From the Department of Cell Biology, Research
Institute for Microbial Diseases, Osaka University, Suita, Osaka
565-0871, Japan and the § Institute of Life Science, Kurume
University, Kurume, Fukuoka 839-0861, Japan
Received for publication, November 20, 2002, and in revised form, February 24, 2003
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ABSTRACT |
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Heparin-binding epidermal growth factor-like
growth factor (HB-EGF) is a critical growth factor for a number of
physiological and pathological processes. HB-EGF is synthesized as a
membrane-anchored form (pro-HB-EGF), and pro-HB-EGF is cleaved at the
cell surface to yield soluble HB-EGF by a mechanism called
"ectodomain shedding." We show here that the ectodomain shedding of
pro-HB-EGF in Vero cells is induced by various stress-inducing stimuli,
including UV light, osmotic pressure, hyperoxidation, and translation
inhibitors. The pro-inflammatory cytokine interleukin-1 Heparin-binding epidermal growth factor
(EGF)1-like growth factor
(HB-EGF) (1), a member of the EGF family, binds to the EGF receptor
(ErbB1) and the related receptor tyrosine kinase (ErbB4) and activates
them (2). HB-EGF, especially the secreted form (sHB-EGF), is a potent
mitogen and chemoattractant for a number of cell types, including
vascular smooth muscle cells, fibroblasts, and keratinocytes (3).
HB-EGF is implicated in a number of physiological and pathological
processes in the body (4), which include wound healing (5, 6), kidney
collecting duct morphogenesis (7), blastocyst implantation (8), cardiac hypertrophy (9-11), smooth muscle cell hyperplasia (12), pulmonary hypertension (13), and oncogenic transformation (14).
HB-EGF, synthesized as a membrane-anchored precursor protein
(pro-HB-EGF) of 208 amino acids, is composed of a signal peptide, a
heparin-binding domain, an EGF-like region, a juxtamembrane domain, a
transmembrane segment, and a cytoplasmic tail (3). Pro-HB-EGF is
cleaved at the cell surface within the juxtamembrane domain through a
process called ectodomain shedding, which results in secretion of a
soluble 75-86-amino acid growth factor (sHB-EGF) (15). Although the
regulated process of pro-HB-EGF ectodomain shedding yields substantial
sHB-EGF, a considerable amount remains uncleaved at the cell surface.
In addition to being a precursor of sHB-EGF, pro-HB-EGF is a
biologically active molecule, forming a complex with both CD9 (16) and
integrin Pro-HB-EGF ectodomain shedding is induced by both physiological and
pharmacological agonists such as
12-O-tetradecanoylphorbol-13-acetate (TPA). TPA, an
activator of protein kinase C (PKC), potently induces the ectodomain
shedding of pro-HB-EGF (15) and of other membrane proteins (20). In
monkey kidney Vero cells, the presence of a constitutively active form
of PKC Lysophosphatidic acid (LPA) and other ligands of the
seven-transmembrane G protein-coupled receptors also stimulate
pro-HB-EGF shedding (23, 24). The activation of shedding by such
ligands is crucial for the transactivation of the EGF receptor by G
protein-coupled receptor ligands (23, 25, 26). In Vero cells, the
Ras-Raf-MEK and Rac signaling pathways are activated in LPA-induced
shedding (27). LPA-induced shedding is inhibited by dnHa-Ras, dnRac1, and the MEK inhibitor PD98059, but not by either dnPKC Pro-HB-EGF expression is enhanced in various tissues upon injury and
inflammation (28-35). The induction of this cellular response under
these conditions suggests that HB-EGF is involved in both physiological
tissue repair, including processes such as wound healing, and
pathological processes, including atherosclerosis, that follow from
inappropriate inflammation. Cellular stress and inflammation may
function as triggers of ectodomain shedding. It remains unclear,
however, if inflammatory cytokines and stimuli inducing cellular stress
responses can trigger pro-HB-EGF ectodomain shedding. We demonstrate
here that various stress-inducing stimuli and inflammatory cytokines
strongly induce ectodomain shedding, acting through a p38 MAPK-mediated
pathway, distinct from both the TPA- and LPA-induced signaling
pathways. Our results suggest that HB-EGF activity is regulated at the
transcriptional level and by ectodomain shedding, which responds to
stress and inflammation.
Reagents--
TPA, Ro-31-8220, and anisomycin were purchased
from Nacelle Tesque Co., Ltd. (Kyoto, Japan). LPA was obtained from
Funakoshi Co., Ltd. (Tokyo, Japan). Recombinant human IL-1 Antibodies--
The goat antibody specific for the C terminus of
human pro-HB-EGF (C-18) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Goat anti-HB-EGF neutralizing antibody was obtained from R&D Systems. A rabbit antiserum against the N terminus of human
pro-HB-EGF (H6) was derived as described (36). Rabbit anti-mouse ADAM9
antibody was raised against a glutathione S-transferase fusion protein containing the mouse ADAM9 C-terminal region (amino acids 718-845). Horseradish peroxidase-conjugated goat anti-rabbit and
unconjugated anti-FLAG (M2) monoclonal antibodies were purchased from
Zymed Laboratories Inc. and Sigma, respectively.
Horseradish peroxidase- and Cy3-conjugated donkey anti-goat antibodies
were obtained from Chemicon International, Inc. (Temecula, CA).
Plasmids--
pEGFP-c1, a plasmid encoding GFP, was purchased
from Clontech. dnPKC Cell Culture and Transfection--
Vero-H cells (15) were
maintained in modified Eagle's medium with nonessential amino acids
supplemented with 10% fetal calf serum. Transfections into Vero-H
cells were performed using the calcium phosphate technique (41) unless
otherwise stated.
