Human epidermal keratinocytes undergo (–)-epigallocatechin-3-gallate-dependent differentiation but not apoptosis

Sivaprakasam Balasubramanian 1, Michael T. Sturniolo, George R. Dubyak 1 and Richard L. Eckert 1–5,     *

1 Department of Physiology and Biophysics, 2 Department of Dermatology, 3 Department of Biochemistry, 4 Department of Reproductive Biology and 5 Department of Oncology, Case School of Medicine, Cleveland, OH 44106-4970, USA

* To whom correspondence should be addressed at: Department of Physiology/Biophysics, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, OH 44106-4970, USA. Tel: +1 216 368 5530; Fax: +1 216 368 5586; Email: rle2{at}po.cwru.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epigallocatechin-3-gallate (EGCG) is an important chemopreventive agent derived from green tea. We recently reported that EGCG treatment enhances keratinocyte differentiation as evidenced by increased human involucrin promoter activity [Balasubramanian,S., Efimova,T. and Eckert,R.L. (2002) J. Biol. Chem., 277, 1828–1836]. In the present paper, we extend these findings and show that EGCG also increases the expression of other differentiation markers—procaspase 14 and type I transglutaminase (TG1). Both TG1 mRNA and protein level, and activity are increased by treatment with EGCG. Increased TG1 activity is evidenced by a direct transglutaminase assay, and by the ability of EGCG to stimulate the covalent incorporation of fluorescein cadaverine substrate into crosslinked intracellular structures. In contrast, type II transglutaminase levels are not altered by EGCG treatment. We also assessed whether EGCG promotes keratinocyte apoptosis. We show that EGCG treatment does not promote the cleavage of procaspase-3, -8, -9 or poly(ADP-ribose) polymerase. Moreover, treatment with the pan-caspase inhibitor, Z-VAD-FMK, does not reverse the EGCG-associated reduction in cell viability. In addition, there is no increase in cells having sub-G1/S DNA content, and no evidence for the release of cytochrome c from the mitochondria. These findings confirm, using several endpoints, that EGCG treatment enhances normal keratinocyte differentiation but does not promote apoptosis.

Abbreviations: COX 4, cytochrome c oxidase; EGCG, epigallocatechin-3-gallate; FC, fluorescein cadaverine; hINV, human involucrin; KSFM, keratinocyte serum-free medium; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered-saline; TG1, type I transglutaminase; TG2, type II transglutaminase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human epidermis is a multilayered stratified squamous epithelium. During keratinocyte (skin cell) differentiation, the skin cells cease proliferating and undergo a programmed set of morphological and biochemical changes that result in the formation of the terminally differentiated dead cell or corneocyte (1). Corneocyte formation represents a specific form of cell death that differs from classical apoptosis, since it does not involve the fragmentation of the cell into apoptotic bodies and subsequent phagocytosis (2,3). Differentiation requires the expression of a specific form of transglutaminase, type I transglutaminase (TG1), which crosslinks protein through the formation of interprotein, covalent isopeptide bonds (47). This enzyme is largely responsible for the assembly of the keratinocyte cornified envelope. Involucrin, loricrin, SPR proteins, and a host of other proteins, serve as transglutaminase substrates (812).

