EGFR-induced cell migration is mediated predominantly by the JAK-STAT pathway in primary esophageal keratinocytes

Article (PDF Available)inAJP Gastrointestinal and Liver Physiology 287(6):G1227-37 · January 2005with50 Reads
DOI: 10.1152/ajpgi.00253.2004 · Source: PubMed
The epidermal growth factor receptor (EGFR) activates several signaling cascades in response to epidermal growth factor stimulation. One of these signaling events involves tyrosine phosphorylation of signal transducer and activator of transcription (STAT), whereas another involves activation of the phosphatidylinositol 3-OH kinase pathway. Two possibilities for STAT activation exist: a janus kinase (JAK)-dependent and a JAK-independent mechanism. Herein, we demonstrate that EGFR overexpression in primary esophageal keratinocytes activates STAT in a JAK-dependent fashion with the functional consequence of enhanced cell migration, which can be abolished by use of a JAK-specific inhibitor, AG-490. We determined the mechanisms underlying the signal transduction pathway responsible for increased cell migration. Stimulation of EGFR induces Tyr701 phosphorylation of STAT1 and initiates complex formation of STAT1 and STAT3 with JAK1 and JAK2. Thereafter, the STATs translocate to the nucleus within 15 min. In addition, we found that activation of this signaling pathway results in matrix metalloproteinase-1 (MMP-1) activity. By contrast, Akt activation does not impact the EGFR-STATs-JAKs complex formation and nuclear translocation of the STATs with subsequent MMP-1 activity, although Akt activation may contribute to cell migration through an independent mechanism. Taken together, we find that the recruitment of the STAT-JAK complex by EGFR is responsible for keratinocyte migration that, in turn, might be mediated by MMP-1 activation.
EGFR-induced cell migration is mediated predominantly by the JAK-STAT
pathway in primary esophageal keratinocytes
Claudia D. Andl, Takaaki Mizushima, Kenji Oyama, Mark Bowser, Hiroshi Nakagawa, and Anil K. Rustgi
Gastroenterology Division, Departments of Medicine and Genetics, Abramson Cancer Center and
Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Submitted 10 June 2004; accepted in final form 21 July 2004
Andl, Claudia D., Takaaki Mizushima, Kenji Oyama, Mark
Bowser, Hiroshi Nakagawa, and Anil K. Rustgi. EGFR-induced
cell migration is mediated predominantly by the JAK-STAT pathway
in primary esophageal keratinocytes. Am J Physiol Gastrointest Liver
Physiol 287: G1227–G1237, 2004. First published July 29, 2004;
doi:10.1152/ajpgi.00253.2004.—The epidermal growth factor receptor
(EGFR) activates several signaling cascades in response to epidermal
growth factor stimulation. One of these signaling events involves tyrosine
phosphorylation of signal transducer and activator of transcription
(STAT), whereas another involves activation of the phosphatidylinositol
3-OH kinase pathway. Two possibilities for STAT activation exist: a
janus kinase (JAK)-dependent and a JAK-independent mechanism.
Herein, we demonstrate that EGFR overexpression in primary esophageal
keratinocytes activates STAT in a JAK-dependent fashion with the
functional consequence of enhanced cell migration, which can be abol-
ished by use of a JAK-specific inhibitor, AG-490. We determined the
mechanisms underlying the signal transduction pathway responsible for
increased cell migration. Stimulation of EGFR induces Tyr701 phosphor-
ylation of STAT1 and initiates complex formation of STAT1 and STAT3
with JAK1 and JAK2. Thereafter, the STATs translocate to the nucleus
within 15 min. In addition, we found that activation of this signaling
pathway results in matrix metalloproteinase-1 (MMP-1) activity. By
contrast, Akt activation does not impact the EGFR-STATs-JAKs com-
plex formation and nuclear translocation of the STATs with subsequent
MMP-1 activity, although Akt activation may contribute to cell migration
through an independent mechanism. Taken together, we find that the
recruitment of the STAT-JAK complex by EGFR is responsible for
keratinocyte migration that, in turn, might be mediated by MMP-1
epidermal growth factor receptor; Akt; cell migration
(EGFR) is involved in diverse cell functions, including regu-
lation of proliferation, differentiation, cell survival, and motil-
ity, but also in processes that are crucial to cancer progression,
including angiogenesis and metastatic spread. These cellular
processes are mediated by a gamut of key molecules in over-
lapping and distinct signal transduction pathways.
Activation of signal transducer and activator of transcription
(STAT) occurs when tyrosine residues are phosphorylated by
janus kinase (JAK) that initiates dimer formation of STATs
(29). The dimers are then translocated into the nucleus, where
they are active in gene transcription. However, STAT activa-
tion can occur independently of JAKs, for example, as medi-
ated directly by EGFR kinase activity (10). Truncation of the
EGFR leads to loss of STAT activation, and specific docking
sites for STAT1 and STAT3 in the cytoplasmic domain of
EGFR have been identified as allowing positive and negative
regulation of STAT activity (39).
EGFR overexpression is found frequently in esophageal,
head and neck, and breast cancers. Induction of STAT signal-
ing has been shown to result from EGFR overexpression in
tumors of different origin. STAT3 activation has been shown
to increase cell proliferation in vitro and tumor growth rates in
vivo (21). This increased proliferation can be abolished by use
of dominant-negative mutants of STAT3 and anti-sense oligo-
nucleotides; however, suppression of STAT1 with the same
approach has no effect in other systems (15). In certain con-
texts, STAT1 has been postulated to be a potential tumor
suppressor gene because of its growth suppressor functions (5).
The effect of STAT1 on cell proliferation and tumorigenesis
still requires reconciliation, and the particular cell type and
tissue-specific context may be critical.
The phosphotidylinositol-3 kinase (PI3-kinase) pathway can
also induce activation of STATs through phosphorylation on
Ser727 in response to interferon (IFN)- stimulation. After
stimulation of the IFN receptor, PI3-kinase and Akt are acti-
vated. STAT is recruited by the activated JAKs and is phos-
phorylated on tyrosine, while a kinase downstream of Akt
phosphorylates Ser727 of STAT (28).
Akt has been known also to be a downstream target of EGFR
signaling. In addition, Akt is an essential element of the
PI3-kinase pathway regulating chemotaxis and motility. Ini-
tially, recruitment and activation of PI3-kinase at the leading
edge of migrating cells results in localized accumulation of
phosphatidylinositol 4,5-trisphosphate (PIP
) (25). PIP
as a docking site for a subclass of PH domain-containing
proteins. Dissection of this pathway in Dictyostelium amoebae
identified Akt as an effector required for cell motility (11).
Experiments in human squamous cancer cell lines support the
important role Akt plays in cell motility, revealing that constitu-
tively active Akt induces epithelial-mesenchymal transition leading
to a loss of cell adhesion and increased motility and invasiveness
(16). Furthermore, use of dominant-negative mutants of Akt
reverses Akt-dependent increased cell motility in fibroblasts (18).
That Akt not only increases cell motility but also promotes cancer
cell invasion has been shown by recruitment of PI3-kinase and
Akt to the leading edge of cells, where Akt kinase activity
increases matrix metalloproteinase (MMP)-9 production (22).
These observations are corroborated by the finding that Akt
activates MMP-2 (30).
This led us to investigate the involvement of the PI3-kinase-
Akt pathway in promoting cell migration and, possibly, regu-
Address for reprint requests and other correspondence: A. K. Rustgi, 600
CRB, Univ. of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104 (E-
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.
Am J Physiol Gastrointest Liver Physiol 287: G1227–G1237, 2004.
First published July 29, 2004; doi:10.1152/ajpgi.00253.2004.
