Keratinocyte Collagenase-1 Expression Requires an Epidermal
Growth Factor Receptor Autocrine Mechanism*
Brian K.
Pilcher
§,
JoAnn
Dumin
,
Michael J.
Schwartz
,
Bruce A.
Mast¶,
Gregory S.
Schultz
,
William C.
Parks
**, and
Howard G.
Welgus
From the
Division of Dermatology, Department of
Medicine, and ** Department of Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
Departments of
Obstetrics/Gynecology and ¶ Surgery,
University of Florida Health Sciences Center,
Gainesville, Florida 32610
 |
ABSTRACT |
In response to cutaneous injury,
expression of collagenase-1 is induced in keratinocytes via
2
1 contact with native type I
collagen, and enzyme activity is essential for cell migration over this
substratum. However, the cellular mechanism(s) mediating integrin
signaling remain poorly understood. We demonstrate here that treatment
of keratinocytes cultured on type I collagen with epidermal growth
factor receptor (EGFR) blocking antibodies or a specific receptor
antagonist inhibited cell migration across type I collagen and the
matrix-directed stimulation of collagenase-1 production. Additionally,
stimulation of collagenase-1 expression by hepatocyte growth factor,
transforming growth factor-
1, and interferon-
was blocked by EGFR
inhibitors, suggesting a required EGFR autocrine signaling step for
enzyme expression. Collagenase-1 mRNA was not detectable in
keratinocytes isolated immediately from normal skin, but increased
progressively following 2 h of contact with collagen. In contrast,
EGFR mRNA was expressed at high steady-state levels in
keratinocytes isolated immediately from intact skin but was absent
following 2 h cell contact with collagen, suggesting
down-regulation following receptor activation. Indeed, tyrosine
phosphorylation of the EGFR was evident as early as 10 min following
cell contact with collagen. Treatment of keratinocytes cultured on
collagen with EGFR antagonist or heparin-binding (HB)-EGF neutralizing
antibodies dramatically inhibited the sustained expression (6-24 h) of
collagenase-1 mRNA, whereas initial induction by collagen alone (2 h) was unaffected. Finally, expression of collagenase-1 in ex
vivo wounded skin and re-epithelialization of partial thickness porcine burn wounds was blocked following treatment with EGFR inhibitors. These results demonstrate that keratinocyte contact with
type I collagen is sufficient to induce collagenase-1 expression, whereas sustained enzyme production requires autocrine EGFR activation by HB-EGF as an obligatory intermediate step, thereby maintaining collagenase-1-dependent migration during the
re-epithelialization of epidermal wounds.
 |
INTRODUCTION |
Efficient repair of a cutaneous wound requires a programmed series
of spatially and temporally regulated events. Among these, effective
proteolytic degradation of extracellular matrix
(ECM)1 macromolecules is
thought to be necessary to remodel the damaged tissue, promote
neovascularization, and facilitate efficient migration of cells during
re-epithelialization (1). Matrix metalloproteinases (MMPs) constitute a
family of zinc-dependent enzymes with the collective
capacity to degrade virtually all ECM components (2). Although most
MMPs can degrade many ECM proteins with overlapping substrate
specificities, degradation of fibrillar type I collagen is initiated
only by the catalytic activity of collagenases, a subgroup of the MMP
gene family.
Previous studies from our group and others have shown that, in both
normally healing wounds and chronic ulcers, basal keratinocytes at the
leading edge of re-epithelialization invariantly express collagenase-1
(3-6). Collagenase-1 expression is rapidly induced in wound-edge
keratinocytes after injury, persists during the healing phase, and
ceases following complete re-epithelialization (7, 8). We demonstrated
that induction of collagenase-1 by basal keratinocytes is mediated via
2
1 interaction with native type I
collagen (9, 10), requires tyrosine kinase activity (11), and that the
activity of this MMP is essential for cell migration over this matrix
protein (10). Although much is known about the role of cell:matrix
interactions regulating collagenase-1 expression by keratinocytes, the
signaling mechanism(s) following collagen binding and integrin receptor
occupancy remain poorly understood.