Shedding Analysis by Western Blotting--
Vero-H cells
(5 × 105 cells/6-cm dish) were cultured for 6-12 h
in serum-free modified Eagle's medium with nonessential amino acids
and then treated with TPA (64 nM), LPA (20 µg/ml),
IL-1 Immunofluorescence Detection of the Shedding of the Pro-HB-EGF
Ectodomain--
Pro-HB-EGF was detected on cells using goat
anti-HB-EGF neutralizing antibody and Cy3-conjugated anti-goat IgG as
described (21). To detect the expression of transfected proteins, fixed cells were permeabilized with 0.1% Triton X-100 for 3 min and then
stained with anti-FLAG antibody or the antibodies indicated, followed
by fluorescein isothiocyanate-conjugated second antibody. To detect
cells transfected with dnPKC Stimuli Inducing Various Stresses and IL-1
Western blot analysis confirmed the ectodomain shedding of pro-HB-EGF
in Vero-H cells (Fig. 1B). Immunoblot analysis of Vero-H cell lysates (upper panel) and the culture medium
(lower panel) following incubation with the indicated
stimuli detected the cytoplasmic domain of pro-HB-EGF and the shed
soluble EGF-like domain, respectively. In cell lysates, bands ranging
from 20 to 30 kDa correspond to pro-HB-EGF, whereas bands between 17.4 and 6.9 kDa are proteolytic fragments composed of the cytoplasmic and
transmembrane domains of pro-HB-EGF (referred to as the "tail
fragment"). Consistent with the appearance of the tail fragment,
sHB-EGF appeared concurrently in the culture medium. These results
indicate that IL-1
We also examined the time course of pro-HB-EGF shedding induced by
IL-1 Stress-induced Pro-HB-EGF Shedding Involves p38 MAPK--
Signals
from pro-inflammatory cytokines and cellular responses to stress are
generally mediated by SAPKs (42). Therefore, we hypothesized that
stress-induced pro-HB-EGF shedding is mediated by a SAPK family kinase.
We examined the activation of p38 MAPK and the various JNKs, major
members of the SAPK family, following stress-inducing stimulation of
Vero-H cells. After treatment with LPA, TPA, IL-1
We next inspected the SB203580 inhibition of pro-HB-EGF ectodomain
shedding induction by IL-1
We examined the requirement for p38 MAPK in stress-induced pro-HB-EGF
shedding using dnp38 MAPK. Following the transfection of FLAG-tagged
dnp38 MAPK, cells were treated with various stimuli and then
double-stained for pro-HB-EGF and dnp38 MAPK. Although treatment with
IL-1
SB203580 (2 µM) reduced the activity of p38 MAPK, but not
that of JNK1/2, as shown in Fig. 3 (A and B). At
the same concentration, SB203580 inhibited stress-induced shedding,
suggesting that JNK is not involved in stress-induced shedding in
Vero-H cells. To examine the involvement of JNK in stress-induced
shedding directly, we tested whether SP600125, an inhibitor of JNK
(46), abrogates stress-induced pro-HB-EGF shedding in Vero-H cells.
SP600125 (5 µM) significantly reduced JNK1/2
phosphorylation caused by TPA, IL-1
In addition to the JNK inhibitor, we used dnJNK1 to examine the
involvement of the JNK pathway in stress-induced pro-HB-EGF shedding.
Following the transfection of FLAG-tagged dnJNK1, cells were treated
with various stimuli as Fig. 4B and then double-stained for
pro-HB-EGF and dnJNK1. Pro-HB-EGF ectodomain shedding induced by each
stimulus was not inhibited in FLAG-positive cells expressing dnJNK1
(Fig. 7). From these results, we conclude
that the JNK pathway is not involved in stress-induced pro-HB-EGF
ectodomain shedding in Vero-H cells.
p38 MAPK-mediated Shedding Cascades Are Independent of the Other
Shedding Cascades--
Using Vero-H cells, we demonstrated that the
LPA-induced shedding cascade involves the Ras-Raf-MEK and small GTPase
Rac pathways, whereas TPA-induced pro-HB-EGF shedding involves PKC Metalloproteases Are Involved in Stress-induced
Shedding--
Metalloprotease inhibitors inhibit both TPA- and
LPA-induced pro-HB-EGF shedding. We treated cells with a hydroxamic
acid-based metalloprotease inhibitor, KB-R8301, to examine the role of
metalloproteases in stress-induced shedding. KB-R8301 (10 µM) inhibited the pro-HB-EGF shedding induced by IL-1
ADAM family metalloproteases are involved in the ectodomain shedding of
a variety of proteins such as TNF- HB-EGF contributes to tissue repair processes acting in response
to various injuries (5, 6, 30, 51). HB-EGF also participates in
pathological processes, including smooth muscle cell hyperplasia (12),
restenosis following balloon injury (52), and cardiac hypertrophy
(9-11). Ectodomain shedding is critical for the biological activity of
this growth factor (53). In addition to regulation of growth factor
activity, pro-HB-EGF ectodomain shedding contributes to the
transactivation of the EGF receptor following ligation by G
protein-coupled receptors and other ligands (23). Recent studies
suggest that pro-HB-EGF ectodomain shedding and transactivation are
involved in the pathological processes of cardiac hypertrophy and
pulmonary hypertension (9, 13). Although a function for HB-EGF in
physiological and pathological processes has been increasingly
reported, information about shedding-inducing stimuli and the
downstream signaling processes involved is limited (54). Two distinct
signaling pathways contribute to pro-HB-EGF shedding in Vero-H cells,
the TPA-induced PKC Stress-induced ectodomain shedding is not specific to pro-HB-EGF.
Osmotic stress induces L-selectin shedding in neutrophils (55). UV
light and osmotic pressure promote the shedding of pro-transforming
growth factor- We have demonstrated that stress- and IL-1 Recent studies suggest that, in addition to ADAM9, ADAM10/Kuzbanian
(13, 62), ADAM12/meltrin- What is the biological significance of stress-induced pro-HB-EGF
shedding? A number of reports indicate that the transcription of
pro-HB-EGF is up-regulated in response to oxidative, ischemic, osmotic,
and mechanical stresses (29-34). The inflammatory cytokines IL-1 also
stimulated the ectodomain shedding of pro-HB-EGF. An inhibitor of p38
MAPK (SB203580) or the expression of a dominant-negative (dn) form of
p38 MAPK inhibited the stress-induced ectodomain shedding of
pro-HB-EGF, whereas an inhibitor of JNK (SP600125) or the expression of
dnJNK1 did not. 12-O-Tetradecanoylphorbol-13-acetate (TPA)
and lysophosphatidic acid (LPA) are also potent inducers of pro-HB-EGF
shedding in Vero cells. Stress-induced pro-HB-EGF shedding was not
inhibited by the inhibitors of TPA- or LPA-induced pro-HB-EGF
shedding or by dn forms of molecules involved in the TPA- or
LPA-induced pro-HB-EGF shedding pathway. Reciprocally, SB203580 or
dnp38 MAPK did not inhibit TPA- or LPA-induced pro-HB-EGF shedding.