Although normal human keratinocytes are a primary target of environmental carcinogens, the effect of chemopreventive agents on the function of normal keratinocytes has not been extensively studied. Green tea polyphenols are important candidate chemopreventive agents in the treatment of a variety of skin diseases, including psoriasis and cancer (13,14). (–)-Epigallocatechin-3-gallate (EGCG) is the major bioactive polyphenol present in green tea. EGCG is an antioxidant, similar to many other chemopreventive agents. We recently reported that EGCG treatment of normal human keratinocytes results in an increased involucrin promoter activity (15,16). Based on this study, we proposed that green tea polyphenols act as pro-differentiation agents to protect the keratinocytes from transforming stimuli. The goal of the present study is to determine whether other differentiation markers are also increased in response to EGCG treatment and whether this agent triggers cell apoptosis as well. We report that EGCG increases procaspase-14, and TG1 mRNA and protein levels in normal human epidermal keratinocytes. The EGCG-dependent increase in TG1 level is associated with a corresponding increase in transglutaminase activity. Parallel studies show that EGCG treatment does not increase the cleavage of procaspase-3, -8, -9 or poly(ADP-ribose) polymerase (PARP), alter the ratio of Bcl2/Bax, or promote cytochrome c release. On balance, these studies suggest that EGCG-treated keratinocytes undergo differentiation and not apoptosis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents
EGCG and dimethyl sulfoxide were obtained from Sigma. EGCG was dissolved as 1000-fold stocks in sterile H2O and stored at –70°C. Keratinocyte serum-free medium (KSFM), trypsin, Hank's balanced salt solution and gentamicin were purchased from Life Technologies. Fluorescein cadaverine (FC) was from Molecular Probes (A-10466). Trypan blue solution was obtained from Sigma (T8154). Z-VAD-FMK, a pan-caspase inhibitor, was purchased from BD PharMingen and prepared as a stock in dimethyl sulfoxide. Fura2-AM was purchased from Molecular Probes.

Detection of transglutaminase activity using FC
Third passage primary human foreskin keratinocytes were grown as described previously (15,16). The cells were grown on coverslips and then treated with or without EGCG (20 µg/ml) for 2 days. During the final 4 h of treatment, fresh medium supplemented with 100 µM FC, a cell permeable fluorescent transglutaminase substrate, was added to the culture (17). After incubation, the cells were washed with phosphate-buffered saline (PBS) to remove non-crosslinked FC, fixed in methanol at –20°C, washed two times with cold methanol and then with PBS. The samples were placed onto slides using N-propyl galate for visualization by fluorescent microscopy, using a Nikon Optiphot fluorescence microscope.

Cell fractionation and transglutaminase assay
Keratinocytes (1 x 100 cm2 dish) were washed with cold PBS, collected by scraping, and sonicated in 1 ml of ice-cold lysis buffer containing 10 mM HEPES, pH 7.6, 1 mM EDTA, 1 mM DTT and 10 µg/ml phenylmethylsulfonyl fluoride (PMSF). The cell extract was centrifuged at 100 000 g for 30 min to obtain the cytosolic fraction. To isolate TG1, the pellet was sonicated in the lysis buffer containing 0.2% Triton X-100 followed by centrifugation at 100 000 g for 30 min at 4°C. An equal amount of protein from each sample was added to the reaction mixture in a total volume of 125 µl of homogenization buffer containing 100 mM Tris–HCl, pH 7.6, 4 mM CaCl2, 5 mM DTT, 10 µg/ml PMSF, 0.5 mg casein and 0.5 µCi [3H] putrescine. The assay mixture was incubated at 37°C for 1 h, precipitated with trichloroacetic acid, washed with ethanol and air dried. Liquid scintillation counting was used to detect the [3H] putrescine incorporation into the pellet.

Preparation of cell lysates and immunoblot analysis
Subconfluent cultured keratinocytes were treated with or without EGCG for 24 or 48 h before the preparation of total cell lysates (15). Equal quantities of protein were electrophoresed on denaturing and reducing polyacrylamide gels and transferred onto nitrocellulose for immunoblot analysis. The membranes were blocked and then incubated with the corresponding primary antibodies followed by appropriate HRP-conjugated secondary antibodies. Secondary antibody binding was visualized using a chemiluminescent detection system (Amersham Pharmacia Biotech).

Antibodies
Mouse monoclonal antibody specific for TG1 was obtained from Biomedical Technologies, Inc. (BT-621, 1:100). Mouse transglutaminase type 2 (TG2) transglutaminase antibody was obtained from NeoMarkers (CUB7402, 1:1000). Rabbit anti-caspase-14 diluted to 1:5000 was kindly provided by Dr Erwin Tschachler, Medical University of Vienna (18). Rabbit anti-caspase 9 (sc-8355, 1:2000 dilution), rabbit anti-Bax (sc-493, 1:8000), mouse monoclonal anti-Bcl-2 (sc-7382, 1:5000), goat polyclonal anti-caspase 8 (sc-6136, 1:3000) and peroxidase-conjugated donkey anti-goat IgG (Sc-2033, 1:5000) were obtained from Santa Cruz Biotechnology. Rabbit polyclonal anti-caspase 3 (AHZ0052, 1:4000) was from Biosource International. Mouse monoclonal antibody anti-PARP (55494, 1:2000) was from BD Pharmingen. Mouse anti-human ß-actin (A5441, 1:10000) was from Sigma. Horseradish peroxidase-conjugated donkey anti-rabbit IgG (NA934, 1:5000) and horseradish peroxidase-conjugated sheep anti-mouse IgG (NA931, 1:5000) secondary antibodies were obtained from Amersham Biosciences.

