 |
INTRODUCTION |
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 |
Keratinocyte Culture--
Normal human keratinocytes were
cultured with MCDB153 medium supplemented with insulin (5 µg/ml),
hydrocortisone (5 × 10
7 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-p21Cip1/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
(GenbankTM/EBI accession number D84476),
866-1133 of transglutaminase-1 (GenbankTM/EBI accession
number D90287), 966-1176 of loricrin (GenbankTM/EBI
accession number M61120), and 74-249 of involucrin
(GenbankTM/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 CycleTESTTM 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 |
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
p21Cip1/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
p21Cip1/WAF1 with differentiation is
consistent with a previous report (17, 18);
p21Cip1/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·p38 KN 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·p38 KN 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 |
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.