The epidermal growth factor receptor (EGFR; c-erbB1/HER1) is a
transmembrane cell-surface tyrosine kinase that, upon ligand binding,
phosphorylates downstream effector molecules, leading to changes in
cell function (12). EGFR-null mice demonstrate involvement of the
receptor in a broad range of developmental processes, and these mice
have pronounced defects in epithelial cell proliferation and
differentiation (13-15). Activation of the EGFR by members of the EGF
family of growth factors (i.e. EGF, TGF-
, HB-EGF, and
amphiregulin) is associated with multiple keratinocyte functions during
wound repair, including cell proliferation, migration, and stimulation
of
2
1 integrin expression (16, 17).
Keratinocyte migration is essential for effective re-epithelialization,
and expression of the EGFR and its ligands is up-regulated following injury in vivo (17, 18). Indeed, exogenously administered EGFR ligands (EGF, TGF-
) stimulate keratinocyte motility in
vitro and re-epithelialization in vivo (19-23), and
wound healing-specific keratins (K6 and K16) contain upstream
regulatory sequences responsive to EGFR activation (24). Furthermore,
EGFR overexpression in cultured keratinocytes enhances ligand-mediated
motility (25).
Autocrine signaling mechanisms often regulate cell function and
behavior, and, in keratinocytes, autocrine activation of the EGFR can
influence epithelial homeostasis and cutaneous repair. Indeed, recent
evidence from Stoll et al. (51) demonstrates that
heparin-binding ligands, namely HB-EGF and amphiregulin, mediate
autocrine activation of the EGFR in a skin organ culture model,
suggesting that these ligands play an important role in the
amplification and transmission of the wound healing signal. Because
collagenase-1 production is tyrosine kinase-dependent (11)
and because keratinocytes express both the EGFR and its various ligands
during wound repair, we reasoned that an autocrine loop mechanism may
regulate keratinocyte collagenase-1 expression following
2
1 integrin-mediated collagen binding. Here, we
report that the initial induction of collagenase-1 by keratinocytes
following contact with type I collagen requires only integrin receptor
activation. However, autocrine activation of the EGFR by HB-EGF is
required for the sustained expression of collagenase-1 by keratinocytes in vitro and during the full re-epithelialization process
in vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials
Recombinant human EGF, HB-EGF, amphiregulin, and polyclonal
neutralizing antiserum to HB-EGF were obtained from R & D Systems, Minneapolis, MN. Recombinant human TGF-
was purchased from
Collaborative Biomedical Products, Becton Dickinson, Inc., Bedford, MA.
Human EGFR antagonist PD153035 was obtained as a generous gift from Dr.
Robert Panek (Parke Davis, Inc., Ann Arbor, MI). Another specific EGFR
tyrosine kinase inhibitor, tyrphostin 1478, was purchased from
Calbiochem, San Diego, CA. Recombinant IL-1
receptor antagonist (IL-1 RA) was a generous gift from Dr. David Carmichael (Synergen, Inc.). This compound is a soluble IL-1
receptor that competitively binds, blocking ligand binding and receptor activation (26). Polyclonal
neutralizing antisera to TGF-
(clone 189-2130.1) (27), a monoclonal
EGFR blocking antibody (clone 528) (28), and polyclonal EGFR Ab-4 (used
for EGFR immunoprecipitation) (28) were purchased from Oncogene
Research Products, Cambridge, MA. Anti-phosphotyrosine monoclonal
antibody (PY-20) and anti-mouse IgG-horseradish peroxidase conjugate
were purchased from Transduction Laboratories, Lexington, KY. Bovine
type I collagen (Vitrogen-100) was obtained from Celltrix Laboratories,
Palo Alto, CA.
Isolation and Culture of Human Keratinocytes
Human keratinocytes were harvested from healthy adult skin from
reduction mammoplasties or abdominoplasties as described (11, 29).