0193-1857/04 $5.00 Copyright
2004 the American Physiological Society G1227
lating STAT activity. This line of investigation has uncovered
that the PI3-kinase-AKT pathway contributes partially to cell
migration and that the main contribution is through EGFR-
mediated recruitment of JAK1/2 to phosphorylate STAT1 and
STAT3. In turn, STAT1 and STAT3 form complexes and are
translocated to the nucleus with possible direct activation of
MMP-1 activity as a critical modulator of cell migration. As a
result, we have focused on the specific functional role of
JAK1/2 and STAT1 and STAT3 in mediating EGFR effects on
cell migration.
Cell lines. Primary esophageal keratinocytes, designated as EPC2,
from normal human esophagus were established and infected with
filtered (0.45-m pore size) retroviral supernatant from an overnight
culture of Phoenix-Ampho cells, producing the pFB-neo retroviruses
encoding EGFR or green fluorescent protein (GFP) as described (1).
EPC2 cells were grown at 37°C and 5% CO
with keratinocyte
serum-free media, with 40 g/ml bovine pituitary extract, 1.0 ng/ml
EGF, 100 U/ml penicillin, and 100 g/ml streptomycin.
When cells were starved, the media used was keratinocyte basic
medium (KBM) and supplemented with 0.5 g/ml hydrocortisone and
0.09 mM calcium chloride (BioWhittaker, Walkersville, MD). Cells
were starved for 48 h without EGF and then stimulated with 10 ng/ml
EGF for designated time periods at 37°C, washed three times with
ice-cold PBS, lysed, and centrifuged for 15 min at 4°C.
Pharmacological inhibitors were added during culture in the fol-
lowing concentrations: 100 nM AG-1478, an EGFR inhibitor; 50 M
AG-490, a JAK inhibitor; and the PI3-kinase inhibitors LY-294002 at
10 M and wortmannin at 50 nM, all purchased from Calbiochem (La
Jolla, CA); and 50 M Fludara, a STAT1 inhibitor (Berlex, CA).
Antibodies. An antibody against EGFR was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA), as were antibodies against
JAK1 and JAK2. Antibodies against phospho-EGFR (Y1173), Akt1,
phospho-STAT1 (Ser727), JAK1, and JAK2 were purchased from
Upstate Biotechnology (Lake Placid, NY). Antibodies specific for Akt
phosphorylated at Ser473, phospho-Tyr701 STAT1, and phospho-
Tyr705 STAT3 were obtained from Cell Signaling (Beverly, MA).
Antibodies against STAT1, STAT3, and PI3-kinase were obtained
from Transduction Laboratories (Lexington, KY). Anti-mouse and
-rabbit horseradish peroxidase-conjugated antibodies were purchased
from Amersham Pharmacia Biotech (Piscataway, NJ).
Immunoprecipitation. Preconfluent cells, starved in KBM and stim-
ulated with EGF (10 ng/ml) for 15 min with or without inhibitors,
were washed with PBS and incubated with 700 l of lysis buffer [1%
Triton X-100, 1% Nonidet P-40, 50 mM Tris, pH 8, and proteinase
inhibitors 2 g/ml Aprotinin, 1 mM phenylmethylsulfonyl fluoride,
10 mM NaF, 2 mM Na
, 5 mM Na-pyrophosphate] for 30 min on
ice. Seventy microliters of 4% BSA and 140 l of 1.5 M NaCl were
added to the extracts, which were then preabsorbed with 10 lof
rProteinG agarose (GIBCO, Gaithersburg, MD) for1hat4°C.
Preabsorbed extracts were incubated with antibodies against STAT1,
STAT3, JAK1, and JAK2. After a 1-h incubation at 4°C, the antigen-
antibody complex was incubated with 10 l of rProteinG agarose for
1 h at 4°C. The precipitates were washed three times with 1 ml of
wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100,
0.5 % deoxycholate, 0.1 % SDS) and boiled with 100 l of lithium
dodecyl sulfate (LDS) buffer (Invitrogen, Carlsbad, CA) containing
DTT for 10 min. Supernatants were used for Western blot analysis as
described in Western blot analysis. Experiments were performed in
Nuclear and cytoplasmic extracts. To generate whole cell lysates,
preconfluent cells, starved in KBM and stimulated with 10 ng/ml EGF
for the indicated time points, were washed with PBS and lysed in
harvest buffer (10 mM HEPES, pH 7.9, 50 mM NaCl, 0.5 M sucrose,
0.1 mM EDTA, 0.5 % Triton X-100, 100 mM DTT). Cytoplasmic/
nuclear extracts were prepared by dousing cells in buffer A (10 mM
HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
DTT) and sedimentation of the nuclei at 14,000 rpm for 15 min. The
supernatant containing the cytoplasmic/membrane extract was re-
moved, and the pellet was resuspended in buffer C (10 mM HEPES,
pH 7.9, 500 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% Nonidet
P-40, 1 mM DTT) to extract nuclear proteins.
Western blot analysis. Subconfluent cells were lysed in lysis buffer
(10 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1%
sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 2 mM sodium
orthovanadate, and a protease inhibitor mixture tablet, Roche Molec-
ular Biochemicals, Indianapolis, IN). All inhibitors were added to the
culture 12 h before harvesting to maintain the conditions employed for
the migration assays. Protein concentration was determined by protein
assay (Bio-Rad, Hercules, CA). The solution was subsequently solu-
bilized in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA)
containing 50 mM DTT. Total protein samples (10 g) were separated
on a 4 –12% SDS-PAGE and transferred to a polyvinylidene difluo-
ride membrane (Immobilon-P, Millipore, Bedford, MA). The mem-
brane was blocked in 5% nonfat milk (Bio-Rad) in TBS (10 mM Tris,
150 mM NaCl, pH 8.0, and 0.1% Tween 20) for 1 h at room
temperature. Membranes were probed with primary antibody diluted
1:1,000 in 5% milk in TBS overnight at 4°C, washed three times in
TBS-Tween 20, incubated with anti-mouse or anti-rabbit horseradish
peroxidase-conjugated antibody diluted 1:3,000 in TBS for1hat
room temperature, and then washed three times in TBS. The signal
was visualized by an enhanced chemiluminescence solution (ECL
Plus, Amersham Pharmacia Biotech) and was exposed to Kodak-X-
Omat LS film (Kodak, New York, NY). Experiments were repeated
Immunofluorescence. EPC2-GFP and EPC2-EGFR cells, cultured
in chamber slides (Nalge Nunc, Naperville, IL) in KBM starvation
medium with and without inhibitors, were stimulated with 10 ng/ml
EGF at the indicated time points. Cells were fixed in 4% paraformal-
dehyde (Fisher) for 10 min at room temperature. After fixation, cells
were treated with 0.1% Triton X-100 in PBS without calcium and
magnesium for 10 min. Objects were washed in PBS and blocked with
1% bovine serum albumin (Sigma, St. Louis, MO) for 1 h. Incubation
with primary antibodies was overnight at 4°C. After being washed
with PBS, cells were incubated with Texas Red-conjugated secondary
antibody (Molecular Probes, Eugene, OR) or Cy2-conjugated second-
ary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h.
Stained cells were examined with a Nikon microscope and imaged
with a digital camera at indicated magnifications.
Cell migration assay. Cell migration assays were performed using
24-well inserts (Falcon cell culture inserts, 8-m pore size, BD,
Franklin Lakes, NJ) according to the manufacturer’s instructions. In
brief, the lower chamber was filled with 0.6 ml of KBM containing 0.5
g/ml hydrocortisone with 10 ng/ml EGF, whereas a 0.5-ml cell
suspension in KBM under serum-starving conditions was plated in the
upper chamber in duplicate or triplicate wells and incubated at 37°C
for 12 h. For inhibition studies, inhibitors were added during the time
of incubation at the concentrations indicated above. Then, cells
attached to the upper side of the membrane were removed gently with
a cotton swab and rinsed. Cells that migrated through the membrane
and attached to the bottom of the membrane were fixed and stained
with reagents from the Diff Quik staining set (Dade Behring, Newark,
DE). Membranes were cut out and photographed such that migrated
cells could be counted. There was no evidence of cell death. All
experiments were performed at least three times in triplicate. Stu-
dent’s t-tests were performed, and P 0.01 was considered statisti-
cally significant.