These results indicate that stress-induced pro-HB-EGF shedding is
mediated by p38 MAPK and that the signaling pathway induced by stress
is distinct from the TPA- or LPA-induced pro-HB-EGF shedding pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1 (17) to transduce biological
signals to neighboring cells in a non-diffusible manner (18). In
contrast to the mitogenic effect of sHB-EGF (4), pro-HB-EGF negatively
regulates cell proliferation (19). These results suggest that strict
control of pro-HB-EGF ectodomain shedding is critical for the proper
regulation of the activity of this growth factor.
results in the ectodomain shedding of pro-HB-EGF, and
dominant-negative (dn) PKC
suppresses TPA-induced shedding,
suggesting that the PKC
isoform contributes to the ectodomain
shedding of pro-HB-EGF. PKC
binds to the cytoplasmic domain of
ADAM9/MDC9/meltrin-
(21), a metalloprotease belonging to the ADAM
(a disintegrin and
metalloprotease) family (22). Overexpression of ADAM9
induces pro-HB-EGF shedding, which can be inhibited by the expression
of the dnADAM9 mutant H347A,H351A in Vero-H cells. Thus, ADAM9 is
thought to act downstream of PKC
in the pro-HB-EGF shedding pathway
(21), although the role of this protease in the direct cleavage of
pro-HB-EGF remains unclear.
or dnADAM9. Conversely, although TPA-induced shedding is inhibited by dnPKC
and
dnADAM9, such shedding is not affected by dnHa-Ras, dnRac1, and the MEK
inhibitor. These results indicate that two distinct signaling pathways
function to regulate pro-HB-EGF ectodomain shedding, here designated
the LPA- and TPA-induced pathways.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, human
TNF-
, and mouse IL-6 were acquired from PeproTech (Rocky
Hill, NJ). PD98059 and SB203580 were purchased from Calbiochem.
SP600125 was obtained from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). H2O2 was acquired from Santoku Chemical
Industries Co., Ltd. (Tokyo). KB-R8301 was obtained from Nippon Organon
K. K. (Osaka, Japan).
(R144A, R145A,
K376R) (37, 38) and the mouse dnADAM9 mutant H347A,H351A (21)
have been described previously. The FLAG-tagged mouse ADAM9 mutant
E348A was generated by site-directed PCR-based mutagenesis. FLAG-tagged
dnRac1 (N17Rac1) has been described previously (27). FLAG-tagged dnp38
MAPK (T180A,Y182F) was kindly provided by H. Hatanaka and Y. Ishikawa
(Institute of Protein Research, Osaka University). pCMV5-JNK1(APF), a
plasmid encoding FLAG-tagged dnJNK1 (39), and a constitutively
active form of MKK6 (EE-MKK6) (40) were kindly provided by
R. J. Davis (University of Massachusetts Medical School) and Y. Gotoh (University of Tokyo), respectively.
(4 ng/ml), anisomycin (10 µg/ml), sorbitol (0.4 M), or H2O2 (0.5 mM).
When indicated, cells were also treated with either TNF-
(50 ng/ml)
or IL-6 (20 ng/ml). For UV irradiation, Vero-H cells in 6-cm dishes in
3 ml of modified Eagle's medium with nonessential amino acids were
exposed to 40 mJ/cm2 UV radiation (254 nm) using a
Spectrolinker (Spectronics Corp.) Following incubation for 30 min with
stimuli, cells were collected and resuspended in 140 µl of lysis
buffer (1% Nonidet P-40, 50 mM Tris-HCl, 0.1 M
NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, 20 µg/ml antipain, and 10 µg/ml chymostatin, pH 7.4).
After 5 min on ice, the lysates were clarified by centrifugation at
15,000 rpm. Western blot analysis was performed using a goat antibody
specific for the human pro-HB-EGF C terminus. The antibody was
visualized with horseradish peroxidase-conjugated anti-goat IgG using
ECL Plus (Amersham Biosciences). To detect sHB-EGF in the culture
medium, sHB-EGF was trapped with heparin-Sepharose beads and then
eluted with SDS gel sample buffer. The eluted materials were subjected
to Western blot analysis and detected with rabbit anti-human HB-EGF
antibody H6. The antibody was visualized with horseradish
peroxidase-conjugated anti-rabbit IgG using ECL Plus.
or vector (pEF-BOS) alone as a control,
EGFP (pEGFP-c1) was cotransfected. Images were captured with FISH ImagerTM system (Carl Zeiss, Inc.). The percentage
of pro-HB-EGF-positive cells was determined by counting the number of
pro-HB-EGF-positive cells among the total cells concomitantly expressing the products of the transfected cDNA. Values were
determined based on the results obtained in at least two independent
transfections from at least 100 independent cells positive for either
the transfected or marker proteins in each experiment. Scoring was
performed in a completely blind manner.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lead to
Pro-HB-EGF Shedding--
Vero-H cells are stable Vero transfectants
overexpressing human pro-HB-EGF (15). This cell line exhibits
consistent responses to stimuli inducing pro-HB-EGF ectodomain shedding
(21, 24, 27). Using Vero-H cells, we examined the effect of
pro-inflammatory cytokines and stress-inducing stimuli on the induction
of pro-HB-EGF ectodomain shedding. First, we examined the ectodomain
shedding of pro-HB-EGF by immunofluorescence microscopy using an
antibody specific for the pro-HB-EGF ectodomain. Vero-H cells were
pretreated with the indicated stimuli and then cultured for 30 min.
Cells were treated with anti-HB-EGF neutralizing antibody and then
fixed and visualized using a secondary antibody. Pro-HB-EGF
fluorescence at the cell surface disappeared following addition of
either LPA (20 µg/ml) or TPA (64 nM) to the culture
medium (Fig. 1A) (21, 24, 27).
In addition, the pro-inflammatory cytokine IL-1
(4 µg/ml) also led
to the disappearance of pro-HB-EGF surface immunofluorescence.
Moreover, stimuli well known to induce a cellular stress response (42),
such as translation inhibitors (anisomycin, 10 µg/ml) (43), exposure
to UV light (40 mJ/cm2), hypertonic osmotic pressure
(sorbitol, 0.4 M), or oxidative stress
(H2O2, 0.5 mM), also induced the
disappearance of pro-HB-EGF immunofluorescence from the cell
surface.
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Fig. 1.
Stress- and inflammatory cytokine-induced
ectodomain shedding of pro-HB-EGF. Vero-H cells were
treated with LPA (20 µg/ml), TPA (64 nM), IL-1 (4 ng/ml), TNF-
(50 ng/ml), IL-6 (20 ng/ml), anisomycin (10 µg/ml),
UV light (40 mJ/cm2), sorbitol (0.4 M), or
H2O2 (0.5 mM) for 30 min, and then
ectodomain shedding was detected by immunostaining of pro-HB-EGF and
Western blot analysis. A, immunofluorescence detection of
pro-HB-EGF at the cell surface. Cells were stained with anti-pro-HB-EGF
neutralizing antibody, followed by Cy3-conjugated anti-goat IgG. The
representative images for the samples treated with LPA, TPA, IL-1
,
anisomycin, UV light, sorbitol, and H2O2 are
shown. The left panels are phase-contrast images, whereas
the right panels are immunofluorescence images.