Briefly, the subcutaneous fat and deep dermis were removed, and the
remaining tissue was incubated in 0.25% trypsin in PBS. After 16 h, the epidermis was separated from the dermis with forceps, and the
keratinocytes were scraped into DMEM. The keratinocyte suspension was
added to fresh DMEM supplemented with 5% fetal calf serum and 0.1%
penicillin/streptomycin. Under these culture conditions, keratinocytes
proliferate, migrate, differentiate, and cornify similarly to cells
in vivo (29). A specified amount of keratinocyte suspension
was then plated onto tissue culture dishes coated with 1 mg/ml type I
collagen, which is necessary for induction of collagenase-1 and
keratinocyte adhesion (5, 9, 11).
In Situ Hybridization
Collagenase-1 mRNA was detected in formalin-fixed tissue
samples by hybridization with 35S-labeled antisense RNA as
described (30, 31). Punch biopsies (2 mm) of human skin were obtained
and grown as explant cultures in serum-containing DMEM for 4 days.
Following treatment, the tissue was fixed in neutral buffered formalin
for 24 h followed by washing in PBS and dehydration in graded
ethanol. Sections of tissue were hybridized with 2.5 × 104 cpm/µl 35S-labeled antisense or sense RNA
overnight at 57 °C. After hybridization the slides were washed under
stringent conditions, including RNase A treatment, and were processed
for autoradiography. After development of the photographic emulsion,
slides were stained with hematoxylin-eosin. The specificity of the
antisense RNA probe for collagenase-1 and the complete lack of
reactivity by the sense probe have been demonstrated in previous
studies (3, 7).
Migration Assays
Colony Dispersion--
Primary human keratinocytes (1 × 104 cells) were plated within a siliconized cloning
cylinder (6-mm inner diameter; BellCo Glass, Inc., Vineland, NJ) onto
collagen-coated dishes. After a 24-h incubation period to allow the
cells to attach and become confluent, the cloning cylinder was removed,
and cell colonies were allowed to migrate for 96 h at 37 °C in
a 5% CO2 humidified incubator. Keratinocytes were fixed in
neutral buffered formalin, stained with 1.5% Coomassie Blue, and the
area of the colony was determined by digitized scanning analysis.
Migration is expressed as the increase in colony area relative to 0-h controls.
Colloidal Gold--
Primary human keratinocytes were plated on
chamber slides precoated with a mixture of 100 µg/ml type I collagen
and colloidal gold particles in serum-containing DMEM. Keratinocytes
(~330 cells) were added to each chamber, and 20 min later,
non-adherent cells were removed and the medium was replaced. Twenty
hours after plating, cultures were fixed in 1× Histochoice tissue
fixative (Amresco, Solon, OH), washed in PBS, and dehydrated through
graded ethanol. Paths of cell migration (phagokinetic tracks) were
identified as areas devoid of gold particles. A migration index was
determined using image analysis software by measuring the area of the
phagokinetic tracks associated with cells in randomly chosen fields
under dark field illumination at 100× magnification. For each
experiment, all conditions were done in triplicate, and all experiments
were repeated at least three times with keratinocytes from individual donors.
Enzyme-linked Immunosorbent Assay (ELISA)
The amount of collagenase-1 accumulated in keratinocyte
conditioned medium was measured by indirect competitive ELISA (32). This ELISA is completely specific for collagenase-1, has nanogram sensitivity, and detects active and zymogen enzyme forms, as well as
collagenase-1 bound to TIMP or bound to substrate. Results were
obtained from triplicate determinations and were normalized to total
cell protein as quantified by the BCA protein assay (Pierce) using
bovine serum albumin as a standard.