Collagen dissolution assay. MMP-1 activity was measured by its
ability to dissolve type 1 collagen as described previously (2). In brief,
six-well cell culture plates were coated with 300 g/ml collagen type
1 (BD), 6 10
cells were applied to the center of the collagen matrix
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
and incubated for 3 h before the medium was added, and plates were
incubated for 2– 6 days. After removal of cells with 0.25% Trypsin
(GIBCO Life Technologies, Grand Island, NY), the collagen gel was
stained with 0.2% Coomassie for 30 min. All experiments were
performed at least three times in triplicate.
Real-time confocal microscopy. EPC2-GFP and EPC2-EGFR cells
infected with GFP-Grp1 PH adenovirus (gift of M. Birnbaum, Uni-
versity of Pennsylvania) were maintained in the TC3 open dish
system, which consists of individual 0.15-mm-thick tissue culture
dishes optimized for use with oil-immersion microscope objectives,
an objective heater, and a stage warmer to maintain the living cells at
37°C throughout the experiment (Bioptechs, Pittsburgh, PA). Images
were acquired as described previously (38) using an Ultraview LCI
Nipkow disc confocal microscope (Perkin-Elmer) attached to a Nikon
model TE300 inverted microscope fitted with a 60 oil-immersion
objective. The GFP-Grp1 PH was visualized using the 488-nm line of
an Argon laser, and the combination set of dichroic mirror (488 nm)
and emission barrier filter (cutoff at 510 nm) was optimized to collect
the GFP signal (Yokogaw). Images were collected at 7.5-s intervals
before and after the addition of 10 ng/ml EGF. Recruitment of
GFP-Grp1 PH to the plasma membrane was measured in response to
EGF stimulation by GFP-Grp1 PH translocation and in vivo PIP
JAK inhibitor AG-490 abolishes migration of EPC2-EGFR
cells. We wanted to investigate which signaling pathway,
PI3-kinase-Akt and/or JAK-STAT, may mediate the enhanced
cell migration in EGFR-overexpressing cells (EPC2-EGFR).
We used canonical PI3-kinase-Akt and JAK-STAT pathway
inhibitors to determine their effects on cell migration in EPC2-
EGFR cells. LY-294002 and wortmannin are two widely used
inhibitors of PI3-kinase. AG-490 is a JAK-specific inhibitor,
with a potential preference for JAK2 (26). Fludara is a reported
STAT1-specific inhibitor (12). The transwell-migration assay
is a useful tool that demonstrated increased migration of
EPC2-EGFR cells compared with GFP-expressing control cells
(Fig. 1). However, because GFP control cells also express
EGFR, it is expected that the inhibitors will have some effect
on GFP control cells. In this assay, the different inhibitors were
added during the total incubation time to test their effects on
cell migration of EPC2-EGFR cells. The JAK inhibitor was the
most potent, essentially abolishing migration. Importantly, ad-
dition of AG-490 had no effect on the viability of the cells.
They were not growth arrested and did not exhibit signs of
apoptosis (data not shown). Inhibitors of the PI3-kinase path-
way only reduced migration of EPC-EGFR cells by 50%
(Fig. 1), with different specificity between LY-294002 and
wortmannin. Fludara was not able to abolish migration to the
same extent as the JAK inhibitor but reduced cell migration to
about one-third (Fig. 1). This led us to postulate that the
JAK-STAT pathway may be preferred in regulating the in-
creased cell migration of EPC-EGFR cells.
PI3-kinase and JAK inhibitors suppress activity of down-
stream molecules in EPC-EGFR cells. To test the ability of the
various inhibitors to efficiently block PI3-kinase-Akt and JAK-
STAT signaling, we performed Western blot analysis using
cell extracts from EPC-EGFR cells after incubation with in-
hibitors for PI3-kinase, JAK, and STAT1 (Fig. 2). Our labo-
ratory has demonstrated previously (1) that EGFR overexpres-
sion induces phosphorylation of STAT1 after stimulation with
EGF when compared with GFP control cells. Akt phosphory-
lation on Ser473 is elevated in EGFR-overexpressing cells,
even in the absence of EGF, compared with GFP control cells
that show no baseline phosphorylation before EGF stimulation.
The use of the two different inhibitors of PI3-kinase activity,
10 M LY-294002 and 50 nM wortmannin, prevents phos-
phorylation of Akt on Ser473 in EPC-EGFR cells, which
should render Akt inactive. In addition, Fludara, a putative
STAT1-specific inhibitor decreases Akt Ser473 phosphoryla-
tion in a manner that is unknown, whereas total Akt levels
remain constant under the different conditions tested. Use of 50
M AG-490, the JAK inhibitor, prevents JAK phosphorylation
and phosphorylation of STAT1 on Tyr701 after EGF stimula-
tion. Total STAT1, JAK1, and JAK2 levels were used as
internal controls and remain essentially unchanged. Tyrosine
phosphorylation of STAT is believed to be mediated by ty-
rosine receptor kinases or JAKs, whereas serine phosphoryla-
tion is induced by serine/threonine kinases such as Akt in
INF- signaling. STAT1 is activated through phosphorylation
on Ser727 in response to EGF stimulation. LY-294002 and
wortmannin prevent phosphorylation of STAT1 on Ser727. In
addition, wortmannin specifically suppresses EGF-induced
phosphorylation of STAT1 on Tyr701. Fludara, although de-
scribed to be a STAT1-specific inhibitor (12) in lymphocytes
and used at the same concentration (50 M) in our study, does
not appear to be efficient in the suppression of STAT1 phos-
phorylation in our cell system, although it reduced cell migra-
tion in the transwell assay. With higher concentrations of
Fludara, we observed a dose-dependent decrease in STAT1
phosphorylation and further inhibition of cell migration (data
not shown), although it did not reach the same effectiveness as
described for lymphocytes.
Increased activity of PI3-kinase at the cell membrane in
EPC-EGFR cells as measured by PIP
production. In experi
ments with Dictyostelium and fibroblasts, it has been described
that PI3-kinase localizes to the leading edge where it recruits
Fig. 1. Janus kinase (JAK) inhibition abolishes cell migration. Migration
assays performed in transwell plates demonstrated that AG-490, a JAK-
specific inhibitor, abolishes migration of endothelial progenitor cell (EPC)-
epitheleal growth factor receptor (EGFR) cells as well as EPC-green fluores-
cent protein (GFP) control cells. Fludara, a signal transducer and activator
transcript (STAT) 1 inhibitor, partially reduces migration in both cell lines. At
the same time, inhibitors of the phosphotidylinositol 3-kinase (PI3-kinase)
pathway, LY-294002 and wortmannin, decreased migration by 50%. Inhibitors
were present during the 12-h incubation time at concentrations described in
MATERIALS AND METHODS. Student’s t-test was carried out comparing EPC-
EGFR cells treated with inhibitors vs. EPC-EGFR cells without inhibitors.
Similar comparisons were done with EPC-GFP control cells treated with
inhibitors vs. EPC-GFP cells without inhibitor. *Significantly different from
control (P 0.001).
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
Akt resulting in an amplification of the signal to migrate (11,
25). Furthermore, a role for JAK and STAT1 for these pro-
cesses in Drosophila could be demonstrated (3, 33). During
oocyte migration, STAT3 could be found directly at the cell
membrane where it colocalizes with PI3-kinase and Akt (20).
We wanted to investigate PI3-kinase activity in our cell system
with real-time confocal microscopy to determine whether the
PI3-kinase-Akt pathway could be regulating STAT activity or
whether there is even evidence for cross talk between the
PI3-kinase-Akt and JAK-STAT pathways.