B, Western blot analysis. The upper panel
illustrates full-length pro-HB-EGF and the tail fragment within cell
lysates, detected using an antibody raised against the pro-HB-EGF C
terminus. The lower panel shows sHB-EGF appearing in the
culture medium, detected using an antibody raised against the
pro-HB-EGF N terminus.
, anisomycin, UV light, and sorbitol, in addition
to LPA and TPA, induce pro-HB-EGF cleavage to generate both the tail
fragment in cell lysates and sHB-EGF in the culture medium. Although
H2O2 generated the tail fragment in cell
lysates and sHB-EGF in the culture medium, this stimulus also reduced
the total pro-HB-EGF protein present through an unknown mechanism.
Centrifuging as a mimic of shearing stress also induced pro-HB-EGF
ectodomain shedding (data not shown). In contrast, the pro-inflammatory
cytokines TNF-
and IL-6 did not affect shedding. Rather, these
cytokines reduced the total pro-HB-EGF protein, although the reduced
level varied depending on the experiments.
, anisomycin, UV light, and sorbitol. As the appearance of
sHB-EGF in the culture medium correlated well with the appearance of
the tail fragment in the cell lysate (Fig. 1B) (27), we
utilized Western blot analysis only for the cell lysates to examine
this event. As shown in Fig. 2,
ectodomain shedding was observed following treatment with each of these
stress stimuli at a time course similar to that observed for
TPA-induced shedding (15, 21).
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Fig. 2.
Time course of stress- and
IL-1 -induced ectodomain shedding. Vero-H
cells were treated with 4 ng/ml IL-1
(A), 10 µg/ml
anisomycin (B), 40 mJ/cm2 UV light
(C), or 0.4 M sorbitol (D). After the
times indicated, the cells were harvested, and the cell lysates were
analyzed by Western blotting with an antibody raised against the
pro-HB-EGF C terminus.
, anisomycin, UV
irradiation, or sorbitol, Vero-H cell lysates were analyzed by Western
blotting with either anti-phospho-p38 MAPK or anti-phospho-JNK antibody
to monitor kinase phosphorylation status. Phosphorylated p38 MAPK was
strongly observed after treatment with anisomycin, UV light, or
sorbitol, with moderate increases in phosphorylation following
treatment with either TPA or IL-1
. SB203580, an inhibitor of p38
MAPK (44), significantly reduced p38 MAPK activity at a concentration
of 2 µM (Fig.
3A). JNK1/2 and p38 MAPK were
activated by treatment with LPA, TPA, IL-1
, anisomycin, UV light,
and sorbitol, although 2 µM SB203580 could not inhibit
JNK1/2 phosphorylation (Fig. 3B). The results confirm that
SB203580 specifically inhibits p38 MAPK activation. TNF-
and IL-6
could not activate either p38 MAPK (Fig. 3A) or JNK (Fig. 3B) in Vero-H cells, concurring with a lack of pro-HB-EGF
cleavage (Fig. 1B).
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Fig. 3.
Effects of SB203580 on the activation of p38
MAPK and JNK1/2 or on pro-HB-EGF ectodomain shedding.
Serum-starved Vero-H cells were pretreated with or without 2 µM SB203580 for 1 h. Cells were then treated with
IL-1 or stress-inducing or other stimuli for 30 min. Cell lysates
were analyzed by Western blotting to detect activation of p38 MAPK and
JNK1/2 and ectodomain shedding. A, activation level of p38
MAPK, detected with anti-phospho-p38 MAPK antibody (upper
panels) and anti-p38 MAPK antibody (lower panels).
B, activation level of JNK1/2, detected with
anti-phospho-JNK1/2 antibody (upper panels) and anti-JNK1/2
antibody (lower panels). C, ectodomain shedding
of pro-HB-EGF, detected by Western blotting using an antibody raised
against the pro-HB-EGF C terminus.
, anisomycin, UV light, or sorbitol to
verify the involvement of p38 MAPK. 2 µM SB203580
drastically inhibited the stress-induced shedding caused by IL-1
,
anisomycin, UV light, or sorbitol (Fig. 3C), whereas TPA-
and LPA-induced shedding was not affected. Even concentrations of
SB203580 in excess of 10 µM could not inhibit TPA- and
LPA-induced shedding (data not shown). These results suggest that
pro-HB-EGF ectodomain shedding induced by IL-1
, anisomycin, UV
light, or sorbitol requires p38 MAPK activation.
, anisomycin, UV light, or sorbitol enhanced pro-HB-EGF shedding
in vector-transfected cells (Fig.
4B), shedding was inhibited in
FLAG-positive cells expressing dnp38 MAPK (Fig. 4, A and
B). We confirmed that LPA- and TPA-induced pro-HB-EGF ectodomain shedding was not inhibited by dnp38 MAPK (Fig.
4B). MKK6 is a specific activator of p38 MAPK (45).
Consistent with the role of the p38 MAPK pathway in shedding,
transfection of a constitutively active form of MKK6 resulted in
pro-HB-EGF ectodomain shedding in the absence of stress-inducing
stimuli in a dose-dependent manner (Fig.
5). These results indicate that p38 MAPK
activation is required for stress-induced pro-HB-EGF shedding in Vero-H
cells.
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Fig. 4.
Inhibition of stress- and
IL-1 -induced ectodomain shedding by dnp38
MAPK. A, Vero-H cells were transfected with a plasmid
encoding FLAG-tagged dnp38 MAPK (20 µg) or with vector (18 µg) plus
EGFP (2 µg) as a control. After 48 h of transfection, the cells
were incubated with LPA, TPA, IL-1
, anisomycin, UV light, or
sorbitol and then double-stained with anti-HB-EGF antibody for the
detection of the pro-HB-EGF ectodomain (red) and with
anti-FLAG antibody for the detection of dnp38 MAPK expression
(green). The right panels are phase-contrast
images. Only the representative images of dnp38 MAPK-transfected cells
are shown. B, shown is the percentage of pro-HB-EGF-positive
cells among transfected cells, determined by immunofluorescence
detection as shown in A. For vector-transfected cells, the
percentage of pro-HB-EGF-positive cells among GFP-positive cells was
determined.
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Fig. 5.