Metabolic Labeling
Post-confluent keratinocytes plated on type I collagen were
cultured for 24 h in the presence of serum-containing DMEM control or experimental solutions. The culture wells were then washed and
replaced with methionine-free DMEM containing 5% dialyzed fetal calf
serum (to remove free amino acids), 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM each of
non-essential amino acids, 50 µCi/ml [35S]methionine
(ICN Radiochemicals, Irvine CA), and the identical concentrations of
experimental reagents. Conditioned medium was collected 24 h later
and analyzed by immunoprecipitation.
Immunoprecipitation and Western Immunoblotting
A specific polyclonal antiserum (33) was used to
immunoprecipitate collagenase-1 from keratinocyte conditioned medium as described (34). To immunoprecipitate the EGFR, cell layers were washed
with PBS and treated for 10 min at room temperature with cell lysis
buffer (1.5% Triton X-100, 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 0.02 mg/ml aprotinin, and
0.01 mg/ml leupeptin). All samples were precleared with protein
A-Sepharose (Zymed Laboratories Inc., San Francisco,
CA), and supernatants were incubated with collagenase-1 or EGFR
polyclonal (35) antibodies for 1 h at 37 °C then overnight at
4 °C. Immune complexes were precipitated with protein A-Sepharose
and washed extensively. For visualization of collagenase-1,
radiolabeled protein was resolved by polyacrylamide gel electrophoresis
and visualized by fluorography as described previously (36).
Immunoprecipitated EGFR was resolved by polyacrylamide gel
electrophoresis and electrophoretically transferred to Immobilon-P polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) using
a semidry blotting apparatus (Bio-Rad). Tyrosine phosphorylation of the
EGFR was then visualized by incubating the membrane with anti-PY-20
(primary) and anti-mouse IgG-horseradish peroxidase (secondary)
antibodies followed by detection using the ECL system (Amersham Corp.,
Arlington Heights, IL) according to the manufacturer's instructions.
Total incorporated radioactivity (new protein synthesis) was determined
from keratinocyte conditioned medium by trichloroacetic acid
precipitation as described previously (37).
RNA Analysis
Collagenase-1, EGFR, EGF, TGF-
, amphiregulin, and GAPDH
mRNAs were detected by modification of previously described reverse transcription-polymerase chain reaction (RT-PCR) assays (9, 38, 39).
HB-EGF mRNA was amplified using primers designed with GeneWorks
software (Oxford Molecular Group Inc., Campbell, CA). The 5'-sense
primer was complementary to bases 791-810 in exon 4, and the
3'-antisense primer was complementary to bases 1017-1036 in exon 6 of
human HB-EGF (40). These primers amplify a fragment across three exons;
thus, the 246-base pair cDNA produced from HB-EGF mRNA would be
easily distinguished from contaminating DNA or preprocessed mRNA.
In addition, all resultant cDNAs from each of the primer pairs
contain restriction sites specific to the mRNA amplified and each
product was subjected to digestion analysis to verify specificity. The
primers used to amplify each mRNA, annealing temperatures, and
resulting product sizes are listed in Table
I.
Total RNA was isolated from cultured keratinocytes by phenol-chloroform
extraction (41) and treated with RQ1 RNase-free DNase (Promega,
Madison, WI) to remove any contaminating DNA as described (42).
DNase-treated RNA was reverse transcribed with random hexamers using
kit reagents and under the manufacturer's recommended conditions
(GeneAmp RNA PCR kit, Perkin Elmer Cetus, Norwalk, CT). Signal strength
for each RT-PCR cDNA product increased exponentially between 21 and
29 cycles using 10 ng of DNase-treated RNA and increased linearly
between 1 and 25 ng of total RNA at 25 cycles. To amplify each
mRNA, we used 10 ng of total RNA and 25 cycles. PCR products were
separated through a 2% agarose gel and visualized by ethidium bromide
staining. Specificity was determined by overnight transfer to Hybond
N+ membrane (Amersham Corp.) followed by Southern
hybridization with a radiolabeled product-specific oligonucleotide
probe (EGF family members and EGFR) or radiolabeled cDNA probes
(collagenase-1 and GAPDH). The probes used to detect EGF family members
and EGFR transcripts following RT-PCR were developed to recognize only specific sequences that were amplified in the PCR reaction. For each
oligonucleotide, a BLAST search was performed, and no sequence similarity to other known cDNAs was found. In addition, a parallel reaction was run without reverse transcriptase to assure that products
were not generated by contaminating DNA. Oligonucleotide probes were
labeled by terminal transferase (Boehringer Mannheim), and cDNA
probes were labeled by random priming (Amersham Corp.) with
[
-32P]dCTP (ICN Radiochemicals). Following
hybridization, the membranes were washed and exposed to x-ray film for
an appropriate duration.
