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J. Biol. Chem., Vol. 276, Issue 2, 999-1004, January 12, 2001

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Apoptosis Signal-regulating Kinase 1 (ASK1) Is an Intracellular Inducer of Keratinocyte Differentiation^*

Koji Sayama^§, Yasushi Hanakawa, Yuji Shirakata, Kenshi Yamasaki, Yasuhiro Sawada^¶, Lin Sun, Kiyofumi Yamanishi^**, Hidenori Ichijo^¶, and Koji Hashimoto

From the  Department of Dermatology, Ehime University School of Medicine, Ehime 791-0295, Japan, ^¶ Laboratory of Cell Signaling, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8519, Japan, the  Department of Dermatology, The Second Affiliated Hospital of Dalian Medical University, Dalian, P.R. China, and the ^** Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan

Received for publication, April 21, 2000, and in revised form, October 9, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells differentiate in response to various extracellular stimuli. This cellular response requires intracellular signaling pathways. The mitogen-activated protein (MAP) kinase cascade is a core signal transduction pathway that determines the fate of many kinds of cell. MAP kinase kinase kinase activates MAP kinase kinase, which in turn activates MAP kinase. Apoptosis signal-regulating kinase (ASK1) was identified as a MAP kinase kinase kinase involved in the stress-induced apoptosis-signaling cascade that activates the SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades. Expression of the constitutively active form of ASK1 (ASK1-N) in keratinocytes induced significant morphological changes and differentiation markers, transglutaminase-1, loricrin, and involucrin. A transient increase in p21^Cip1/WAF1 reduced DNA synthesis, and cell cycle analysis verified the differentiation. p38 MAP kinase inhibitors, SB202190 and SB203580, abolished the induction of differentiation markers, transglutaminase-1, loricrin, and involucrin. In turn, the induction of differentiation with ceramide in keratinocytes caused an increase in ASK1 expression and activity. Furthermore, normal human skin expresses ASK1 protein in the upper epidermis, implicating ASK1 in in vivo keratinocyte differentiation. We propose that the ASK1-p38 MAP kinase cascade is a new intracellular regulator of keratinocyte differentiation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mitogen-activated protein (MAP)¹ kinase cascade has been characterized as a core signal transduction pathway that is conserved from yeast to humans. It determines cell fate and regulates fundamental cellular functions in response to a wide range of extracellular stimuli (1-4). This cascade plays an essential role in diverse intracellular signaling processes including cell growth, cell cycle regulation, differentiation, and apoptosis. The MAP kinase cascade consists of three distinct protein kinase families, including MAP kinase, MAP kinase kinase (MAPKK), and MAP kinase kinase kinase (MAPKKK). MAPKKK activates MAPKK, which in turn activates MAP kinase. The MAP kinase superfamily consists of the classical MAP kinase (extracellular signal-regulating kinase, ERK) family, the c-Jun N-terminal kinase (JNK, also known as stress-activated protein kinase; SAPK) family, and the p38 MAP kinase family. Each MAP kinase family is activated by different stimuli via distinct upstream kinases and controls many aspects of cellular physiology.

ASK1 was identified as a MAPKKK involved in the stress-induced apoptosis-signaling cascade that activates the SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades (5). In addition to stresses, Daxx, a Fas adapter protein, and TNF receptor-associated factor 2 activate ASK1 (6, 7), suggesting a possible role of ASK1 in the Fas- and TNF receptor-mediated signaling cascades. Moreover, the ASK1/JNK cascade phosphorylates and inactivates antiapoptotic Bcl-2 (8).

The epidermis is a self-renewing tissue maintained by the precise regulation of keratinocyte proliferation, differentiation, and cell death. Differentiation is one of the most important ways by which keratinocytes form a multilayered epidermis, and the MAP kinase cascade may be integrated into this process. The ASK1 p38 MAP kinase cascade is a candidate pathway as the regulator. To prove this, the constitutively active form ASK1 (N terminus-deleted mutant, ASK1-N) was introduced into cultured normal human keratinocytes using adenovirus vector (Ad).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Keratinocyte Culture-- Normal human keratinocytes were cultured with MCDB153 medium supplemented with insulin (5 µg/ml), hydrocortisone (5 × 10⁷ M), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), bovine hypothalamic extract (100 µg/ml), and Ca²⁺ (0.1 mM) as described previously (9).