Keratinocytes treatment with UVB
Near-confluent cells were exposed to 40 mJ/cm2 with Kodacel-filtered UVB light. The cells were harvested at various times after treatment.

RT–PCR analysis
Near-confluent keratinocytes were treated in the presence or absence of EGCG (20 µg/ml) for 48 h followed by RNA isolation using RNeasy Mini Kit (Catalog no #74104 from Qiagen). The Titan one tube RT–PCR system (catalog no # 1 855 476, Roche) was utilized for the RT–PCR analysis according to the manufacturer's protocol. The primers used were as follows: Caspase-14 (5'-ATATGATATGTCAGGTGCCCG-3' and 5'-CTTTGGTGACACACAGTATTAG-3'), human involucrin (hINV) (5'-CTCCACCAAAGCCTCTGC-3' and 5'-CTGCTTAAGCTGCTGCTC-3'), TG1 (5'-TGAATAGTGACAAGGTGTACTGGCA-3' and 5'-GTGGCCTGAGACATTGAGCAGCAT-3'), TG2 (5'-TCACCCACACCTACAAATACCCAG-3' and 5'-TGATTTCTGGATTCTCCAGGTAGAG-3'), ß-actin (5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'). The denaturation, annealing and elongation steps of hINV, ß-actin, TG1 and TG-2 were conducted at 94°C for 10 s, 55°C for 30 s, and 68°C for 90 s for 25 cycles. The procaspase-14 specific PCR involved 35 cycles of DNA denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 75 s. PCR products were detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide.

Cell proliferation
Keratinocytes were seeded at 10 000 cells/cm2 in 35 mm dishes in KSFM and allowed to grow for 2 days. The cells were then treated by an addition of fresh KSFM in the absence or presence of EGCG and/or 40 µM Z-VAD-FMK. Additional cells were treated with 40 mJ/cm2 UVB in the absence or presence of 40 µM Z-VAD-FMK. After 48 h, the cells were harvested and viable cell number was determined by the trypan blue exclusion assay (17).

Flow cytometry
For flow cytometric analysis, subconfluent keratinocytes, grown on 50 cm2 dishes, were treated with increasing concentrations of EGCG for 24–48 h. The cells were trypsinized, washed with PBS fixed in methanol and processed for propidium iodide staining. The cells were then analyzed by flow cytometry.

Determination of cytochrome c release from mitochondria
Keratinocytes were treated with or without EGCG (20 µg/ml) for 24 h, followed by a mitochondrial and cytosolic fraction preparation using the ApoAlert cell fractionation kit (BD Biosciences/Clontech). We monitored the levels of cytochrome c oxidase subunit 4 (COX4, a mitochondrial marker) and ß-actin, a cytosolic marker, which are indices of the purity of the subcellular fractions.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
EGCG regulation of cell morphology
We began by assessing the effects of EGCG treatment on keratinocyte morphology. As shown in Figure 1, untreated keratinocytes proliferate in a relatively loose non-structured pattern. In contrast, cells treated for 48 h with 20 µg/ml EGCG produce adherent colonies, which fuse to form web-like arrays. This change is apparent as early as 3 h after the initiation of EGCG treatment (not shown) and occurs before any EGCG-dependent reduction in cell number.



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Fig. 1. EGCG alters keratinocyte colony morphology. Normal human keratinocytes were treated for 48 h in the presence or absence of 20 µg/ml EGCG. The cells were then photographed using a Nikon inverted brightfield microscope. At this point, the cell number in control versus EGCG-treated cultures is relatively similar; however, the EGCG treated cells form more compact colonies. A similar morphologic response is observed as early as 3 h after EGCG addition of EGCG.