Transient Transfection
Expression constructs contained the chloramphenicol
acetyltransferase (CAT) reporter gene with a 2.2-kilobase pair fragment of the human collagenase-1 promoter, pCLCAT (provided by Dr. Steven Frisch, Burnham Institute, La Jolla, CA), and pSV-
-galactosidase (Promega, Madison, WI). Primary keratinocytes were transfected at 50%
confluence with 2 µg/well of each construct using 10 µl/well LipofectAMINE (LifeTechnologies, Inc.). After 16 h, the medium was
replaced, and the cells were incubated with control or experimental solutions for an additional 24 h and harvested. Detection of
-galactosidase and CAT activity was performed as described
previously (37). Under the culture conditions used, we routinely
achieve 85% transfection efficiency of keratinocytes (9, 11, 37).
In Vivo Studies
Partial thickness thermal burns were created on the dorsal skin
of pigs as described (43). Briefly, two domestic male pigs, each
weighing 35 pounds, were anesthetized with ketamine and xylazine, and
anesthesia was maintained with halothane inhalation. The dorsal skin
was chemically depilitated, and six partial thickness burns measuring
3 × 3 cm were created by contact for 10 s with a solid brass
block weighing 714 g heated previously in a water bath maintained at 70 °C. The six burns were arranged in two rows of three burns on
each side of the spine. Blister roofs were removed and two burns were
treated topically with 3 ml of Silvadene® cream (Marion
Laboratories, Kansas City, MO) containing 20 µg/ml tyrphostin 1478 (Calbiochem) (44). Two burns were treated with Silvadene®
cream alone, and two burns were left untreated. Each burn was individually covered with a 6 × 6-cm2 adhesive
occlusive dressing (Steri-DrapeTM 2, 3M, St. Paul, MN). Dressings were
removed daily, the burns were retreated, and fresh dressings were
applied. Five days after injury, the pigs were sacrificed and a
full-thickness biopsy was taken diagonally across each burn, fixed in
10% buffered formalin, and embedded in paraffin. Sections were stained
with hematoxylin and eosin. The extent of epithelial healing for each
burn was calculated by measuring the distance between intact epithelial
edges divided by the total length of the wound and was expressed as
percentage of re-epithelialization. Epithelial healing for each of the
three treatment groups was averaged and compared for statistical
significance by analysis of variance and Tukey's HSD post-test.
 |
RESULTS |
Blockade of EGFR Signaling Inhibits Migration on Type I
Collagen--
We used specific inhibitors of EGFR occupancy and
tyrosine kinase activity to determine if signaling through this
receptor was related to collagenase-1 induction and keratinocyte
migration across type I collagen. Migration was assessed using
colloidal gold and colony-dispersion motility assays as described (10). In the colloidal gold assay, keratinocytes were plated on chamber slides coated with a colloidal gold type I collagen mixture, and migration was quantified 20 h later. In the colony dispersion assay, cells were cultured within cloning cylinders for 24 h. After cell attachment to the matrix, the cloning cylinders were removed
and migration was assessed at 96 h. As we have shown (10), keratinocytes migrated efficiently over type I collagen in both migration assays (Fig. 1, A
and D).

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Fig. 1.