Western Blotting-- The analysis was performed as described previously (9) using a Vistra ECF kit (Amersham Pharmacia Biotech). Rabbit anti-human involucrin (Biomedical Technologies Inc., Stoughton, MA), monoclonal anti-p21^Cip1/WAF1 (6B6, PharMingen Co., San Diego, CA), and rabbit anti-ASK1 (DAV, Ref. 10) were used as the first antibodies at a dilution of 1:1000. The intensity of each band was quantified with ImageQuant (Molecular Dynamics Inc.), referring to the control signal as one unit.

Northern Blotting-- Total RNA was prepared using Isogen (Nippon Gene Co., Tokyo, Japan). Ten µg of total RNA were separated in a 1.2% formaldehyde/agarose gel, transferred to a nylon membrane, and probed with ³²P-labeled cDNA corresponding to transglutaminase-1 (11) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard.

Reverse Transcriptase-PCR Analysis-- The epidermis was separated from normal human skin by incubation in phosphate-buffered saline at 60 °C for 1 min, immediately followed by immersion in ice-cold phosphate-buffered saline. Total RNA was prepared with Isogen and treated with 50 units/ml DNase 1 (CLONTECH Laboratories, Inc., Palo Alto, CA) at 37 °C for 30 min. Specific primers for ASK1 was produced by selecting specific nucleotide sequences from previously published sequences (5). The reverse transcriptase-PCR was performed using RT-PCR High Plus (Toyobo Co., Ltd., Osaka, Japan). The sequences of the primer pair, product size, annealing temperature, and number of cycles were as follows: 5'-TGACAGAGTCGTTTTAGGAA-3' and 3'-ACAAGCAAGTCGTTAGCACA-5', 759 base pairs, 57 °C, and 25 cycles. The PCR products were sequenced to confirm the mRNA expression.

RNase Protection Assay-- Analysis was performed using the multi-probe RNase protection assay system (PharMingen Co.) according to the manufacturer's instructions. Oligonucleotide probes were prepared by inserting the PCR-amplified human cDNA corresponding to oligonucleotides 2659-2874 of ASK1 (Genbank^TM/EBI accession number D84476), 866-1133 of transglutaminase-1 (Genbank^TM/EBI accession number D90287), 966-1176 of loricrin (Genbank^TM/EBI accession number M61120), and 74-249 of involucrin (Genbank^TM/EBI accession number M13903) into the EcoRI and HindIII sites of pPMG vector. 5 µg of total RNA were hybridized with ³²P-labeled riboprobe and digested with RNase. The hybridization products were separated on a 5% polyacrylamide/8 M urea gel and exposed to film. GAPDH is shown as an internal standard. The intensity of each band was quantified using NIH Image, referring to the signal of the control as one unit.

Luciferase Assay-- A reporter plasmid containing the involucrin promoter and firefly luciferase was constructed (pINV-Luc) as follows. The involucrin promoter cassette was a generous gift from Dr. Taichman (12). The coding region of firefly luciferase was digested from pGL3 basic (Promega Co., Madison, WI) and subcloned into the involucrin promoter cassette. The correct insertion and orientation were confirmed by sequencing. To normalize the transfection efficiency, a plasmid containing Renilla luciferase driven by herpes simplex virus thymidine kinase promoter (pRL-TK Promega Co.) was included in the assay. The reporter plasmids were introduced into the keratinocytes using FuGENE6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. In each transfection, 1 µg of pINV-Luc and 0.5 µg of pRL-TK were introduced into 2 × 10⁵ keratinocytes in 6-well plates. After 24 h, the cells were infected with the indicated Ad at an MOI of 5 and were incubated for an additional 24 h. Then the cells were harvested with 250 µl of lysis buffer (Promega Co.), and luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega Co.) with a luminometer (Luminescencer JNR AB-2100; Atto Co., Osaka, Japan). Transfection was performed in triplicate. The relative luciferase activity was calculated by normalizing to the Renilla luciferase activity. Statistical analysis was performed using Student's t test.