 
Regulation of differentiation marker gene expression
Our previous study showed that EGCG treatment of normal human keratinocyte cultures results in a markedly increased involucrin promoter activity (15,16). To determine whether EGCG produces a generalized increase in differentiation, we examined the effect of EGCG treatment on the expression of several markers of keratinocyte differentiation. Keratinocytes were treated for 24 or 48 h with 20 µg/ml EGCG and the extracts were assayed for the expression of TG1, TG2, procaspase-14 and involucrin. We first monitored involucrin levels as a standard. As shown in Figure 2A, involucrin levels are markedly increased by treatment with EGCG—a finding consistent with our previous reports (15,16). In addition, EGCG treatment increased the level of procaspase-14, a procaspase that is known to be expressed in differentiating keratinocytes (1822). Figure 2B shows, as measured by RT–PCR, that the mRNA levels encoding each of these proteins, increases in parallel with the increase in protein level. The level of ß-actin mRNA, in contrast, is not altered.



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Fig. 2. EGCG increases keratinocyte differentiation marker expression. Normal human keratinocytes were treated in the absence or presence of 20 µg/ml EGCG for 24 or 48 h. (A) Total extracts were prepared and equivalent amounts of protein were electrophoresed on a 10% denaturing polyacrylamide gel and transferred onto nitrocellulose membrane. Procaspase-14 and involucrin were detected using rabbit polyclonal antibodies (68) and the ß-actin was detected using a commercially available anti-ß-actin mouse monoclonal antibody. Primary antibody binding was detected by incubation with a corresponding HRP-conjugated secondary antibody and addition of chemiluminescence detection reagents. Normal human epidermal extract was used as a positive control for marker protein expression (EPI). (B) Cells were treated as above and mRNA was prepared for RT–PCR detection of involucrin, procaspase-14 and ß-actin mRNA. A similar increase in hINV and procaspase-14 mRNA was observed at 24 h (data not shown).

 
We then measured the effects of EGCG on transglutaminase level and activity. Transglutaminases are key enzymes required for keratinocyte cell death and terminal differentiation. TG1 is the primary enzyme responsible for catalysis of the crosslinks that assemble the cornified envelope [4,7,23], but several other transglutaminases are known to be expressed in keratinocytes. Moreover, different transglutaminase isoforms are known to be selectively expressed in response to regulatory agents (2426). A previous study indicated that total transglutaminase activity is increased in EGCG-treated keratinocytes; however, the specific transglutaminase involved was not identified (27). To identify the EGCG responsive isoform, cells were treated with EGCG for 48 h and the total cell extracts were fractionated into soluble and particulate fractions. Figure 3A shows that the total transglutaminase activity is increased by 6-fold in EGCG-treated keratinocytes. The increase is observed in both the soluble and particulate phases; however, a majority of the activity is associated with the particulate phase. Based on past experience, we believe that the increased soluble phase activity is due to the contaminating particulate fraction. The presence of activity in the particulate phase is consistent with the activity being membrane-anchored TG1 [6,28,29]. To provide in situ visual evidence for EGCG-dependent activation of transglutaminase in cells, keratinocytes were incubated with or without EGCG for 48 h and during the final 4 h a cell permeable transglutaminase substrate, FC, was added to the cultures. As shown in Figure 3B, control cultures showed a minimal FC incorporation into cell structures, but the level was substantially increased in EGCG-treated cells. Figure 3C and D shows that the EGCG-dependent increase in transglutaminase activity is associated with a parallel increase in TG1 mRNA and protein level. Keratinocytes were treated for 48 h with EGCG, and TG1 protein level was then monitored by immunoblot and mRNA level by RT–PCR. The 6-fold increase in TG1 protein and mRNA level mirrors the increase in transglutaminase activity shown in Figure 3A. In contrast, EGCG treatment does not increase the level of mRNA or protein encoding TG2, indicating that TG2 is unlikely to account for any of the increase in transglutaminase activity. As expected, ß-actin levels were not regulated by treatment with EGCG.