Activation of PI3-kinase generates increased 3-phosphoi-
nositides at the cell membrane. PIP
and phosphatidylinositol
4,5-diphosphate bind to the PH domain of Akt and recruit it to
the plasma membrane. Use of an adenovirus encoding for the
grp1 PH domain and GFP enables us to measure PI3-kinase
activity in EGFR-overexpressing EPC cells compared with
parental cells (Fig. 3A). After transduction with 10 MOI
adenovirus expressing the GFPgrp1PH fusion protein
(Ad5GFP grp1PH), we starved the cells overnight and stimu-
lated them with 10 ng/ml EGF. Before stimulation, the GFP
signal can be detected in the cytoplasm and the nucleus, the
latter due to a cryptic nuclear localization signal. After stimu-
lation with EGF, the fusion protein is translocated to a greater
extent to the cell membrane in EGFR-overexpressing cells
(white arrows in Fig. 3B, and supplemental data at http://
Inhibition of the JAK-STAT pathway abolishes MMP-1 ac-
tivity. As previously shown by our laboratory (1), EGFR
overexpression in primary esophageal keratinocytes induces
upregulation of MMP-1 mRNA and enhanced secretion of
MMP-1 compared with GFP-expressing control cells. This
effect is specific to MMP-1 and not other MMPs. To link
MMP-1 overexpression in response to EGFR activation and
enhanced cell migration, we performed migration assays in the
presence of two different MMP inhibitors, designated MMP
inhibitor-II and MMP inhibitor-III (data not shown). The MMP
inhibitors inhibit a broad spectrum of MMPs with different
specificities and IC
. MMP inhibitor-III (suppresses MMP-1,
MMP-2, MMP-3, MMP-7, and MMP-13) appears more effec-
tive in suppressing MMP-1-mediated migration than MMP
inhibitor-II (suppresses MMP-1, MMP-3, MMP-7, and MMP-
9). Using different concentrations of MMP inhibitor-III, we
could demonstrate a dose-dependent decrease in migration,
although the level of reduced migration was modest (data not
shown). It is conceivable that the functional effects of MMP-1
activation may have been initiated already or progressed suf-
ficiently, such that pharmacological inhibition is only partial.
MMP-1 is a collagenase, and its specific activity can be
detected by plating cells on a type I collagen matrix (2).
However, MMP-1 cannot be detected using zymography. To
test our hypothesis that increased migration of EPC-EGFR
cells is due to MMP-1 activity, we used the collagen dissolu-
Fig. 2. Effects on EPC-EGFR cells with
PI3-kinase and JAK/STAT inhibitors.
Western blot analysis of EPC-EGFR cells
treated without inhibitor and with inhibitors
for 12 h at concentrations described in ex-
perimental procedures against the PI3-ki-
nase-Akt and JAK-STAT pathways were
performed to test their effectiveness in sup-
pressing activation of these targets. Phos-
phorylation of EGFR was not inhibited by
any of the inhibitors used. Wortmannin and
LY-294002 inhibitors prevent Akt phos-
phorylation at Ser473 as does Fludara, a
STAT1-specific inhibitor, whereas total Akt
levels remain unchanged. AG-490, a JAK-
specific inhibitor, and wortmannin decrease
STAT1 phosphorylation at Tyr701, whereas
wortmannin and LY-294002 prevent STAT1
phosphorylation on Ser727. Fludara seems
to have minimal effect on Tyr phosphoryla-
tion of STAT1. Total STAT1 expression
remains constant, and total JAK1 and JAK2
remain essentially unchanged. After the
membrane was probed with phosphotyrosine
antibody to determine the phosphorylation
status of JAK, the membrane was stripped of
signal, and total JAK levels were deter-
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
tion assay that enables us to assay MMP-1 activity. Analysis of
cells treated with inhibitors of different key pathways during
their growth on collagen matrices permits us to gain insights
into which signaling pathway may regulate MMP-1 activity.
After 5 days, the cells were removed by trypsin treatment and
the collagen was stained with Coomassie blue. In wells where
MMP-1 digested the collagen, white halos can be observed,
whereas in wells in which addition of the inhibitors prevented
collagen dissolution by MMP-1, the white halos were absent.
The addition of AG-490, the JAK inhibitor, prevents the
dissolution of collagen, confirming that MMP-1 activity is
completely abolished by the JAK inhibitor (Fig. 4). At the
same time, use of the EGFR inhibitor AG-1478 and the STAT1
inhibitor Fludara suppresses collagen dissolution by MMP-1.
EPC-EGFR cells without inhibitor and EPC-EGFR cells grow-
ing in the presence of PI3-kinase inhibitors reveal MMP-1
activity as detected by the presence of collagen dissolution
(Fig. 4, small black arrows). EPC-GFP cells do not exhibit
MMP-1 activity, demonstrating that MMP-1 activity is induced
specifically by EGFR overexpression. The suppression of
MMP-1 activity with JAK and STAT inhibitors leads us to
believe that the JAK-STAT pathway is crucial in regulating
MMP-1 activity and that the PI3-kinase-Akt pathway has little
or no effect on MMP-1-mediated cell migration.
EGFR activates STAT through complex formation with
JAK1 and JAK2. We hypothesized that EGFR leads to the
activation of STAT1, possibly mediated by JAKs, and thereby
induces a signaling cascade that results in MMP-1 activation.
To answer the question of whether EGFR interacts with STAT
or whether JAKs are involved in the activation of STAT, we
performed immunoprecipitations with antibodies against
STAT1, STAT3, JAK1, and JAK2. In IFN- signaling, acti-
Fig. 3. PI3-kinase/Akt activity at the cell
membrane in EPC-EGFR cells. Still photo-
graphs of real-time confocal microscopy
demonstrate stronger PIP
production at the
cell membrane of EGFR-overexpressing
cells compared with parental or control cells
(A) infected with the GFP-grp1 virus. After
EGF stimulation, the GFP signal increases at
the site of PI3-kinase activity (B; arrows).
Fig. 4. JAK and STAT are necessary for
matrix metalloproteinase (MMP)-1 activity.
EPC-GFP and EPC-EGFR cells are grown
on collagen matrices in the presence and
absence of inhibitors. EPC-GFP cells have
no MMP-1 activity. EPC-EGFR cells dem-
onstrate MMP-1 activity, as detected by col-
lagen dissolution or white halos (small black
arrows), which is not suppressed by PI3-
kinase inhibitors. AG-490, the JAK inhibi-
tor, Fludara, and AG-1478, the EGFR inhib-
itor, block collagen dissolution mediated by
MMP-1. wort, Wortmannin.
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
vation of JAK1 and JAK2 is known to phosphorylate STAT1
as well as STAT3 on tyrosine residues. We can show here that
EGFR forms a complex with STAT1 and STAT3 in EPC-
EGFR cells after stimulation with EGF (Fig. 5A, arrow). The
enhanced signal found for STAT1 and STAT3 after complex
formation induced by EGF stimulation could imply that there
is heterodimerization. Phosphorylation of STAT1 on Tyr701
and STAT3 on Tyr705 appears necessary, since the interaction
is only observed in EPC-EGFR cells after EGF stimulation.
Immunoprecipitation with antibodies against JAK1 and
JAK2 demonstrates that complex formation of JAK1 with
STAT1 and STAT3 occurs exclusively in EPC-EGFR-overex-
pressing cells after stimulation with EGF. Although EPC-
EGFR is present in a complex with JAK1 and JAK2 in GFP
control cells and before EGF stimulation, the recruitment of
STAT1 and STAT3 to this complex is only observed after EGF
stimulation in EPC-EGFR cells (Fig. 5B).
Inhibition of JAK activity suppresses EGFR-JAK-STAT
complex formation. To prove that the observed complex for-
mation is indeed initiated by EGFR and mediated by the
JAK-STAT pathway, we performed coimmunoprecipitations
in the presence of EGFR, JAK, and STAT1 inhibitors (Fig. 6).