Enhanced ectodomain shedding by a
constitutively active form of MKK6. Vero-H cells were transfected
with a plasmid encoding constitutively active (ca) MKK6 by
electroporation (Bio-Rad, Gene Pulser) according to the manufacturer's
instruction. The empty vector (pEF-BOS) and a plasmid encoding EGFP
were also cotransfected at the amounts indicated. The percentage of
pro-HB-EGF-positive cells among GFP-positive cells was
determined.
, anisomycin, and sorbitol
treatment (Fig. 6A), whereas
the same concentration of SP600125 could not inhibit p38 MAPK
phosphorylation caused by IL-1
, anisomycin, and sorbitol treatment
(Fig. 6B). Based on these results, we tested whether
SP600125 inhibits stress-induced ectodomain shedding in Vero-H cells.
SP600125 (5 µM) did not inhibit pro-HB-EGF ectodomain
shedding induced by any stimulus (Fig. 6C). In UV
light-irradiated cells, JNK1/2 phosphorylation was strong and was not
significantly reduced with 5 µM SP600125. SP600125 (20 µM) inhibited JNK1/2 phosphorylation caused by UV light,
but SP600125 scarcely inhibited UV light-induced shedding even at this
concentration.
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Fig. 6.
Effects of the JNK inhibitor SP600125 on
stress-induced ectodomain shedding. Serum-starved Vero-H cells
were pretreated with or without SP600125 at the indicated
concentrations for 1 h and then treated with IL-1 or
stress-inducing or other stimuli for 30 min. Cell lysates were analyzed
by Western blotting to detect activation of JNK1/2 and p38 MAPK and
ectodomain shedding. A and B, activation levels
of JNK1/2 and p38 MAPK, respectively. C, ectodomain shedding
of pro-HB-EGF.
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Fig. 7.
Effects of dnJNK1 on stress-induced
ectodomain shedding. Vero-H cells were transfected with a plasmid
encoding FLAG-tagged dnJNK1 (20 µg) or with vector (pEF-BOS; 18 µg)
plus EGFP (2 µg). After 48 h of transfection, the cells were
incubated with LPA, TPA, IL-1 , anisomycin, UV light, or sorbitol and
then stained with anti-HB-EGF antibody for the detection of the
pro-HB-EGF ectodomain. The expression of dnJNK1 was detected by
anti-FLAG antibody. The percentage of pro-HB-EGF-positive cells among
FLAG-positive cells was determined. For cells transfected with the
empty vector, the percentage of pro-HB-EGF-positive cells among
GFP-positive cells was determined.
and ADAM9. IL-1
, anisomycin, and additional stresses induce shedding
through a pathway involving the activation of p38 MAPK. We next
analyzed the connection of the p38 MAPK-mediated pathway with either
the LPA- or TPA-induced pathway. We tested the effect of the MEK
inhibitor PD98059 (47) and the PKC inhibitor Ro-31-8220 (48) on p38 MAPK-mediated pro-HB-EGF ectodomain shedding. PD98059 inhibited LPA-induced, but not TPA-induced, shedding (Fig.
8A), whereas Ro-31-8220
inhibited TPA-induced, but not LPA-induced, shedding (Fig.
8B), confirming previous results (27). p38 MAPK-mediated stress-induced shedding was not inhibited by either PD98059 or Ro-31-8220. We also transfected dnPKC
or dnRac1. Neither dnPKC
nor dnRac1 inhibited p38 MAPK-mediated shedding, with the exception of
partial inhibition observed for sorbitol-induced shedding by dnRac1
(Fig. 9). These data indicate that the
stress-induced shedding cascade mediated by p38 MAPK activation
functions independently of the LPA- and TPA-induced shedding
cascades.
View larger version (36K):
[in a new window]
Fig. 8.
Effects of the MEK inhibitor PD98059 and the
PKC inhibitor Ro-31-8220 on the ectodomain shedding of pro-HB-EGF.
Serum-starved Vero-H cells were pretreated with 25 µM
PD98059 (A) or 5 µM Ro-31-8220 (B)
for 1 h. As a control, 0.1% Me2SO, the solvent of
these inhibitors, was added. The cells were treated with each stimulus
for 30 min. Ectodomain shedding was detected by Western blot analysis
of cell lysates using an antibody raised against the pro-HB-EGF C
terminus.
View larger version (32K):
[in a new window]
Fig. 9.
Effects of dnRac1 and
dnPKC on the ectodomain shedding of
pro-HB-EGF. Vero-H cells were transfected with plasmids encoding
FLAG-tagged dnRac1 (20 µg) and dnPKC
(18 µg) plus EGFP (2 µg)
or with vector (18 µg) plus EGFP (2 µg) as a control. After 48 h of transfection, the cells were incubated with LPA, TPA, IL-1
,
anisomycin, UV light, or sorbitol and then stained with anti-HB-EGF
antibody for the detection of the pro-HB-EGF ectodomain. The expression
of dnRac1 was detected by anti-FLAG antibody. The percentage of
pro-HB-EGF-positive cells among FLAG-positive cells was determined. For
cells transfected with either dnPKC
or the empty vector, the
percentage of pro-HB-EGF-positive cells among GFP-positive cells was
determined. ca, constitutively active.
,
anisomycin, UV light, H2O2, and sorbitol (Fig.
10A), indicating that
metalloproteases are required for p38 MAPK-mediated pro-HB-EGF shedding
as well.
View larger version (49K):
[in a new window]
Fig. 10.
Inhibition of stress- and
IL-1 -induced pro-HB-EGF shedding by the
metalloprotease inhibitor KB-R8301, but not dnADAM9. A,
effect of the metalloprotease inhibitor KB-R8301. Vero-H cells were
pretreated with 10 µM KB-R8301 or 0.1% Me2SO
for 1 h and then treated with each stimulus for 30 min. Ectodomain
shedding was detected by Western blot analysis of cell lysates using an
antibody raised against the pro-HB-EGF C terminus. B, effect
of dnADAM9. Vero-H cells were transfected with plasmids encoding the
dominant-negative mutants H347A,H351A (20 µg) and E348A (20 µg) or
with vector alone (18 µg) plus EGFP (2 µg) as a control. After
48 h of transfection, cells were incubated with LPA, TPA, IL-1
,
UV light, or sorbitol and then double-stained with either anti-HB-EGF
or anti-ADAM9 antibody. The percentage of pro-HB-EGF-positive cells
among ADAM9-positive cells was determined. wt,
wild-type.