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Fig. 3. EGCG increases transglutaminase activity. (A) Near-confluent normal human keratinocytes were treated for 48 h with EGCG (20 µg/ml). The cells were harvested, fractionated into total, soluble and particulate fractions, and each fraction was assayed for transglutaminase activity. (B) Human keratinocytes were grown on cover slips and treated with EGCG as above for 48 h. The medium was then supplemented with 100 µM FC during the last 4 h of treatment. The cells were then washed with PBS and fixed in methanol at –20°C to remove free FC, washed twice with 100% methanol, and then with PBS before placing onto slides using N-propyl galate for visualization by fluorescent microscopy, using a Nikon Optiphot fluorescence microscope. (C) Total extracts were prepared from keratinocytes treated as above, and equivalent amounts of protein were electrophoresed on a 10% denaturing polyacrylamide gel and transferred onto a nitrocellulose membrane. TG1 and TG2, and ß-actin protein levels were detected using specific antibodies. Primary antibody binding was detected by incubation with an appropriate HRP-conjugated secondary antibody and addition of chemiluminescence detection reagents. (D) Cells were treated with EGCG for 48 h followed by the preparation of mRNA and detection of TG1, TG2 and ß-actin mRNA levels by RT–PCR. Similar changes were observed as early as 24 h after the initiation of EGCG treatment (data not shown).

 
EGCG does not promote the accumulation of cells in sub-G1/S
The above findings suggest that EGCG increases the differentiation of keratinocytes. Differentiation is generally associated with an obligatory loss of proliferative capacity. Thus, we monitored the effects of EGCG treatment on cell number. Figure 4A shows that the cell number increases 2-fold over 48 h in untreated cells, and that this increase is completely inhibited by treatment with 20 µg/ml EGCG.



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Fig. 4. EGCG treatment does not promote the accumulation of sub-G1/S phase cells. (A) Near-confluent cultures of keratinocytes were treated for with or without 20 µg/ml EGCG for 48 h and the cell number was assessed. The cell count is expressed as percent cell number as compared with the cell number present at the time (0 h) of initiation of treatment. (B) Cells were grown as described above, and DNA content was assayed by flow cytometry of propidium iodide stained cells. The percentage of sub-G1 cells is indicated. This experiment was repeated four times with a similar result wherein EGCG does not profoundly alter the distribution of cells within the cell cycle.

 
We then performed studies to assess whether EGCG specifically regulates differentiation or if it has effects on apoptosis as well. Apoptosis is frequently associated with an accumulation of cells of sub-G1/S DNA content. We therefore examined the effects of a 48 h EGCG treatment on the distribution of cells in the cell cycle following treatment with EGCG. As shown in Figure 4B, there is no change in the cell cycle distribution when comparing untreated and EGCG-treated cells. Moreover, the fraction of cells having sub-G1/S DNA complement is ~1% in control cells and this percentage is not altered by treatment with EGCG. In addition, we have not observed any substantial change in the distribution of cells in the other cell cycle phases, suggesting that the reduced cell number observed may be due to a generalized slowing of cell cycle progression.

Z-VAD-FMK does not reverse EGCG-dependent reduction in keratinocyte cell number
We then determined whether the apoptosis inhibitor, Z-VAD-FMK, could reverse the EGCG-dependent responses. Keratinocytes were treated with 20 µg/ml of EGCG for 48 h in the presence or absence of Z-VAD-FMK, a pan-caspase inhibitor. As shown in Figure 5A, the survival of untreated cells is not affected by treatment with Z-VAD-FMK. Moreover, treatment with Z-VAD-FMK does not reverse the EGCG-dependent reduction in viability cell number. In contrast, UVB-dependent cell death, which is known to be caspase dependent (30), is partially reversed by treatment with Z-VAD-FMK. To assess this further, we determined whether treatment with EGCG alters the procaspase level. Figure 5B shows that treatment with EGCG does not reduce the procaspase-3, -8 or -9 level. Moreover, the level of PARP, a downstream target of caspase-3, is not reduced. Although not shown in these gels, there was no evidence for the presence of procaspase or PARP cleavage products. We also monitored the ratio of Bax and Bcl2. The Bax/Bcl2 ratio has been reported to increase in cells undergoing apoptosis (31). As shown in Figure 5C, EGCG does not alter the level of these apoptosis/survival regulatory proteins.