The presence of JAK inhibitor AG-490 prevents complex
formation of EGFR and STAT1/STAT3. In addition, there is
loss of complex formation after use of the EGFR inhibitor
AG-1478, whereas the STAT1 inhibitor Fludara only reduces
complex formation partially (Fig. 6A). This shows that JAK is
essential in the recruitment of STATs to the EGFR-induced
complex formation, but inhibition of EGFR prevents complex
formation of STAT1 and STAT3. The JAK inhibitor and the
EGFR inhibitor prevent JAK1 and JAK2 tyrosine-phosphory-
lation, demonstrating that EGFR activity is necessary to acti-
vate JAK1 and JAK2 (Fig. 6B), whereas JAK phosphorylation,
in turn, is necessary to recruit STATs to the complex.
pSTAT1 and pSTAT3 translocate to the nucleus after EGF
stimulation. Phosphorylation of STATs induces dimer forma-
tion and initiates their translocation to the nucleus. To analyze
the kinetics of this event after EGF stimulation in our cell
system, we utilized cytoplasmic/nuclear extractions of EPC-
GFP and EPC-EGFR cells to document the translocation of the
STAT dimer (Fig. 7A). Even before stimulation with EGF,
EPC-EGFR cells have some basally activated STAT1 and
STAT3 in the cytoplasmic fraction when compared with EPC-
GFP cells that show no signal in Western blot analysis with
antibodies against pSTAT1 and pSTAT3 (Fig. 7A). After 5 min
of EGF stimulation, the total pool of activated STAT1 and
Fig. 5. A: complex formation of EGFR with
STAT1 and STAT3 after stimulation in
EPC-EGFR cells. Immunoprecipitation with
antibodies against STAT1 and STAT3 dem-
onstrates the presence of EGFR in a complex
with STAT1 and STAT3 and interaction
between STAT1 and STAT3 only after EGF
stimulation of EPC-EFGR cells. This com-
plex formation occurs as STAT1 is phos-
phorylated on Tyr701 and STAT3 is phos-
phorylated on Tyr705 after EGF stimulation
of EPC-EGFR cells. B: JAK1 and JAK2 are
found in the EGFR-STAT complex after
EGF stimulation in EPC-EGFR cells. Immu-
noprecipitation with JAK1 and JAK2 anti-
bodies shows weak complex formation with
EGFR in EPC-GFP cells as well as EPC-
EGFR cells. However, the complex forma-
tion is enhanced after stimulation with EGF
of EPC-EGFR cells, and STAT1 and STAT3
are increasingly coprecipitated in EGF stim-
ulated EPC-EGFR cells. Total JAK1 and
JAK2 levels remain essentially unchanged.
IP, immunoprecipitation.
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
STAT3 has translocated from the cytoplasm to the nucleus in
EPC-EGFR cells, whereas EPC-GFP cells have only a very
weak signal at this time point. The delayed translocation of
pSTAT1 and pSTAT3 in EPC-GFP cells results in a strong
signal in the nuclear fraction at 30 min. Although phosphory-
lated forms of STAT1 and STAT3 can be detected in the
nucleus of EPC-EGFR cells 5 min after stimulation, the stron-
gest signals are detected after 15 min, and there is sustained
activity up to 30 min (Figure 7B), indicating that EGFR
overexpression facilitates and hastens the nuclear translocation
of pSTAT1 and pSTAT3.
To further dissect the kinetics of the STAT1/STAT3 trans-
location in EGFR-overexpressing cells, we extracted cytoplas-
mic/nuclear fractions at time points of 5, 15, 30, and 60 min
(Fig. 7B). At 5 min, STAT1 and STAT3 are phosphorylated at
their respective tyrosine residues, and translocation to the
nucleus has already occurred. Translocation is complete after
15 min when the strongest signal for activated STAT1 and
STAT3 can be detected in the nucleus after 30 min of EGF
stimulation. At 60 min, the signal is very weak and the
activation cycle appears to have been completed. The parallel
kinetics of pSTAT1 and pSTAT3 and the data obtained with
the coimmunoprecipitation of STAT1 and STAT3 in the pres-
ence of EGFR lead us to conclude that STAT1 and STAT3
could form heterodimers.
To analyze the translocation of STATs in situ, EPC-GFP and
EPC-EGFR cells were starved overnight and stimulated with
EGF for the same time periods as indicated above. Cells were
fixed in paraformaldehyde, and double immunofluorescence
was performed to localize total STAT1, STAT3, and phosphor-
ylated forms of STATs (Fig. 8). Phosphorylated STATs are
detected by use of a Texas Red-conjugated antibody, and thus
appear as red signals, whereas total STATs are detected with a
green signal due to a Cy2-conjugated antibody. Before EGF
stimulation, anti-pSTAT1 and anti-pSTAT3 yield a diffuse
signal in GFP cells with little signal in the nucleus. EPC-EGFR
cells exhibit strong staining for the phosphorylated forms at the
cell membrane and in the cytoplasm with some staining in the
nucleus (data not shown). In accordance with the cytoplasmic/
nuclear extraction data, pSTAT1 and pSTAT3 can be localized
almost exclusively to the nucleus at 15 min after EGF stimu-
lation (Fig. 8). Stainings for total STAT1 and total STAT3
demonstrate similar levels in the cytoplasm and the nucleus.
Cell migration is a critical cellular function that is important
for normal cellular homeostasis but necessary for tumor cells to
invade through the extracellular matrix. Insights into underly-
ing molecular mechanisms have been gained through investi-
gation of lower organisms.
The PI3-kinase pathway has recently been implicated in cell
migration in Drosophila melanogaster and Dictyostelium
amoebae. In this context, cell migration is a response to
chemotactic signals. Exposure to a chemotactic gradient in-
duces activation of PI3-kinase and the accumulation of its lipid
product PIP
to the leading edge (13, 32, 37). Our observation
that Akt phosphorylation occurs in EPC-EGFR cells at a high
basal level without EGF stimulation, compared with GFP
control cells, led us to investigate the role of this particular
pathway in our model system. Real-time confocal microscopy
confirmed the enhanced PI3-kinase activity and demonstrated
increased PIP
production in EGFR-overexpressing cells com
pared with parental cells, as well as spiking and blebbing at the
cell membrane after EGF stimulation (see supplemental data at
However, experiments using inhibitors against the PI3-kinase-
Akt pathway led us to conclude that the PI3-kinase pathway,
although active in our cell system, has only a partial effect on
cell migration and no effect on MMP-1 activity. Furthermore,
in mammalian cells, the activation of the PI3-kinase-AKT
pathway leads to remodeling of the cytoskeleton, which in turn
influences cell migration through mechanisms independent of
MMP-1 activation (11).
Although we found STAT1 activation by Akt through phos-
phorylation on Ser727 in response to EGF stimulation as
observed also in the IFN- pathway (28), this activation did not
appear to be necessary for MMP-1 activation and modulation
of cell migration. This led us to focus upon the interaction of
EGFR with the JAK-STAT pathway. Observations by other
groups (9, 39) that the EGFR cytoplasmic tail contains docking
sites for STAT1 and can directly regulate STAT1 activity
prompted us to postulate that there is direct interaction of
EGFR with STAT1 and STAT3. In addition, experiments with
Fig. 6. A: inhibition of complex formation with AG-490. Coimmunoprecipi-
tation with antibodies against STAT demonstrates that, in the presence of the
JAK inhibitor AG-490 and the EGFR inhibitor AG-1478, complex formation
is abolished, whereas Fludara, the inhibitor for STAT1, reduces the complex
formation. B: inhibition of JAK1 and JAK2 tyrosine phosphorylation with
AG-490 and AG-1478. Immunoprecpiations of JAK1 and JAK2 show com-
parable levels of JAK1 and JAK2 with the different inhibitors. Tyrosine
phosphorylation is inhibited by the Jak inhibitor AG-490 and by the EGFR
inhibitor AG-1478 but not by Fludara, the STAT1 inhibitor.
