g/ml EGCG, and completely inhibited at 20 g/ml EGCG. Fig. 11B shows that the number of cornified cells was markedly increased by EGCG treatment. This percentage of cornified cells increased from 1% in the FIG. 8. ECGC-dependent regulation of hINV gene expression requires p38 MAPK activity. A, primary keratinocytes were grown in KSFM until 70% confluent and then were transfected with 1 g of pINV-241 in the presence of 1 g of empty expression vector (EV) or vector encoding dominant-negative ERK1 or ERK2 (A), or JNK or p38␣ (B). The transfected cells were maintained for 24 h in the presence or absence of 20 g/ml EGCG, and then extracts were prepared for assay of luciferase activity. Identical results were observed in each of three experiments. The error bars represent the mean Ϯ S.E. C, keratinocytes were transfected with 1 g of empty expression plasmid (Ϫ) or plasmid encoding dnJNK, dnERK1 or dnERK2 (ϩ), and dnp38. After 24 h, extracts were prepared and assayed by immunoblot with rabbit antibodies specific for JNK1/2 (Sigma, J4500, diluted 1:2000), ERK1/2 (Sigma, M5670, diluted 1:5000), and p38 (Sigma, M0800, diluted 1:5000), respectively. ␤-Actin levels were monitored as a protein loading control. Expression of endogenous MAPK proteins in nontransfected cells (Ϫ) was detected upon longer exposure of the membranes (data not shown). D, postulated cascade whereby EGCG regulates hINV gene expression in normal human keratinocytes.
FIG. 9. EGCG activates p38␦ MAPK. A, keratinocytes were treated with 20 g/ml EGCG. At the indicated times, JNK1/2, ERK1/2, and p38 MAPKs were immunoprecipitated, respectively, using c-Jun fusion beads (New England Biolabs 9811), rabbit polyclonal anti-phospho-ERK1/2 (New England Biolabs 9109), or rabbit polyclonal anti-phospho-p38␣, -␤, -␦, and -␥ (New England Biolabs 9219). Phosphorylation of substrates was monitored by immunoblot using rabbit anti-phospho-c-Jun (New England Biolabs 810), rabbit anti-phospho-ELK1 (New England Biolabs 9181), or rabbit anti-phospho-ATF-2 (New England Biolabs 9221S, diluted 1:1000). B, p38, JNK1/2, and ERK1/2 levels were assayed in by immunoblot using rabbit anti-p38 (Sigma M0800 diluted 1:5000), rabbit anti-JNK1/2 (Sigma J4500 diluted 1:2000), and rabbit anti-ERK1/2 (Sigma M5670 diluted 1:5000). Primary antibody binding was detected using horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham NA934 diluted 1:10,000). C, to measure the extent of ERK1 and ERK2 phosphorylation, keratinocytes were treated with 20 g/ml EGCG for 0 -24 h. Equivalent quantities of protein were electrophoresed and immunoblotted with a mouse monoclonal antiphospho-ERK1/2 (Santa Cruz, sc-8383) diluted 1:1000. Binding of the primary antibody was detected using anti-mouse IgG. D, preconfluent primary keratinocytes, growing in KSFM, were infected with empty adenovirus (EV) or adenovirus encoding individual wild-type, FLAGtagged p38 isoforms. At 48 h, the cells were treated with 20 g EGCG/ml for 0 -24 h. The cells were then harvested, and extracts were prepared. Equal amounts of protein (200 g) were precipitated using mouse monoclonal anti-FLAG (Sigma, F3165, 5 g) and protein A/Gagarose. Activity of the precipitated kinase was monitored based on the ability to phosphorylate ATF-2 fusion protein using an antibody specific for phospho-ATF-2 (New England Biolabs 9221S). No FLAG-related immunoreactivity was detected in empty vector-infected cells (EV) at any time point. E, after EGCG treatment, cell extracts were prepared, and p38␦ was detected by immunoblot using goat anti-p38␦ (Santa Cruz, sc7585). In parallel, p38␦ activity was immunoprecipitated using the same antibody, and the ability of the precipitated kinase to phosphorylate ATF2 was measured as outlined above. absence of EGCG treatment to 15% in the presence of 20 g/ml EGCG. DISCUSSION EGCG Inhibits AP1 Factor Expression and Activity in Immortalized Keratinocytes-AP1 transcription factor activity in immortalized and/or transformed keratinocytes is increased by carcinogenic stimuli. This change is thought to be part of a pathway that leads to increased cell proliferation. EGCG has been shown to inhibit this increase (5-7, 14, 44). For example, in JB6 mouse epidermal cells, EGCG inhibits epidermal growth factor and phorbol ester-associated cell transformation, and this inhibition is correlated with a reduction in AP1 factor expression and DNA binding (44). In addition, EGCG and other bioactive polyphenols inhibit the increase in AP1 level that is associated with exposure to UVB radiation (5,14). In HaCaT cells, an immortalized human keratinocyte cell line, UVB-dependent induction of AP1 activity is inhibited by EGCG (7). In addition, EGCG inhibits transcriptional activation of the gene for c-Fos and accumulation of c-Fos protein in this cell line (6).