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Fig. 5. EGCG does not promote caspase or PARP cleavage or alter Bcl2/Bax expression ratio. (A) Keratinocytes were seeded at 10 000 cells/cm2 in 9.5 cm2 dishes in KSFM medium. After attachment, the keratinocytes were treated with 20 µg/ml EGCG for 48 h in the presence or absence of Z-VAD-FMK (40 µM). Parallel cultures were exposed to 40 mJ/cm2 UVB with or without Z-VAD-FMK co-treatment. The cells were harvested with Hank's balanced salt solution containing 0.025% trypsin and 1 mM EDTA, and cell viability was assessed using trypan blue exclusion. (B) and (C) Human epidermal keratinocytes were treated for 24 or 48 h with EGCG (20 µg/ml). Cells lysates were then prepared and equivalent amounts of protein were electrophoresed for immunoblot detection of the indicated proteins. ß-actin antibody was used to confirm whether equal amounts of protein were loaded in each lane. The results are representative of three independent experiments.

 
EGCG treatment does not promote cytochrome c release from mitochondria
As an additional measure of apoptotic status, we monitored the effects of EGCG treatment on mitochondrial cytochrome c release. Keratinocytes were treated with EGCG for 48 h before the preparation of cytosolic and mitochondrial extracts. As shown in Figure 6, comparable levels of cytochrome c were observed in both the mitochondrial and cytosolic fractions prepared from the control or EGCG-treated cells. In contrast, UVB treatment (40 mJ/cm2), which is known to increase keratinocyte apoptosis, results in the release of mitochondrial cytochrome c to the cytoplasm. Detection of COX4, a mitochondrial marker that is not released by apoptosis, and ß-actin, a cytoplasmic marker, reveals that the subcellular fractions were successfully prepared.



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Fig. 6. EGCG does not promote mitochondrial release of cytochrome c. Keratinocytes were treated with or without 20 µg/ml EGCG for 24 h. The cells were then harvested and cytosolic and mitochondrial fractions were prepared and cytochrome c level was monitored by immunoblot. Successful separation of the mitochondrial and cytosolic fractions was confirmed by detection of COX4 (a mitochondrial marker) and ß-actin (a cytosolic marker). Similar changes were observed after 48 h of EGCG treatment (data not shown).

 
EGCG does not cause increases in intracellular calcium
Addition of extracellular calcium to cultured murine keratinocytes triggers an increase in intracellular calcium levels and enhanced cell differentiation (32,33). This increase in calcium is observed within seconds after the addition of calcium and elevated calcium levels persist for hours (34,35). In addition, calcium is a necessary cofactor for transglutaminase activation (3638). Thus, it is possible that EGCG enhances keratinocyte differentiation by increasing the intracellular calcium level. To test this, we monitored the ability of EGCG to alter intracellular calcium, using Fura2. Cells were equilibrated with Fura2-AM before treatment with the indicated agents. Cells were treated with 3 mM ATP as a positive control. As shown in Figure 7A, treatment with ATP results in a transient increase in intracellular free calcium that dissipates within several minutes. Addition of 20 µg/ml EGCG, in contrast, causes a small but sustained increase in fluorescence. Cell permeabilization, by treatment with 20 µg/ml digitonin, reveals the full detectable intracellular calcium pool.



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Fig. 7. EGCG treatment does not increase the intracellular calcium level. (A) Ligand-dependent mobilization of intracellular calcium in keratinocytes. Suspensions of trypsin-detached primary human keratinocytes were prepared for the measurement of intracellular calcium by incubation in calcium and magnesium-supplemented basal salt solution (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 20 mM Na-HEPES, pH 7.5, 5 mM glucose and 0.1% bovine serum albumin) containing 1 µM Fura2 acetoxymethyl ester (Fura2-AM) at 25°C for 45 min. The cells were then washed with basal salt solution, resuspended at 1 x 106 cells/ml in basal salt solution, and permitted to recover for 10 min at 25°C before transfer to a stirred cuvette for measurement of Fura2 fluorescence (339 nm excitation, 500 nm emission) (69). The final treatment concentrations were 3 mM ATP, 20 µg/ml EGCG and 20 µg/ml digitonin. Similar changes were observed irrespective of whether EGCG or ATP was added first. The results are representative of three separate experiments. (B) EGCG autofluorescence. Basal salt solution was added to a stirred cuvette and permitted to equilibrate at 25°C for 10 min. EGCG was then added at a final concentration of 20 µg/ml and fluorescence level was monitored.