MAP Kinase Activity-- MAP kinase activity was measured with JNK and p38 kinase assay kits (BioLabs Inc., Beverly, MA). The lysate of 1 × 10⁶ keratinocytes was immunoprecipitated with a 1:100 dilution of rabbit antibody to p38 MAP kinase and protein G-Sepharose (Amersham Pharmacia Biotech). The resulting immunoprecipitate was then incubated with ATF-2 fusion protein at 30 °C for 30 min in the presence of 200 µM ATP. JNK was precipitated from the cell lysates with c-Jun fusion protein bound to glutathione-Sepharose beads and incubated with 100 µM ATP at 30 °C for 30 min. The phosphorylation of ATF-2 at Thr-71 and c-Jun at Ser-63 was detected by Western blotting using a 1:100 dilution of phosphospecific ATF-2 or c-Jun. To show that equal amounts of JNK and p38 MAP kinase were precipitated, the beads were incubated with SDS sample buffer at 97 °C for 3 min and then subjected to Western blot analysis using a 1:1000 dilution of antibody to JNK and p38 MAP kinase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Immune Complex-coupled Kinase Assay for ASK1-- The immune complex-coupled kinase assay has been described previously (7). In brief, cells were lysed in the lysis buffer containing 20 mM Tris-HCl, pH 7.5, 12 mM -glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1.5% aprotinin. The lysates of 1 × 10⁶ cells were immunoprecipitated with a 1:1000 dilution of anti-ASK1 (DAV, Ref. 10) using protein A-Sepharose. The beads were washed with washing buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EGTA, and 1 mM dithiothreitol and was subjected to kinase assay. GST·MKK6 (0.2 µg) was first incubated with the immune complex for 10 min at 30 °C in a final volume of 10 µl in a solution containing 20 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, and 100 µM ATP. Thereafter, the activated complex was incubated with 0.3 µCi of [-³²P]ATP and 1 µg of GST·p38^KN in the same solution (final volume 20 µl) for 10 min at room temperature. Kinase reactions were stopped by adding SDS sample buffer and were analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. Phosphorylation of GST·p38^KN was analyzed using a Fuji BAS2000 image analyzer.

Cell Sorter Analysis-- Involucrin-positive cells were analyzed with a cell sorter. Keratinocytes were harvested from the dishes with trypsin and fixed with 3.7% formaldehyde at room temperature for 8 min with methanol at 20 °C for 4 min and then with acetone at 20 °C for 2 min (13). After washing with Tris-buffered saline, pH 7.4, containing 0.2% Tween 20, the cells were reacted with a 1:100 dilution of rabbit anti-human involucrin antibody (Biomedical Technologies, Inc.) and a 1:100 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. The labeled cells were analyzed with a flow cytometer (Becton Dickinson Co.).

The cell cycle distribution was analyzed using a CycleTEST^TM PLUS DNA reagent kit (Becton Dickinson Immunocytometry Systems) according to the manufacturer's instructions. Nuclei isolated by trypsinization were stained with propidium iodide and then run on a flow cytometer (Becton Dickinson Co.).

TdT-mediated dUTP Nick End Labeling (TUNEL)-- Apoptotic cells were stained on chamber slides with an in situ cell death detection kit (Roche Molecular Biochemicals GmbH) according to the manufacturer's instructions. After treatment with 4% paraformaldehyde and 0.1% Triton X in sodium citrate, the cells were incubated with fluorescein-labeled nucleotides and terminal deoxynucleotidyl transferase for 1 h at 37 °C. Fluorescent specimens were observed by fluorescence microscopy.

BrdUrd Incorporation-- BrdUrd incorporation was assessed with a cell proliferation kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Cells were incubated with BrdUrd in thymidine-free culture medium for 60 min at 37 °C. BrdUrd incorporated into cellular DNA was stained with monoclonal anti-BrdUrd diluted 1:100, peroxidase anti-mouse IgG diluted 1:70, and 3,3'-diaminobenzidine. Two hundred cells were counted in randomly selected fields to quantify the positive cells. Each experiment was repeated three times.

Immunohistochemical Staining-- Paraffin-embedded normal human skin sections were stained immunohistochemically with rabbit anti-ASK1 (DAV, Ref. 10; diluted 1:1000) and normal rabbit IgG, using a streptavidin-biotin-peroxidase staining kit (Nichirei Co. Inc., Tokyo, Japan) according to the manufacturer's instructions.