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
EGFR-overexpressing cells defective in JAK1 and JAK2 have
revealed that EGFR-induced phosphorylation activates JAKs
but that JAKs are not necessary for STAT activation as
measured by c-fos induction (23). Instead, only a kinase-dead
mutant of EGFR abolished STAT activation consistent with a
JAK-independent pathway in which the intrinsic kinase do-
main of the EGFR is crucial (23). Earlier in vitro kinase studies
using EGFR, JAK1, JAK2, and STAT1 purified from insect
cells demonstrated STAT1 phosphorylation on Tyr701 by
JAK1 and JAK2 but not by a catalytic inactive mutant of
JAK2. However, EGFR alone could also activate STAT1 and
its DNA-binding activity in vitro (31). Tyrosine phosphoryla-
tion of JAK1 is not necessary for STAT activation by EGFR
and amphiregulin (10). By contrast, our data demonstrate the
novel finding that JAKs are essential to mediate interaction of
EGFR with STAT1 and STAT3. Complex formation of JAKs
with STAT1 and STAT3 initiates a signaling cascade that
involves tyrosine phosphorylation of STAT1 and possible
dimerization with STAT3, leading to translocation into the
nucleus after 15 min.
Given the fact that STAT1 plays a role in growth restraint
(7), whereas STAT3 is frequently constitutively activated in a
variety of cancers in response to EGFR and is sufficient for
cellular transformation (4, 8), questions have been raised by the
frequent coactivation of STAT1 and STAT3 by the same
ligand and the often observed STAT1-STAT3 heterodimer.
EGFR-dependent activation of STAT3, but not STAT1, has
been described for squamous cell carcinoma (15). By contrast,
we observed coactivation of STAT1 and STAT3 as a conse-
quence of EGFR-JAK complex formation with subsequent
translocation of STAT1/STAT3 from the cytoplasm to the cell
nucleus. These findings may point to a balancing effect of
STAT1 and STAT3 on each other (6). Indeed, the interplay
between STAT1 and STAT3 that is fostered by EGFR and the
JAKs may modulate cues to either reside in the native cellular
environment or initiate cellular migration. Examination of
Fig. 7. A: cytoplasmic fractions contain more STAT1/3 in
EPC-EGFR-overexpressing cells compared with EPC-GFP
cells. Western blot analysis of cytoplasmic (cyto) and nuclear
(nucl) fractions from EPC-GFP and EPC-EGFR cells were
performed using anti-STAT1 and anti-STAT3 antibodies to
detect total STAT, and anti-phospho-STAT1 and anti-phospho-
STAT3 antibodies to analyze activated STAT1 and STAT3 in
their respective fractions. At time 0, STAT1 and STAT3 are
found in the cytoplasm with almost no proteins detected in the
nuclear fraction. EPC-EGFR cells not only have more STAT1
and STAT3 in the cytoplasmic fraction before stimulation with
EGF than GFP cells, but also pSTAT1 and pSTAT3 antibodies
demonstrate that the STATs are activated. After 5 min of EGF
stimulation, the amount of total STAT1 and STAT3 in the
cytoplasmic fraction is equal to the nuclear fraction, but a shift
of activated STATs from the cytoplasmic to the nuclear fraction
occurred in EPC-EGFR cells. After 30 min of EGF stimulation,
pSTAT1 and pSTAT3 can now be detected in the nuclear
fraction of EPC-GFP cells, demonstrating a delayed response to
EGF stimulation. Lamin B serves as a control for fractionation
of the cytoplasmic and nuclear extracts. B: phosphorylated
STAT dimers translocate to the nucleus after EGF stimulation.
Western blot analysis of cytoplasmic and nuclear fractions of
EPC-EGFR cells were stimulated with EGF for 5, 15, 30, and
60 min. Although the total STAT1 and STAT3 amount stays
constant from 5 to 30 min after stimulation and is equal in both
fractions, their levels decrease at 60 min in the nuclear fraction.
Activated STAT1 and STAT3 are detected by anti-phospho-
STAT1 and anti-phospho-STAT3 antibodies and are translo-
cated to the nucleus 15 min after stimulation and with sustained
activity for 30 min after EGF stimulation. Thereafter, the signal
for the activated STATs decreases.
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
downstream genes regulated by each STAT will be important
in evaluating such a possibility.
A role of STATs in cell migration is described in lower
organisms such as Drosophila (14), namely border cell migra-
tion during oogenesis (3), and Dictyostelium (20, 27). How-
ever, how the STATs are involved in cell migration in mam-
malian cell migration remains to be elucidated. STAT1 has
been identified as a downstream target of focal adhesion
kinase, and inactivation of focal adhesion kinase abolished
STAT1 activation, correlating with decreased migration (40).
In addition, depletion of STAT1 resulted in enhanced cell
adhesion and a decrease in cell migration. Phosphorylation of
STAT3 in response to VEGFR stimulation results in nuclear
translocation and induction of endothelial cell migration (41).
A dominant-negative mutant of STAT3 not only abolished
nuclear translocation but inhibited endothelial migration com-
pletely (41).
The STAT COOH terminus contains an autonomously func-
tioning transcriptional activation domain, and alternatively
spliced isoforms of STAT1, STAT3, and STAT4 lacking this
domain have attenuated transcriptional activity. Factors shown
to bind to STATs include CBP/p300, c-Jun, MCM5, and
BRCA-1 (19). STATs also interact with a wide variety of other
factors, such as nuclear factor-B, surfactant protein-1, and
SMAD-1. Analysis of the rabbit MMP-1 promoter identified a
STAT-binding site in close proximity to the TATA box (36)
that upregulates MMP-1 transcription as a result of v-src
signaling through STAT. This may lead to activation of the
human MMP-1 promoter by STATs.
In mammalian cells, MMPs modulate cell migration through
complicated interactions with components of the extracellular
matrix, which provides a platform for tumor cell invasion.
Unlike classical oncogenes, MMPs are generally not upregu-
lated by gene amplification or activating mutations. The only
Fig. 8. Double immunofluorescence of EPC-
EGFR cells fixed with paraformaldehyde 15
min after EGF stimulation. Cells were
stained with antibodies against total STAT1
and STAT3 (green), and the activated forms
were detected with antibodies against phos-
pho-STAT1 and phospho-STAT3 (red). Af-
ter 15 min of EGF stimulation, the activated
forms of pSTAT1 and pSTAT3 are localized
to the nucleus in EPC-EGFR cells, whereas
total STAT1 and STAT3 are equally distrib-
uted between the cytoplasm and nucleus.
4,6-diamidino-2-phenylindole (DAPI; blue)
serves as a counter stain.
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
two reported alterations in cancer cells are translocation of
MMP23 gene in neurobastoma (17) and amplification of the
MMP24 gene (24). Thus increased MMP expression in tumors
is likely due to transcriptional induction rather than genetic
alterations. Transcription of MMP-1 and MMP-13 has been
demonstrated to be repressed by the p53 tumor suppressor gene
(34, 35). Targeting extracellular factors or their cognate cell-
surface receptors, signal transduction pathways, and nuclear
factors that activate expression of these genes can inhibit MMP
gene transcription. A greater understanding of regulatory
mechanisms that control MMP transcription and activation
provides new avenues for therapeutic intervention. To that end,
abrogation of STAT activation may be a meaningful venue in
which to abrogate MMP-1-mediated cell migration and tumor
cell invasion.
In summary, we demonstrate in a novel fashion that JAK1
and JAK2 are necessary for activation of STAT1 and STAT3
in response to EGFR overexpression and that this signaling
pathway has a novel regulatory function for cell migration and
is correlated with MMP-1 activity, whereas the PI3-kinase-Akt
pathway is not involved directly and may modulate migration
through independent mechanisms.