and Delta (49, 50). ADAM9 is
known to be involved in TPA-induced pro-HB-EGF shedding in Vero-H cells
(21). To examine the role of ADAM9 in p38 MAPK-mediated shedding, we
examined the effect of overexpression of dnADAM9 on shedding. Two ADAM9
mutants were used, one carrying the modification of the conserved
histidine residues in the catalytic domain to alanines (H347A,H351A)
and the other carrying a modification of the conserved glutamic acid in
the catalytic domain to alanine (E348A). We investigated the influence
of these mutants on stress- and IL-1
-induced shedding. Both mutants
inhibited TPA-induced shedding, but did not affect LPA-, stress-, and
IL-1
-induced shedding (Fig. 10B). These results indicate
that ADAM9 is not required for stress- and IL-1
-induced pro-HB-EGF
shedding in Vero-H cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and ADAM9-mediated pathway and the LPA-induced
MEK- and Rac-mediated pathway. Using Vero-H cells, we determined that
various stress-inducing and inflammatory stimuli also trigger
ectodomain shedding in a p38 MAPK-mediated manner. As this cascade does
not require the activation of MEK, Rac, PKC
, or ADAM9, it appears to
function independently of the TPA- and LPA-induced pathways. Thus, the
p38 MAPK-mediated pathway is a third signal cascade that can induce
pro-HB-EGF shedding in these cells.
and proneuregulin in Chinese hamster ovary cells
(56). Although stress activates both p38 MAPK and JNK, this and
previous studies (55-57) suggest that p38 MAPK, rather than JNK, is
the primary contributor to the stress-induced ectodomain shedding of a
number of membrane proteins. Inflammatory cytokines, including TNF-
,
IL-1
, and IL-6, also stimulate membrane protein shedding (58-60).
In Vero-H cells, however, neither ectodomain shedding nor p38 MAPK
activation was observed following treatment with either TNF-
or IL-6
(Figs. 1B and 3A). Although it is possible that
Vero-H cells do not bear the receptors ligating TNF-
and IL-6, it
appears that signaling pathways induced by TNF-
and IL-6 do not
contribute to pro-HB-EGF shedding.
-induced shedding requires
protease(s) abrogated by metalloprotease inhibitors. This result is in
agreement with data obtained for TPA- and LPA-induced shedding and
for additional shedding cascades of transmembrane molecules (61).
Therefore, a metalloprotease such as matrix metalloprotease or
ADAM that works in close proximity to the membrane may participate in
pro-HB-EGF shedding. The ADAM family metalloproteases contains a
conserved sequence (HEXXH) that is a putative zinc-binding motif within the catalytic domain. We previously constructed a mutant
form of ADAM9 (H347A,H351A, in which the conserved histidines are
replaced with alanines) that was found to inhibit TPA-induced pro-HB-EGF shedding (21). Thus, ADAM9 is involved in pro-HB-EGF shedding induced by treatment of Vero-H cells with TPA. In this study,
we constructed another dnADAM9 mutant (E348A) to examine its effect on
TPA-induced pro-HB-EGF shedding. As E348A exhibited a subcellular
localization similar to that of native ADAM9 when expressed in Vero-H
cells, E348A may be a more suitable dnADAM9 mutant than the H347A,H351A
mutant, which exhibited an altered localization pattern. Both
dominant-negative mutants inhibited TPA-induced shedding, confirming
the role of ADAM9 in TPA-induced shedding. Neither H347A, H351A nor
E348A inhibited LPA-, stress-, or IL-1
-induced pro-HB-EGF shedding
in Vero-H cells. From these results, we conclude that ADAM9 does not
participate in LPA-, stress-, or IL-1
-induced pro-HB-EGF shedding.
(9), and ADAM17/TACE (TNF-
converting enzyme) (63) function in pro-HB-EGF shedding in various cell
systems. We therefore constructed putative dnADAM10, dnADAM12, and
dnADAM17 mutants by substituting the conserved glutamic acid within the
catalytic domain with an alanine (Glu mutants). These mutants were
tested for their ability to inhibit stress- and IL-1
-induced
shedding in Vero-H cells. They did not prevent stress- and
IL-1
-induced pro-HB-EGF shedding in Vero-H
cells.2 Although we have not
confirmed the loss of catalytic activity by these constructed Glu
mutants, these results suggest that ADAM10, ADAM12, and ADAM17 are not
sheddases for stress- and IL-1
-induced pro-HB-EGF shedding in
Vero-H cells. Thus, the protease responsible for p38 MAPK-mediated
pro-HB-EGF shedding in Vero-H cells remained to be identified.
and TNF-
also markedly increase pro-HB-EGF gene expression (35). The
release of sHB-EGF from the membrane by ectodomain shedding is a
prerequisite for the mitogenic activity as a paracrine and autocrine
growth factor. Therefore, stress and inflammatory cytokines may also
induce pro-HB-EGF shedding in addition to their transcriptional
up-regulation. In response to stress and inflammation, HB-EGF activity
may be regulated at both the transcriptional and ectodomain shedding
levels. Considering the role of HB-EGF in tissue repair, the rapid
release of sHB-EGF following stress-inducing stimuli may facilitate the
repair of wounded tissues. These results also suggest that excess
release of sHB-EGF by continual exposure of tissues to stress and
inflammation may result in the pathological hyperplasia of cells such
as smooth muscle and cardiac cells.
![]() |
FOOTNOTES |
---|
* This work was supported by the Research for the Future Program of the Japan Society for the Promotion of Science and by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology (to E. M.).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.
¶ Present address: Radioisotope Research Center, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan.
To whom correspondence should be addressed. Tel.:
81-6-6879-8286; Fax: 81-6-6879-8289; E-mail:
emekada@biken.osaka-u.ac.jp.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M211835200
2 A. Yamazaki and E. Mekada, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
EGF, epidermal
growth factor;
HB-EGF, heparin-binding EGF-like growth factor;
sHB-EGF, soluble HB-EGF;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
PKC, protein kinase C;
dn, dominant-negative;
LPA, lysophosphatidic acid;
MAPK, mitogen-activated protein kinase;
MEK, MAPK/extracellular
signal-regulated kinase kinase;
MKK, MAPK kinase;
IL, interleukin;
TNF-, tumor necrosis factor-
;
GFP, green
fluorescent protein;
EGFP, enhanced GFP;
JNK, c-Jun N-terminal kinase;
SAPK, stress-activated protein kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Higashiyama, S., Abraham, J. A., Miller, J., Fiddes, J. C., and Klagsbrun, M. (1991) Science 251, 936-939[Medline] [Order article via Infotrieve] |
2. |
Elenius, K.,
Paul, S.,
Allison, G.,
Sun, J.,
and Klagsbrun, M.
(1997)
EMBO J.
16,
1268-1278 |
3. |
Higashiyama, S.,
Lau, K.,
Besner, G. E.,
Abraham, J. A.,
and Klagsbrun, M.