Other Reagents-- SB202190, SB202474, and SB203580 were purchased from Calbiochem-Novabiochem International Co. (San Diego, CA), dissolved in dimethyl sulfoxide (Me₂SO) at 2 mM and stored at 20 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Induction of Keratinocyte Differentiation with ASK1-N-- Ad carrying ASK1-N (Ad-ASK1-N) was constructed as described previously (14). In this study, Ad expressing a bacterial -galactosidase gene (Ad--gal) and no exogenous gene (Ad-1W) were used as controls to exclude the effect of Ad itself. Gene expression was found in almost all of the keratinocytes with Ad (data not shown). We infected normal human keratinocytes with Ad-ASK1-N or control Ad at an MOI of 5 or 50. As expected from our previous reports, infection of Ad-ASK1-N but not Ad--gal at a higher MOI (50) strongly induced apoptosis as determined by morphology and TUNEL staining (Fig. 1). Surprisingly however, infection of Ad-ASK1-N at a lower level (MOI of 5) induced dramatic morphological changes without any sign of apoptotic phenotypes. The cells became enlarged and flattened, showing a differentiated phenotype 48 h after infection with Ad-ASK1-N. There were no morphological changes in keratinocytes infected with Ad--gal and Ad-1W at an MOI of 5 compared with no-vector (data not shown). Because, the morphology is apparently different from apoptotic cells, and the TUNEL staining was negative, we conclude that the morphological change induced by the infection of Ad-ASK1-N at an MOI of 5 is not apoptosis. In the following experiments, keratinocytes were infected with Ad at an MOI of 5. 

View larger version (55K): [in this window] [in a new window]   Fig. 1.   The morphological change of keratinocytes with Ad-ASK1-N. After infection with Ad-ASK1-N or Ad--gal at an MOI of 5 or 50, normal human keratinocytes were cultured for 48 h. The morphological change was observed under phase-contrast microscopy. Apoptotic cells were stained with TUNEL.

In addition to the morphological changes, ASK1-N expression induced differentiation markers including involucrin protein and transglutaminase-1 mRNA (Fig. 2A) as seen with 10% FCS, a potent inducer of keratinocyte differentiation (15). Transglutaminase-1 enzymatically cross-links its substrate proteins including involucrin and loricrin, forming a cornified envelope in terminally differentiated keratinocytes (16). Similar results were obtained with three keratinocyte strains (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Induction of differentiation markers with Ad-ASK1-N. Keratinocytes were infected with Ad-ASK1-N or Ad--gal at MOI of 5. Ad--gal is a control. A, induction of involucrin protein and transglutaminase-1 mRNA 48 h after Ad infection was analyzed by Western and Northern blotting, respectively. The treatment with 10% FCS is a positive control for differentiation. B, time course for the expression of ASK1-N, involucrin, and p21Cip/WAF1 protein after Ad infection, analyzed by Western blotting. C, time course for the expression of transglutaminase-1, loricrin, and involucrin mRNA after Ad infection, analyzed by RNase protection assay. D, involucrin-positive keratinocytes 48 h after infections with Ad, analyzed with a cell sorter. The treatment with 10% FCS is a positive control for differentiation. The proportions of positive cells with Ad-1W or without vector were 5.2 and 3.7%, respectively (data not shown). The solid line indicates anti-involucrin. The dotted line indicates control IgG.