We thank Drs. Meenhard Herlyn, Wafik El-Deiry, Hideki Harada, Therese
Deramaudt, and Ben Rhoades for discussions and Cameron Johnstone for the
statistical analysis. We are grateful for reagents provided by Dr. Morris
This work was supported by National Institutes of Health Grants PO1-
CA-098101 (to A. K. Rustgi, C. D. Andl, K. Oyama, and H. Nakagawa) and
R21-DK-64249 (to H. Nakagawa), an AGA/FDHN Research Scholar Award
(to H. Nakagawa), NRSA Award (to C. D. Andl), and Center for Molecular
Studies in Digestive and Liver Diseases (P30-DK-050306) and its Morphol-
ogy, Molecular Biology, and Cell Culture Cores.
1. Andl CD, Mizushima T, Nakagawa H, Oyama K, Harada H, Chruma
K, Herlyn M, and Rustgi AK. Epidermal growth factor mediates in-
creased cell proliferation, migration and aggregation in esophageal kera-
tinocytes in vitro and in vivo. J Biol Chem 278: 1824 –1830, 2003.
2. Aznavoorian S, Moore BA, Alexander-Lister LD, Hallit SL, Windsor
LJ, and Engler JA. Membrane type I-matrix metalloproteinase-mediated
degradation of type I collagen by oral squamous cell carcinoma cells.
Cancer Res 61: 6264 6275, 2001.
3. Beccari S, Teixeira L, and Rorth P. The JAK/STAT pathway is required
for border cell migration during Drosophila oogenesis. Mech Dev 111:
115–123, 2002.
4. Berclaz G, Altermatt HJ, Rohrbach V, Siragusa A, Dreher E, and
Smith PD. EGFR dependent expression of STAT3 (but not STAT1) in
breast cancer. Int J Oncol 19: 1155–1160, 2001.
5. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 109: 1139
1142, 2002.
6. Bromberg J and Darnell JE. The role of STATs in transcriptional
control and their impact on cellular function. Oncogene 19: 2468 –2478,
7. Bromberg JF, Fan Z, Brown C, Mendelsohn J, and Darnell JE Jr.
Epidermal growth factor-induced growth inhibition requires Stat1 activa-
tion. Cell Growth Differ 9: 505–512, 1998.
8. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestelll RG,
Albanese C, and Darnell JE Jr. Stat3 as an oncogene. Cell 98: 295–303,
9. Coffer PJ and Kruijer W. EGF receptor deletions define a region
specifically mediating STAT transcription factor activation. Biochem Bio-
phys Res Commun 210: 74 81, 1995.
10. David M, Wong L, Flavell R, Thompson SA, Wells A, Larner AC, and
Johnson GR. STAT activation by epidermal growth factor (EGF) and
amphiregulin. Requirement for the EGF receptor kinase, but not for
tyrosine phosphorylation sites or JAK1. J Biol Chem 271: 9185–9188,
11. Firtel RA and Chung CY. The molecular genetics of chemotaxis: sensing
and responding to chemoattractant gradients. Bioessays 22: 603– 615,
12. Frank DA, Mahajan S, and Ritz J. Fludarabine-induced immunosup-
pression is associated with inhibition of STAT1 signaling. Nat Med 5:
444 447, 1999.
13. Funamoto S, Meili R, Lee S, Parry L, and Firtel RA. Spatial and
temporal regulation of 3-phosphoinositides by PI3-kinase and PTEN
mediates chemotaxis. Cell 109: 611– 623, 2002.
14. Ghiglione C, Devergne O, Georgenthum E, Carballes F, Medioni C,
Cerezo D, and Noselli S. The Drosophila cytokine receptor Domeless
controls border cell migration and epithelial polarization during oogenesis.
Development 129: 5437–5447, 2002.
15. Grandis JR, Drenning SD, Chakraborty A, Zhou MY, Zeng Q, Pitt
AS, and Tweardy DJ. Requirement of Stat3 but not Stat1 activation for
epidermal growth factor receptor-mediated cell growth in vitro. J Clin
Invest 102: 1385–1392, 1998.
16. Grille SJ, Bellacosa A, Upson J, Klein-Szanto AJ, van Roy F, Lee-
Kwon W, Donowitz M, Tsichlis PN, and Larue L. The protein kinase
Akt induces epithelial mesenchymal transition and promotes enhanced
motility and invasiveness of squamous cell carcinoma lines. Cancer Res
63: 2172–2178, 2003.
17. Gururajan R, Lahti JM, Grenet J, Easton J, Gruber I, Ambros PF,
and Kidd VJ. Duplication of a genomic region containing the Cdc2L1–2
and MMP21–22 genes on human chromosome 1p36.3 and their linkage to
D1Z2. Genome Res 8: 929 –939, 1999.
18. Higuchi M, Masuyama N, Fukui Y, Suzuki A, and Gotoh Y. Akt
mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated
cells and in invasive PTEN knockout cells. Curr Biol 11: 1958–1962,
19. Horvath CM. STAT proteins and transcriptional responses to extracellu-
lar signals. Trends Biochem Sci 25: 496 450, 2000.
20. Hou SX, Zheng Z, Chen X, and Perrimon N. The Jak/STAT pathway in
model organisms: emerging roles in cell movement. Dev Cell 3: 765–778,
21. Kijima T, Niwa H, Steinman RA, Drenning SD, Gooding WE, Went-
zel AL, Xi S, and Grandis JR. STAT3 activation abrogates growth factor
dependence and contributes to head and neck squamous cell carcinoma
tumor growth in vivo. Cell Growth Differ 13: 355–362, 2002.
22. Kim D, Kim S, Koh H, Yoon S, Chung A, Cho KS, and Chung J.
Akt/PKB promotes cancer cell invasion via increased motility and metal-
loproteinase production. FASEB J 15: 1953–1962, 2001.
23. Leaman DW, Pisharody S, Flickinger TW, Commane MA, Schless-
inger J, Kerr IM, Levy DE, and Stark GR. Roles of JAKs in activation
of STATs and stimulation of c-fos gene expression by epidermal growth
factor. Mol Cell Biol 16: 369 –375, 1996.
24. Llano E, Pendas AM, Freije JP, Nakano A, Knauper V, Murphy G,
and Lopez-Otin C. Identification and characterization of human MT5-
MMP, a new membrane-bound activator of progelatinase is overexpressed
in brain tumors. Cancer Res 59: 2570 –2576, 1999.
25. Merlot S and Firtel RA. Leading the way: directional sensing through
phosphatidylinositol 3-kinase and other signaling pathways. J Cell Sci
116: 3471–3478, 2003.
26. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z,
Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, and Roifman
CM. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor.
Nature 379: 645– 648, 1996.
27. Mohanty S, Jermyn KA, Early A, Kawata T, Aubry L, Ceccarelli A,
Schaap P, Williams JG, and Firtel RA. Evidence that the Dictyostelium
Dd-STATa protein is a repressor that regulates commitment to stalk cell
differentiation and is also required for efficient chemotaxis. Development
126: 3391–3405, 1999.
28. Nguyen H, Ramana CV, Bayes J, and Stark GR. Roles of phosphati-
dylinositol 3-kinase in interferon--dependent phosphorylation of STAT1
on serine 727 and activation of gene expression. J Biol Chem 276:
33361–33368, 2001.
29. O’Shea JJ, Gadina M, and Schreiber RD. Cytokine signaling in 2002:
new surprises in the Jak/Stat pathway. Cell 109: 121–131, 2002.
30. Park BK, Zeng X, and Glazer RI. Akt1 induces extracellular matrix
invasion and matrix metalloproteinase-2 activity in mouse mammary
epithelial cells. Cancer Res 61: 7647–7653, 2001.