(1992)
J. Biol. Chem.
267,
6205-6212 |
4. | Raab, G., and Klagsbrun, M. (1997) Biochim. Biophys. Acta 1333, F179-F199[CrossRef][Medline] [Order article via Infotrieve] |
5. | Marikovsky, M., Breuing, K., Liu, P. Y., Eriksson, E., Higashiyama, S., Farber, P., Abraham, J., and Klagsbrun, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3889-3893[Abstract] |
6. |
Tokumaru, S.,
Higashiyama, S.,
Endo, T.,
Nakagawa, T.,
Miyagawa, J. I.,
Yamamori, K.,
Hanakawa, Y.,
Ohmoto, H.,
Yoshino, K.,
Shirakata, Y.,
Matsuzawa, Y.,
Hashimoto, K.,
and Taniguchi, N.
(2000)
J. Cell Biol.
151,
209-220 |
7. |
Takemura, T.,
Hino, S.,
Kuwajima, H.,
Yanagida, H.,
Okada, M.,
Nagata, M.,
Sasaki, S.,
Barasch, J.,
Harris, R. C.,
and Yoshioka, K.
(2001)
J. Am. Soc. Nephrol.
12,
964-972 |
8. |
Das, S. K.,
Wang, X. N.,
Paria, B. C.,
Damm, D.,
Abraham, J. A.,
Klagsbrun, M.,
Andrews, G. K.,
and Dey, S. K.
(1994)
Development
120,
1071-1083 |
9. | Asakura, M., Kitakaze, M., Takashima, S., Liao, Y., Ishikura, F., Yoshinaka, T., Ohmoto, H., Node, K., Yoshino, K., Ishiguro, H., Asanuma, H., Sanada, S., Matsumura, Y., Takeda, H., Beppu, S., Tada, M., Hori, M., and Higashiyama, S. (2002) Nat. Med. 8, 35-40[CrossRef][Medline] [Order article via Infotrieve] |
10. | Fujino, T., Hasebe, N., Fujita, M., Takeuchi, K., Kawabe, J., Tobise, K., Higashiyama, S., Taniguchi, N., and Kikuchi, K. (1998) Cardiovasc. Res. 38, 365-374[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Perrella, M. A.,
Maki, T.,
Prasad, S.,
Pimental, D.,
Singh, K.,
Takahashi, N.,
Yoshizumi, M.,
Alali, A.,
Higashiyama, S.,
and Kelly, R. A.
(1994)
J. Biol. Chem.
269,
27045-27050 |
12. | Miyagawa, J., Higashiyama, S., Kawata, S., Inui, Y., Tamura, S., Yamamoto, K., Nishida, M., Nakamura, T., Yamashita, S., Matsuzawa, Y., and Taniguchi, N. (1995) J. Clin. Invest. 95, 404-411[Medline] [Order article via Infotrieve] |
13. | Lemjabbar, H., and Basbaum, C. (2002) Nat. Med. 8, 41-46[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Fu, S.,
Bottoli, I.,
Goller, M.,
and Vogt, P. K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5716-5721 |
15. | Goishi, K., Higashiyama, S., Klagsbrun, M., Nakano, N., Umata, T., Ishikawa, M., Mekada, E., and Taniguchi, N. (1995) Mol. Biol. Cell 6, 967-980[Abstract] |
16. | Mitamura, T., Iwamoto, R., Umata, T., Yomo, T., Urabe, I., Tsuneoka, M., and Mekada, E. (1992) J. Cell Biol. 118, 1389-1399[Abstract] |
17. | Nakamura, K., Iwamoto, R., and Mekada, E. (1995) J. Cell Biol. 129, 1691-1705[Abstract] |
18. | Higashiyama, S., Iwamoto, R., Goishi, K., Raab, G., Taniguchi, N., Klagsbrun, M., and Mekada, E. (1995) J. Cell Biol. 128, 929-938[Abstract] |
19. |
Iwamoto, R.,
Handa, K.,
and Mekada, E.
(1999)
J. Biol. Chem.
274,
25906-25912 |
20. |
Arribas, J.,
Coodly, L.,
Vollmer, P.,
Kishimoto, T. K.,
Rose-John, S.,
and Massagué, J.
(1996)
J. Biol. Chem.
271,
11376-11382 |
21. |
Izumi, Y.,
Hirata, M.,
Hasuwa, H.,
Iwamoto, R.,
Umata, T.,
Miyado, K.,
Tamai, Y.,
Kurisaki, T.,
Sehara-Fujisawa, A.,
Ohno, S.,
and Mekada, E.
(1998)
EMBO J.
17,
7260-7272 |
22. | Weskamp, G., Kratzschmar, J., Reid, M. S., and Blobel, C. P. (1996) J. Cell Biol. 132, 717-726[Abstract] |
23. | Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., and Ullrich, A. (1999) Nature 402, 884-888[CrossRef][Medline] [Order article via Infotrieve] |
24. | Hirata, M., Umata, T., Takahashi, T., Ohnuma, M., Miura, Y., Iwamoto, R., and Mekada, E. (2001) Biochem. Biophys. Res. Commun. 283, 915-922[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Eguchi, S.,
Dempsey, P. J.,
Frank, G. D.,
Motley, E. D.,
and Inagami, T.
(2001)
J. Biol. Chem.
276,
7957-7962 |
26. |
Fujiyama, S.,
Matsubara, H.,
Nozawa, Y.,
Maruyama, K.,
Mori, Y.,
Tsutsumi, Y.,
Masaki, H.,
Uchiyama, Y.,
Koyama, Y.,
Nose, A.,
Iba, O.,
Tateishi, E.,
Ogata, N.,
Jyo, N.,
Higashiyama, S.,
and Iwasaka, T.
(2001)
Circ. Res.
88,
22-29 |
27. |
Umata, T.,
Hirata, M.,
Takahashi, T.,
Ryu, F.,
Shida, S.,
Takahashi, Y.,
Tsuneoka, M.,
Miura, Y.,
Masuda, M.,
Horiguchi, Y.,
and Mekada, E.
(2001)
J. Biol. Chem.
276,
30475-30482 |
28. | Blotnick, S., Peoples, G. E., Freeman, M. R., Eberlein, T. J., and Klagsbrun, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2890-2894[Abstract] |
29. | Koh, Y. H., Che, W., Higashiyama, S., Takahashi, M., Miyamoto, Y., Suzuki, K., and Taniguchi, N. (2001) J. Biochem. (Tokyo) 130, 351-358[Abstract] |
30. |
Fang, L.,
Li, G.,
Liu, G.,
Lee, S. W.,
and Aaronson, S. A.