To further confirm that Ad-ASK1-N infection at an MOI of 5 induces keratinocyte differentiation, the time course for protein and mRNA expression, the percentage of involucrin-positive cells, 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and the cell cycle were analyzed (Figs. 2 and 3). ASK1-N protein appeared within 3 h of infection with Ad-ASK1-N, reaching a maximum level at 48 h, which lasted until 72 h (Fig. 2B). The expression of mRNA of differentiation markers including transglutaminase-1, loricrin, and involucrin significantly increased at 24 h (Fig. 2C). The increase of p21^Cip1/WAF1, the cyclin-dependent kinase inhibitor, occurred 6 h after infection, which was earlier than the transglutaminase-1, loricrin, and involucrin induction (Fig. 2, B and C), and reached a maximum at 24 h. The transient increase of p21^Cip1/WAF1 with differentiation is consistent with a previous report (17, 18); p21^Cip1/WAF1 expression is up-regulated in the early stage of keratinocyte differentiation and then declines in the late stages of differentiation. ASK1-N expression increased the percentage of involucrin-positive cells from 5.5 to 29.1% (Fig. 2D), which is quite similar to that seen with 10% FCS (13). Cell growth is suppressed in differentiated cells. ASK1-N expression suppressed the percentage of BrdUrd-positive cells from 38.7 (control) to 13.8% 48 h after the infection (Fig. 3A), indicating that cell growth declined. The cell cycle distribution of the keratinocytes 48 h after Ad infection (Fig. 3B) indicates that ASK1-N expression causes G₀/G₁ and G₂/M arrest. Differentiated epidermal keratinocytes in vivo are in G₀/G₁ arrest (19). However, in cultured keratinocytes the induction of differentiation by suspension culture and FCS results in G₀/G₁ and G₂/M arrest (15), which is consistent with our results. G₀/G₁ and G₂/M arrest by differentiation stimuli suggests that in cultured keratinocytes differentiation signals are not restricted to the cell cycle stage.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Suppression of cell growth with Ad-ASK1-N. A, incorporation of BrdUrd into keratinocytes after Ad-ASK1-N infection. Keratinocytes were studied 0, 24, and 48 h after infection with Ad-ASK1-N (triangle), Ad--gal (circle), Ad-1W (diamond), and no-vector (square). The BrdUrd-positive cells were stained and counted under a microscope. Photomicrographs show positive cells 48 h after infection with Ad-ASK1-N and Ad--gal. Values are the mean ± S.D. of triplicate determinations. Two hundred cells were counted in randomly selected fields to quantify the positive cells. B, cell cycle distribution of keratinocytes 48 h after infection with Ad-ASK1-N and Ad--gal, analyzed with a cell sorter.

To investigate the role of ASK1 in the regulation of involucrin expression, a luciferase assay was performed. Keratinocytes transfected with an involucrin promoter-luciferase reporter plasmid (pINV-Luc) were infected with Ad at an MOI of 5. The reporter activity increased 5.5-fold with Ad-ASK1-N, whereas Ad--gal had no effect (Fig. 4), suggesting a positive role of ASK1 in the regulation of involucrin expression. All of these data (Figs. 2-4) verify that ASK1-N differentiates keratinocytes.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Activation of the involucrin promoter by ASK1-N. Keratinocytes cultured to 2 × 105 in 6-well plates were transfected with 1 µg of involucrin reporter plasmid (pINV-Luc) and 0.5 µg of pRL-TK (Renilla luciferase) as a standard, using FuGENE6. After 24 h, the cells were infected with Ad-ASK1-N or Ad--gal at an MOI of 5 and cultured for an additional 24 h. Luciferase activity was measured using the Dual-Luciferase reporter assay system. Transfection was performed in triplicate. The relative luciferase activity was calculated by normalizing to the Renilla luciferase activity. Statistical analysis was performed using Student's t test. * statistically significant (p < 0.0001).

Involvement of p38 MAP Kinase in ASK1-induced Keratinocyte Differentiation-- ASK1 is a MAPKKK that activates SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades. Therefore, we examined whether the expression of ASK1-N enhanced the JNK and p38 MAP kinase activities in keratinocytes. The activities of both JNK and p38 MAP kinase started to increase 6-12 h after Ad-ASK1-N infection (Fig. 5A). This increase paralleled the level of ASK1-N protein shown in Fig. 2A, suggesting that ASK1-N activates the SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades in keratinocytes.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Involvement of p38 MAP kinase in ASK1-induced differentiation. A, the time course for the JNK and p38 MAP kinase activities after infecting keratinocytes with Ad-ASK1-N. The JNK and p38 MAP kinase activities are shown as phospho-c-Jun and phospho-ATF-2, respectively. Keratinocytes were infected with Ad-ASK1-N or Ad--gal at an MOI of 5 and cultured for the indicated period. p38 MAP kinase was immunoprecipitated with a rabbit antibody to p38 MAP kinase and protein G-Sepharose. The resulting immunoprecipitate was then incubated with ATF-2 fusion protein at 30 °C for 30 min in the presence of 200 µM ATP. JNK was precipitated from the cell lysates with c-Jun fusion protein bound to glutathione-Sepharose beads and incubated with 100 µM ATP at 30 °C for 30 min. The phosphorylation of ATF-2 at Thr-71 and c-Jun at Ser-63 was detected by Western blotting using phospho-specific ATF-2 or c-Jun. Equal amounts of JNK and p38 MAP kinase were precipitated as shown by Western blot analysis using antibody to JNK and p38 MAP kinase. B, effect of p38 MAP kinase inhibitors on ASK1-induced differentiation. After infection with Ad-ASK1-N at an MOI of 5, keratinocytes were cultured for 24 h in the presence of p38 MAP kinase inhibitors, SB202190 and SB203580 at a concentration of 200 µM. Transglutaminase-1, loricrin, and involucrin mRNA and ASK1 protein were analyzed by RNase protection assay and Western blotting, respectively. SB202474 is a negative control. The intensity of each band was quantified using NIH Image, referring to the signal of the control as one unit.