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
31. Quelle FW, Thierfelder W, Witthuhn BA, Tang B, Cohen S, and Ihle
JN. Phosphorylation and activation of the DNA binding activity of
purified Stat1 by the Janus protein-tyrosine kinases and the epidermal
growth factor receptor. J Biol Chem 270: 20775–20780, 1995.
32. Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, and Bourne
HR. Polarization of chemoattractant receptor signaling during neutrophil
chemotaxis. Science 287: 1037–1040, 2000.
33. Silver DL and Montell DJ. Paracrine signaling through the JAK/STAT
pathway activates invasive behavior of ovarian epithelial cells in Dro-
sophila. Cell 107: 831– 841, 2001.
34. Sun Y, Cheung JM, Martel-Pelletier J, Pelletier JP, Wenger L,
Altman RD, Howell DS, and Cheung HS. Wild type and mutant p53
differentially regulate the gene expression of human collagenase-3
(hMMP-13). J Biol Chem 275: 11327–11323, 2000.
35. Sun Y, Sun Y, Wenger L, Rutter JL, Brinckerhoff CE, and Cheung
HS. p53 Down-regulates human matrix metalloproteinase-1 (collage-
nase-1) gene expression. J Biol Chem 274: 11535–11540, 1999.
36. Vincenti MP, Schroen DJ, Coon CI, and Brinckerhoff CE. v-src
Activation of the collagenase-1 (matrix metalloproteinase-1) promoter
through PEA3 and STAT: requirement of extracellular signal-regulated
kinases and inhibition by retinoic acid receptors. Mol Carcinog 21:
194 –204, 1998.
37. Weiner OD. Regulation of cell polarity during eukaryotic chemotaxis: the
chemotactic compass. Curr Opin Cell Biol 14: 196 –202, 2002.
38. Whiteman EL, Chen JJ, and Birnbaum MJ. Platelet-derived growth
factor (PDGF) stimulates glucose transport in 3T3–L1 adipocytes over-
expressing PDGF receptor by a pathway independent of insulin receptor
substrates. Endocrinology 144: 3811–3820, 2003.
39. Xia L, Wang L, Chung AS, Ivanov SS, Ling MY, Dragoi AM, Platt A,
Gilmer TM, Fu XY, and Chin YE. Identification of both positive and
negative domains within the epidermal growth factor receptor COOH-
terminal region for signal transducer and activator of transcription (STAT)
activation. J Biol Chem 277: 30716 –30723, 2002.
40. Xie B, Zhao J, Kitagawa M, Durbin J, Madri JA, Guan JL, and Fu
XY. Focal adhesion kinase activates Stat1 in integrin-mediated cell mi-
gration and adhesion. J Biol Chem 276: 19512–19523, 2001.
41. Yahata Y, Shirakata Y, Tokumaru S, Yamasaki K, Sayama K,
Hanakawa Y, Detmar M, and Hashimoto K. Nuclear translocation of
phosphorylated STAT3 is essential for VEGF-induced human dermal
microvascular endothelial cell migration and tube formation. J Biol Chem
278: 40026 40031, 2003.
AJP-Gastrointest Liver Physiol VOL 287 DECEMBER 2004
    • "The solutions found highlight cross-talk among enriched pathways, mainly among the JAK/STAT signalling pathway, EGF receptor signalling pathway, Gonadotropin releasing hormone receptor pathway and p38 MAPK pathway (see Additional file 2: Figures S9, S10 and S11). The EGF receptor signalling pathway acts by phosphorylating the Janus kinases (JAK) resulting in the activation of Signal Transducer and Activator of Transcription proteins (STATs) and plays a role in regulating inflammation, in particular during colitis [24, 25]. Although the exact role of STAT3 in the pathogenesis of CD is not understood, mice with tissue-specific disruption of Stat3 show CD-like pathogenesis and constitutively phosphorylated STAT3 is found in intestinal T cells from patients with CD. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Inflammatory bowel disease (IBD) consists of two main disease-subtypes, Crohn’s disease (CD) and ulcerative colitis (UC); these subtypes share overlapping genetic and clinical features. Genome-wide microarray data enable unbiased documentation of alterations in gene expression that may be disease-specific. As genetic diseases are believed to be caused by genetic alterations affecting the function of signalling pathways, module-centric optimisation algorithms, whose aim is to identify sub-networks that are dys-regulated in disease, are emerging as promising approaches. Results In order to account for the topological structure of molecular interaction networks, we developed an optimisation algorithm that integrates databases of known molecular interactions with gene expression data; such integration enables identification of differentially regulated network modules. We verified the performance of our algorithm by testing it on simulated networks; we then applied the same method to study experimental data derived from microarray analysis of CD and UC biopsies and human interactome databases. This analysis allowed the extraction of dys-regulated subnetworks under different experimental conditions (inflamed and uninflamed tissues in CD and UC). Optimisation was performed to highlight differentially expressed network modules that may be common or specific to the disease subtype. Conclusions We show that the selected subnetworks include genes and pathways of known relevance for IBD; in particular, the solutions found highlight cross-talk among enriched pathways, mainly the JAK/STAT signalling pathway and the EGF receptor signalling pathway. In addition, integration of gene expression with molecular interaction data highlights nodes that, although not being differentially expressed, interact with differentially expressed nodes and are part of pathways that are relevant to IBD. The method proposed here may help identifying dys-regulated sub-networks that are common in different diseases and sub-networks whose dys-regulation is specific to a particular disease. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-0886-z) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • "Furthermore, one of the signaling events implicates tyrosine phosphorylation and activation of STAT3 protein [82]. This is in line with the previous demonstration that EGFR-induced cell migration is mediated predominantly by the STAT-pathway in keratinocytes and that STAT3 plays an essential role for skin remodeling and wound healing [82,83]. Esculentin-1a(1-21)NH 2 Stimulates Migration of Keratinocytes PLOS ONE | DOI:10.1371/journal.pone.0128663 "
    [Show abstract] [Hide abstract] ABSTRACT: One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025-4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.
    Full-text · Article · Jun 2015
    • "First, we aimed to identify the central mechanism through which pan-EGFR inhibitors inhibits MUC4 protein expression in pancreatic cancer cells. It has been previously shown that EGFR signaling can directly activate STAT and mediate cell migration in esophageal cancer keratinocyte cells [31]. STAT phosphorylation at serine 727 is essential for the transcriptional activity of different genes in cancer cells [32]. "
    [Show abstract] [Hide abstract] ABSTRACT: Transmembrane proteins MUC4, EGFR and HER2 are shown to be critical in invasion and metastasis of pancreatic cancer. Besides, we and others have demonstrated de novo expression of MUC4 in ~70-90% of pancreatic cancer patients and its stabilizing effects on HER2 downstream signaling in pancreatic cancer. Here, we found that use of canertinib or afatinib resulted in reduction of MUC4 and abrogation of in vitro and in vivo oncogenic functions of MUC4 in pancreatic cancer cells. Notably, silencing of EGFR family member in pancreatic cancer cells decreased MUC4 expression through reduced phospho-STAT1. Furthermore, canertinib and afatinib treatment also inhibited proliferation, migration and survival of pancreatic cancer cells by attenuation of signaling events including pERK1/2 (T202/Y204), cyclin D1, cyclin A, pFAK (Y925) and pAKT (Ser473). Using in vivo bioluminescent imaging, we demonstrated that canertinib treatment significantly reduced tumor burden (P=0.0164) and metastasis to various organs. Further, reduced expression of MUC4 and EGFR family members were confirmed in xenografts. Our results for the first time demonstrated the targeting of EGFR family members along with MUC4 by using pan-EGFR inhibitors. In conclusion, our studies will enhance the translational acquaintance of pan-EGFR inhibitors for combinational therapies to combat against lethal pancreatic cancer.
    Full-text · Article · Dec 2014
Show more