EGCG also suppresses signal transduction cascades that lead to AP1 factor activation (6,44) and decreases c-Jun phosphorylation in transformed and immortalized human bronchial epithelial cell lines (45).
p38 MAPK Mediates the EGCG-dependent Regulation of hINV Gene Expression-In contrast to the above studies, we show that AP1 factor levels are markedly increased when normal keratinocytes are treated with EGCG, suggesting a fundamentally different mechanism of EGCG action in normal keratinocytes. To explore this mechanism, we examined the EGCGassociated signaling pathways that regulate AP1 factor level and involucrin gene expression. Functional studies, using dominant-negative kinases and pharmacologic inhibitors, suggest that a Ras, MEKK1, MEK3 cascade mediates this regulation. These cascades can target a variety of MAPK isoforms, including ERK1/2, JNK1/2, and p38 MAPKs. A role for JNK1/2 in this regulation is ruled out by the absence of activation of JNK following EGCG treatment and the finding that dnJNK does not inhibit the response. We also considered the possibility that ERK1/2 may convey the signal, as ERK1/2 activity is transiently elevated at 15-30 min after EGCG treatment. However, it is unlikely that ERK1 mediates this regulation, as functional studies show that expression of dnERK1 results in an increase FIG. 10. EGCG regulates cell proliferation. A, normal human keratinocytes were plated at low density in 9.5-cm 2 surface area dishes and then treated with the indicated concentration of EGCG (0 -30 g/ml). After 4 days, the cells were harvested and counted using a Coulter Counter. The open bar (control) indicates the number of cells present on day 0 when the EGCG treatment was initiated. B, normal keratinocytes were plated and allowed to become confluent prior to a 4-day treatment with 0 -30 g/ml EGCG. The open bar (control) indicates the number of cells present on day 0 when the EGCG treatment was initiated. Cell number was determined using a Coulter Counter. The error bars represent the standard error of the mean. This experiment was repeated three times with essentially identical results.
FIG. 11. EGCG enhances cornified envelope formation. Normal human keratinocytes were plated in 9.5-cm 2 surface area dishes and then treated with 0 -20 g/ml EGCG for 4 days. A, following EGCG treatment, the cells were harvested and the number of trypan blueexcluding cells were determined using a hemocytometer. The open bar indicates the cell count at the time treatment was initiated. The viable cell number after 4 days of EGCG treatment is indicated by the crosshatched bars. B, after EGCG treatment, the cells were harvested directly into Laemmli sample buffer and boiled (67). Cornified envelopes were counted as described previously (32,33). Values in parentheses indicate the ratio of the number of envelopes divided by the viable cell number expressed as a percentage. Error bars indicate the standard error of the mean. in promoter activity. This suggests that ERK1 suppresses EGCG-dependent promoter activity. Parallel studies show that dnERK2 does not influence the regulation.
In contrast, the EGCG-dependent response is associated with prolonged activation of p38 MAPK activity. There are four known p38 MAPK isoforms, p38␣, -␤, -␦, and -␥. Each isoform is thought to produce different effects on downstream responses (46 -50). Only p38␣, -␤, and -␦ are expressed in keratinocytes (28). Kinase activity studies show that p38␦ is the sole p38 MAPK isoform activated in response to EGCG treatment. In addition, treatment with 10 M SB203580, an agent that inhibits p38␣ and p38␤ but not p38␦ or p38␥ activity at concentrations less than 1-2 M (51), does not inhibit the EGCG response (data not shown). Taken together, these results strongly implicate p38␦ as the major MAPK conveying the signal downstream of MEK3.