The involvement of p38 MAP kinase in ASK1-N-induced differentiation was shown using p38 MAP kinase inhibitors: SB202190 and SB203580 (Fig. 5B). They significantly reduced the induction levels of transglutaminase-1, loricrin, and involucrin mRNA mediated by ASK1-N but not the negative control SB202474. In SB202190-treated cells, the levels of transglutaminase-1, loricrin, and involucrin mRNA declined to 0.13, 0.02, and 0.38-fold compared with the controls, respectively. Because the inhibitors did not affect the level of ASK1-N protein expression, the suppressive effect of p38 MAP kinase inhibitors on the induction of transglutaminase-1 and involucrin mRNA was caused by the blockade of p38 MAP kinase. Therefore, p38 MAP kinase is necessary for the downstream signal transduction of ASK1-induced differentiation.

Induction of ASK1 with Differentiation and ASK1 Localization in Vivo-- We next studied whether the induction of differentiation caused ASK1 expression. Inducing differentiation with C2 ceramide, a potent differentiation inducer (22), enhanced ASK1 mRNA expression (Fig. 6A) and activity (Fig. 6B). This experiment was repeated more than three times with essentially identical results. In vivo, normal human epidermis expressed ASK1 mRNA (Fig. 7A). Moreover, an immunohistochemical study revealed that the expression of ASK1 in the upper epidermis paralleled keratinocyte differentiation (Fig. 7B), implicating ASK1 in in vivo differentiation.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Induction of ASK1 with differentiation. Keratinocytes were cultured with 5 µM C2 ceramide (C2) for 24 h. C2 dihydroceramide (C2D) is a negative control. A, ASK1 mRNA expression, analyzed by RNase protection assay. GAPDH is an internal standard. B, ASK1 activity, analyzed by immune complex-coupled kinase assay (13). Briefly, lysates of 1 × 106 cells were immunoprecipitated with anti-ASK1 (DAV, Ref. 10) and protein A-Sepharose. The beads were first incubated with 0.2 µg of GST·MKK6 and 100 µM ATP for 10 min at 30 °C. Thereafter, the activated complex was incubated with 0.3 µCi of [-32P]ATP and 1 µg of GST·p38KN for 10 min at room temperature. Kinase reactions were stopped by adding SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Phosphorylation of GST·p38KN was analyzed by a Fuji BAS2000 image analyzer. Preimmune serum (pre) is a negative control for anti-ASK1.

View larger version (69K): [in this window] [in a new window]   Fig. 7.   Expression of ASK1 in vivo. A, expression of ASK1 mRNA in normal human epidermis, analyzed with reverse transcriptase-PCR. Total RNA was treated with DNase 1. PCR was performed with or without reverse transcriptase (RT). B, expression of ASK1 protein in normal human epidermis. Paraffin-embedded normal human skin sections were immunohistochemically stained with anti-ASK1 antibody (DAV) or control rabbit IgG.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The regulation of keratinocyte differentiation by extracellular stimuli has been studied primarily; studies include suspension culture, high Ca²⁺, FCS, 1-25-dihydroxyvitamin D₃, 12-O-tetradecanoylphorbol-13-acetate (TPA), and ceramide (15, 22-27). However, the intracellular signaling mechanisms of differentiation are poorly understood. We propose that the ASK1-p38 MAP kinase cascade is a newly discovered signaling cascade for keratinocyte differentiation.