 
Some ligands can autofluoresce. To determine whether the small EGCG-dependent increase in fluorescence observed in Figure 7A is due to EGCG autofluorescence, we added EGCG, at a final concentration of 20 µg/ml, to a cell-free assay medium and monitored the fluorescence output. As shown in Figure 7B, EGCG addition to assay medium alone results in an increase in signal identical to that observed in the presence of cells (Figure 7A), indicating that the increase is due to EGCG autofluorescence and not to an increase in intracellular calcium level.


    Discussion
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 Abstract
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 Materials and methods
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 Discussion
 References
 
EGCG increases keratinocyte differentiation
In a previous study, we showed that involucrin gene expression is increased by treatment with EGCG (15,16). Involucrin is a precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of epidermis and other surface epithelia (8,39). This study showed that EGCG increases hINV promoter activity using a mechanism that has been described for other differentiating agents—through activation of a PKC, Ras, MEKK1, MEK3 pathway that targets p38{delta}/ERK1/2 to increase p38{delta} activity and decrease ERK1/2 activity (15). Activation of this pathway results in increased C/EBP factor (16) and AP1 factor (15) binding to the hINV promoter and increased transcriptional activity. These previous findings clearly suggest that EGCG treatment increases keratinocyte differentiation; however, it was not clear whether EGCG would regulate other differentiation-associated responses and whether it could also promote apoptosis. A major goal of the present study was to extend these observations to determine whether other markers of keratinocyte differentiation are also increased by treatment with EGCG. Procaspase-14 is a caspase that, unlike the killer caspases, is increased during keratinocyte differentiation [18,20,21,40]. It appears to have a role in keratinocyte maturation, as its level and activity are increased in epidermis in vivo. Our present experiments show that procaspase-14 level, both protein and mRNA, are increased by EGCG treatment. However, we did not detect any procaspase-14 cleaved forms that would suggest procaspase-14 activation. These findings confirm a recent report by Hsu et al. (41), which demonstrates that EGCG treatment increases procaspase-14 gene expression.

In addition, we monitored transglutaminase activity. A previous study showed that EGCG treatment of primary keratinocytes increases total transglutaminase activity, as determined by an activity assay using total cell extracts (42). However, this study did not identify the responsive transglutaminase isoform. The major transglutaminase form responsible for cornified envelope assembly, TG1, is expressed in a differentiation-dependent manner (43). However, keratinocytes are known to express several types of transglutaminase, each encoded by a distinct gene (29,4446). Moreover, different isoforms are induced by different agents. For example, TG2, which is not associated with differentiation, has been reported to be induced in retinoic acid-treated murine keratinocytes (25). Our present studies show that EGCG treatment selectively increases the level of mRNA and protein encoding TG1 and that TG2 levels are not altered by EGCG treatment. In addition, we show that the increased transglutaminase activity is preferentially associated with the membrane (particulate) phase. Association with the membrane is a property of TG1, which is anchored to the membrane through a myristal linkage (6,29). Thus, the present studies demonstrate that the increase in transglutaminase activity is due to increased levels and activity of TG1.