(2001)
EMBO J.
20,
1931-1939 |
31. | Miyazaki, Y., Hiraoka, S., Tsutsui, S., Kitamura, S., Shinomura, Y., and Matsuzawa, Y. (2001) Gastroenterology 120, 108-116[Medline] [Order article via Infotrieve] |
32. | Sakai, M., Tsukada, T., and Harris, R. C. (2001) Exp. Nephrol. 9, 28-39[CrossRef][Medline] [Order article via Infotrieve] |
33. | Nguyen, H. T., Adam, R. M., Bride, S. H., Park, J. M., Peters, C. A., and Freeman, M. R. (2000) Am. J. Physiol. 279, C1155-C1167 |
34. | Morita, T., Yoshizumi, M., Kurihara, H., Maemura, K., Nagai, R., and Yazaki, Y. (1993) Biochem. Biophys. Res. Commun. 197, 256-262[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Yoshizumi, M.,
Kourembanas, S.,
Temizer, D. H.,
Cambria, R. P.,
Quertermous, T.,
and Lee, M. E.
(1992)
J. Biol. Chem.
267,
9467-9469 |
36. | Iwamoto, R., Higashiyama, S., Mitamura, T., Taniguchi, N., Klagsbrun, M., and Mekada, E. (1994) EMBO J. 13, 2322-2330[Abstract] |
37. | Hirai, S., Izumi, Y., Higa, K., Kaibuchi, K., Mizuno, K., Osada, S., Suzuki, K., and Ohno, S. (1994) EMBO J. 13, 2331-2340[Abstract] |
38. |
Ohno, S.,
Mizuno, K.,
Adachi, Y.,
Hata, A.,
Akita, Y.,
Akimoto, K.,
Osada, S.,
Hirai, S.,
and Suzuki, K.
(1994)
J. Biol. Chem.
269,
17495-17501 |
39. | Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037[Medline] [Order article via Infotrieve] |
40. |
Fujishiro, M.,
Gotoh, Y.,
Katagiri, H.,
Sakoda, H.,
Ogihara, T.,
Anai, M.,
Onishi, Y.,
Ono, H.,
Funaki, M.,
Inukai, K.,
Fukushima, Y.,
Kikuchi, M.,
Oka, Y.,
and Asano, T.
(2001)
J. Biol. Chem.
276,
19800-19806 |
41. | Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752[Medline] [Order article via Infotrieve] |
42. |
Kyriakis, J. M.,
and Avruch, J.
(2001)
Physiol. Rev.
81,
807-869 |
43. | Cano, E., Hazzalin, C. A., and Mahadevan, L. C. (1994) Mol. Cell. Biol. 14, 7352-7362[Abstract] |
44. | Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young, P. R. (1994) Nature 372, 739-746[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Han, J.,
Lee, J. D.,
Jiang, Y.,
Li, Z.,
Feng, L.,
and Ulevitch, R. J.
(1996)
J. Biol. Chem.
271,
2886-2891 |
46. |
Bennett, B. L.,
Sasaki, D. T.,
Murray, B. W.,
O'Leary, E. C.,
Sakata, S. T.,
Xu, W.,
Leisten, J. C.,
Motiwala, A.,
Pierce, S.,
Satoh, Y.,
Bhagwat, S. S.,
Manning, A. M.,
and Anderson, D. W.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
13681-13686 |
47. | Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689[Abstract] |
48. | Bradshaw, D., Hill, C. H., Nixon, J. S., and Wilkinson, S. E. (1993) Agent Actions 38, 135-147[Medline] [Order article via Infotrieve] |
49. | Blobel, C. P. (2000) Curr. Opin. Cell Biol. 12, 606-612[CrossRef][Medline] [Order article via Infotrieve] |
50. | Black, R. A., and White, J. M. (1998) Curr. Opin. Cell Biol. 10, 654-659[CrossRef][Medline] [Order article via Infotrieve] |
51. | Michalsky, M. P., Kuhn, A., Mehta, V., and Besner, G. E. (2001) J. Pediatr. Surg. 36, 1130-1135[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Igura, T.,
Kawata, S.,
Miyagawa, J.,
Inui, Y.,
Tamura, S.,
Fukuda, K.,
Isozaki, K.,
Yamamori, K.,
Taniguchi, N.,
Higashiyama, S.,
and Matsuzawa, Y.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
1524-1531 |
53. | Iwamoto, R., and Mekada, E. (2000) Cytokine Growth Factor Rev. 11, 335-344[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Gechtman, Z.,
Alonso, J. L.,
Raab, G.,
Ingber, D. E.,
and Klagsbrun, M.
(1999)
J. Biol. Chem.
274,
28828-28835 |
55. |
Rizoli, S. B.,
Rotstein, O. D.,
and Kapus, A.
(1999)
J. Biol. Chem.
274,
22072-22080 |
56. | Montero, J. C., Yuste, L., Diaz-Rodriguez, E., Esparis-Ogando, A., and Pandiella, A. (2002) Biochem. J. 363, 211-221[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Fan, H.,
and Derynck, R.
(1999)
EMBO J.
18,
6962-6972 |
58. |
Franzke, C. W.,
Tasanen, K.,
Schacke, H.,
Zhou, Z.,
Tryggvason, K.,
Mauch, C.,
Zigrino, P.,
Sunnarborg, S.,
Lee, D. C.,
Fahrenholz, F.,
and Bruckner-Tuderman, L.
(2002)
EMBO J.
21,
5026-5035 |
59. | Levine, S. J., Logun, C., Chopra, D. P., Rhim, J. S., and Shelhamer, J. H. (1996) Am. J. Respir. Cell Mol. Biol. 14, 254-261[Abstract] |
60. |
Yabkowitz, R.,
Meyer, S.,
Black, T.,
Elliott, G.,
Merewether, L. A.,
and Yamane, H. K.
(1999)
Blood
93,
1969-1979 |
61. | Hooper, N. M., Karran, E. H., and Turner, A. J. (1997) Biochem. J. 321, 265-279[Medline] [Order article via Infotrieve] |
62. |
Yan, Y.,
Shirakabe, K.,
and Werb, Z.
(2002)
J. Cell Biol.
158,
221-226 |
63. |
Sunnarborg, S. W.,
Hinkle, C. L.,
Stevenson, M.,
Russell, W. E.,
Raska, C. S.,
Peschon, J. J.,
Castner, B. J.,
Gerhart, M. J.,
Paxton, R. J.,
Black, R. A.,
and Lee, D. C.
(2002)
J. Biol. Chem.
277,
12838-12845 |