EGCG-dependent Regulation of AP1 Factor Levels-Two previous studies suggest that EGCG can increase AP1 factors levels. EGCG treatment increases JunD, c-Fos, and FosB mRNA expression in BALB/3T3 cells (52) and c-Fos and c-Jun expression in HepG2 cells (53). Moreover, previous studies indicate that a Ras, MEKK1, MEK3, p38 targets AP1 factors in keratinocytes (17,34). We therefore examined the effects of EGCG on AP1 factor expression and AP1 factor DNA binding. Different homo-or heterodimeric combinations of AP1 factors can produce differential effects on cell proliferation, differentiation, and apoptosis (54 -61). Our analysis reveals that EGCG treatment increases c-Fos, FosB, Fra-1, and Fra-2 levels. c-Jun, JunB, and JunD levels are also increased. Gel mobility shift experiments indicate that EGCG causes a substantial increase in AP1 DNA binding, a result opposite to that observed in transformed skin cells (44). We show that the complex that forms at the hINV promoter AP1 site includes Fra-1 and JunD. This is similar to previous experiments showing that the differentiating agent, TPA, increases binding at AP1-1 (18). Thus, EGCG and TPA are likely to activate differentiation-associated gene expression via similar mechanisms. This activation clearly requires a specific AP1 site, AP1-1, located within the hINV promoter proximal regulatory region (18). It should be noted that our study is not the first to document the effects of EGCG via a specific transcriptional element. EGCG reduces androgen receptor expression, via suppression of Sp1 binding to the androgen receptor promoter (62), and also regulates NFB transcription factor function (63).
Relationship to Other hINV Regulatory Mechanisms-Other agents also regulate hINV gene expression. For example, phorbol ester, a known keratinocyte differentiating agent, increases hINV gene expression via a MAPK cascade that includes Ras and MEKK1. MEKK1, in turn, activates MEK1, MEK3, and MEK7 (17,34). Ultimately these enzymes regulate p38 activity; MEK7-dependent activation of hINV gene expression, for example, is associated with activation of p38␣ (28). Thus, although the EGCG-dependent regulation of AP1 factor and hINV gene expression uses signaling kinases in common with other agents that regulate hINV gene expression, there are also distinct differences. An important difference, for example, is that EGCG appears to specifically activate p38 via a MEK3dependent mechanism. It is also interesting that the response is conveyed via p38␦ and not the other isoforms.
Green Tea Polyphenol Enhances Keratinocyte Differentiation-Normal keratinocytes are the major surface epithelial cell type exposed to environmental carcinogens. Therefore, it is important to identify how individual chemopreventive agents effect normal keratinocyte function. This is an area that has not been adequately studied. Our present results suggest that EGCG may protect normal keratinocytes from transforming stimuli by promoting keratinocyte differentiation. There are three lines of evidence that support this thesis. First, as noted above, EGCG enhances the expression of involucrin, an established marker of keratinocyte differentiation (15). Second, EGCG markedly increases the conversion of undifferentiated keratinocytes into corneocytes, the terminal products of keratinocyte differentiation (42). One percent of the cells were cornified in control cultures, compared with 15% after EGCG treatment. Third, EGCG inhibits keratinocyte proliferation. Moreover, this inhibition is not caused by toxicity, as although EGCG inhibits proliferation, only the highest concentration tested, 30 g/ml, reduced cell number below the number present at the beginning of treatment. In addition, when confluent cultures are treated with EGCG, cell number is only slightly reduced at EGCG concentrations ranging to 30 g/ml. It is important to note that this EGCG level is similar or less than the concentration used to inhibit cancer cell proliferation (7,44,64,65). Based on these studies, we propose that EGCG regulates hINV gene expression via a pathway that includes Ras, MEKK1, MEK3, p38␦, and AP1 (Fig. 8D). Additional studies, designed to further understand the role of EGCG in regulating the signal transduction cascades that lead to enhanced keratinocyte differentiation and activation of hINV gene expression, are in progress.