Does EGCG regulate differentiation through effects on intracellular calcium?
The increase in transglutaminase is particularly interesting, as activity is increased by EGCG when the cells are maintained in low calcium (0.09 mM)-containing medium. Previous studies show that cells maintained in a medium containing 0.09 mM calcium display minimal, if any, transglutaminase activity and that the activity increases only when calcium levels exceed ≥0.3 mM (37,47). Addition of extracellular calcium to cultured murine keratinocytes triggers an increase in intracellular calcium (35). This increase in intracellular calcium is observed within seconds after the extracellular addition of calcium and the increased calcium level persists for hours (35). We therefore tested the possibility that EGCG may increase intracellular free calcium, and thereby regulate differentiation. It is interesting that we failed to detect an increase in intracellular calcium level in response to EGCG treatment, suggesting that a change in free calcium level is not necessary for the EGCG-dependent enhanced differentiation. Alternatively, it may be that a minimal increase in calcium is responsible for the differentiation response, or that EGCG acts through a calcium-independent mechanism. It is already known that EGCG can activate involucrin gene transcription through a novel PKC, Ras, MEKK1m, MEK3 pathway that regulates p38{delta} and ERK1/2 MAPK activity [16,48,49]. Activation of this pathway, in turn, increases the level and activity of transcription factors that regulate differentiation-associated target genes (5053). Thus, it is possible that EGCG acts directly on this signal transduction regulatory pathway without altering the intracellular calcium level.

EGCG treatment does not increase keratinocyte apoptosis
In addition to differentiation, keratinocytes can also die by apoptosis (2,3). Apoptosis is characterized by cell shrinkage, condensation of chromatin and the cytoplasm, internucleosomal DNA cleavage, membrane blebbing and cell fragmentation into apoptotic bodies. Apoptotic cell death is associated with either FADD adaptor protein-mediated or mitochondrial lysis-associated caspase activation (54). EGCG has been reported to promote apoptosis in other cell systems (5558). Mitochondria-associated apoptosis is activated by a range of stimuli, including cytokine deprivation, DNA damage, anoxia and cytotoxic drugs. This occurs through the activation of initiator caspases and their adaptors. Caspase-9 and its activators, Apaf-1, and cytochrome c are part of this pathway (54). Activation of procaspases-9, -8 and -3 leads to the cleavage of PARP (54). Ultimately these events lead to the destruction of cell organelles, nuclear condensation and DNA fragmentation. Apoptosis is activated by ultraviolet radiation, production of hydrogen peroxide and other stimuli in keratinocytes (5964).

A goal of the present studies was to determine whether EGCG, in addition to promoting differentiation, also promotes apoptosis. To test this hypothesis, we monitored the ability of EGCG to activate apoptotic markers. A recent study suggested that EGCG does not promote keratinocyte apoptosis. This study showed that EGCG treatment did not activate procaspase-3 as measured by the activity assay (42); however, these investigators did not measure the impact on other procaspases. Our present studies extend these findings and show that EGCG does not promote the cleavage of procaspase-9, -8, or -3, or change the level of Bcl2 or Bax. In addition, Z-VAD-FMK, a pan-caspase inhibitor, does not inhibit the EGCG-dependent reduction in keratinocyte viability. Finally, EGCG treatment did not increase the number of sub-G1 cell fragments and did not promote the release of cytochrome c from the mitochondria.

Biological role of EGCG
EGCG has been proposed as a potential chemopreventive agent in skin cancer (65,66). In most cases, this has been attributed to its potent antioxidant activity. Our studies further confirm a second possible mode of chemoprevention—removal of compromised cells through enhanced keratinocyte differentiation. Our present studies suggest that in cultured keratinocytes, EGCG acts to enhance keratinocyte differentiation without triggering apoptosis. A recent report suggests that topical application of EGCG on human skin results in increased cell proliferation and reduced keratinocyte apoptosis (67). These authors further suggest that apoptosis is inhibited due to an increased Bcl-2/Bax ratio. Unfortunately, this paper did not examine the effects of EGCG treatment on differentiation. It is possible that in vivo, EGCG has both anti-apoptotic and pro-differentiation effects. The response may also depend upon the concentration of EGCG applied. Additional studies will be required to assess these possibilities.


    Acknowledgments
 
This work utilized the facilities of the Skin Diseases Research Center of Northeast Ohio (NIH, AR39750) and was supported by a grant from the National Institutes of Health (R.L.E.). S.B. is a recipient of O-CHA(tea) pioneer academic research grant award from World Green Tea Association.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 12, 2004; revised February 1, 2005; accepted February 6, 2005.





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