Protein kinase C (PKC) is an intracellular signal transduction molecule that regulates keratinocyte differentiation (20, 21), and epidermal keratinocytes express , , , , and  isoforms of PKC (20, 28-31). We examined whether PKC isoforms were involved in the ASK1 signaling cascade. Activation of PKC isoforms was determined by analyzing the subcellular distribution of PKC isoforms using Western blotting. The redistribution of PKC from the soluble fraction to a particulate fraction is a useful indicator of PKC activation (32). However, the expression of ASK1-N did not affect the subcellular distribution of PKCs (data not shown), indicating that PKCs were not activated by ASK1-N. Therefore, PKCs are not localized in the downstream signaling of the ASK1-p38 MAP kinase cascade. A recent study showed that PKC regulates involucrin promoter activity via p38 MAP kinase (33) as well as ASK1. Therefore, the ASK1-p38 MAP kinase cascade may cross-talk with the PKC cascade at the level of p38 MAP kinase.

NF-B is another candidate for a molecular regulator of keratinocyte differentiation (34-37). The epidermis of mice lacking the inhibitor of B kinase  (IKK) shows abnormal differentiation (35). In normal epidermis, NF-B is localized in the cytoplasm of basal cells and then translocates to the nucleus in suprabasal cells, suggesting a possible role for commitment to differentiate (34). In contrast to NF-B, ASK1 protein is found in the upper epidermis and has a different distribution from that of NF-B. Furthermore, the expression of ASK1-N does not activate NF-B as analyzed with a gel shift mobility assay (data not shown), indicating that NF-B is not involved in the ASK1-induced keratinocyte differentiation. One suggested role for NF-B is that it prevents premature apoptosis before the final step by regulating the expression of the anti-apoptotic molecules TRAF1, TRAF2, c-IAP1, and c-IAP2 (37). On the other hand, ASK1 is an apoptosis inducer and appears in the late stages of differentiation in vivo. Therefore, NF-B and ASK1 have distinct roles in the regulation of keratinocyte differentiation.

Although, ASK1 has been identified as an apoptosis inducer (5), we have shown that ASK1 induces keratinocyte differentiation. We also found that introducing ASK1 induced apoptosis. However, it required 10-fold higher (MOI of 50) levels of ASK1-N expression than those required for differentiation (MOI of 5). These results suggest that ASK1 has dual physiological functions in keratinocytes; weak or strong activation of ASK1 may lead to differentiation or apoptosis of keratinocytes, respectively. The biological activity of ASK1 depends on the cell type and conditions. In the pheochromocytoma cell line PC12, moderate expression of ASK1-N induces neuronal differentiation and survival rather than apoptosis (38). Thus, ASK1 is not only an apoptosis inducer but also mediates a wide range of cellular functions.

In continuously self-renewing tissues, such as the gastrointestinal tract, epithelial cells are shed by terminal differentiation and apoptosis. Epidermal keratinocytes also differentiate and are ultimately shed from the epidermis after cell death. However, the morphology of cells dying in terminal differentiation is different from that of cells undergoing pathological apoptosis induced by ultraviolet light and Fas. This physiological cell death is suggested to be a specialized form of apoptosis (39). In ASK1-induced apoptosis, involucrin and transglutaminase-1 are strongly induced before apoptosis (MOI of 50, data not shown), whereas ultraviolet B irradiation does not enhance the expression of transglutaminase-1 mRNA (data not shown). Therefore, the two apoptosis mechanisms are different. Because ASK1 induces apoptosis with differentiation markers, and its expression is localized in the upper epidermis, ASK1 may be involved in the mechanism of apoptosis in terminal differentiation.

	FOOTNOTES

^* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and a SHISEIDO Grant for Skin Aging Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Dermatology, Ehime University School of Medicine, Shigenobucho, Onsengun, Ehime 791-0295, Japan. Tel.: 81 (89) 960-5350; Fax:. 81 (89) 960-5352; E-mail: sayama@m.ehime-u.ac.jp.

Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M003425200

	ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; ASK1, apoptosis signal-regulating kinase; Ad, adenovirus vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; MOI, multiplicity of infection; GST, glutathione S-transferase; PCR, polymerase chain reaction; TK, thymidine kinase; INV, involucrin; FCS, fetal calf serum; PKC, protein kinase C; TUNEL, TdT-mediated dUTP nick end labeling; BrdUrd, bromodeoxyuridine; KN, kinase negative.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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