Blockade of EGFR signaling inhibits
keratinocyte migration on type I collagen. A-C,
primary human keratinocytes were plated on culture slides coated with a
mixture of type I collagen (1.0 mg/ml) and colloidal gold particles,
treated with vehicle control (collagen alone) (A), PD153035
(500 nM) (B), or EGFR blocking antibody 528 (1.0 µg/ml) (C), and fixed after 20 h. D,
colloidal gold. Primary human keratinocytes were plated on culture
slides coated with colloidal gold and type I collagen (1.0 mg/ml) in
the presence of vehicle control (collagen alone), affinity-purified
collagenase-1 antiserum (1:4 dilution), PD153035 (500 nM),
EGFR blocking Ab 528 (1.0 µg/ml), or IL-1 RA (500 ng/ml) and were
fixed 20 h later. Keratinocyte migration was quantified as
described under "Experimental Procedures," and the data are shown
as means ± S.D. of triplicate samples from three experiments. For
colony dispersion, primary human keratinocytes were cultured for
24 h within cloning cylinders on type I collagen (1.0 mg/ml).
After 24 h, the cloning cylinders were removed and the cells were
allowed to migrate for an additional 96 h in the presence of
vehicle control (collagen alone), collagenase-1 antiserum (1:4
dilution), PD153035 (500 nM), EGFR blocking Ab 528 (1.0 µg/ml), or IL-1 RA (500 ng/ml). The data presented are means ± S.D. of values from three separate wells per treatment group, and
migration is expressed as arbitrary units relative to 0-h
controls.
|
|
As we reported (10), treatment of keratinocytes with an
affinity-purified collagenase-1 antibody (1:4 antiserum dilution), which blocks enzymatic activity, markedly inhibited cell migration across type I collagen, reconfirming that collagenase-1 activity is
required for motility over this matrix (Fig. 1D). Similar to collagenase-1 antiserum, the addition of PD153035 (500 nM),
a highly specific EGFR tyrosine kinase antagonist (45), inhibited keratinocyte migration by about 80% (Fig. 1, A
versus B and D). EGFR phosphorylation
and EGFR-dependent cellular functions in vitro
are inhibited by this compound at concentrations of 40-300 nM, whereas other tyrosine kinases are unaffected below 10 µM (45). In addition, anti-EGFR mAb 528 (1.0 µg/ml),
which blocks ligand binding and subsequent receptor activation, was
equally effective at inhibiting keratinocyte motility on collagen (Fig. 1, C and D). Addition of an IL-1
receptor
antagonist (IL-1 RA) (500 ng/ml) did not affect keratinocyte migration
(Fig. 1D). Results for all conditions paralleled one another
in each migration assay (Fig. 1D). Thus, inhibition of EGFR
function blocks keratinocyte motility across a type I collagen matrix.
Blockade of EGFR Function Inhibits Keratinocyte Collagenase-1
Production--
Because keratinocyte migration across type I collagen
is dependent on both collagenase-1 activity and EGFR function, we
determined if EGFR signaling was required for collagen-mediated
collagenase-1 production. Human keratinocytes were plated on native
type I collagen (all conditions shown except for "gelatin") or
heat-denatured collagen (gelatin) and treated with inhibitors of EGFR
signaling, and accumulation of collagenase-1 protein in the conditioned
medium was quantified by ELISA. We used the IL-1 RA as a control in
these experiments because signaling through this receptor is required for induction of collagenase-1 expression in fibroblasts (46, 47).
Treatment with PD153035 or EGFR Ab 528 inhibited collagen-mediated collagenase-1 production in a dose-dependent manner,
reducing enzyme levels to those detected in cells plated on gelatin
(negative control) (Fig. 2A).
In contrast, the IL-1 RA did not reduce collagenase-1 expression but
had a slight stimulatory effect. Keratinocyte viability was not
affected by treatment with either EGFR inhibitor as assessed by total
protein synthesis (data not shown).

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Fig. 2.
Blockade of EGFR signaling inhibits matrix-
and soluble factor-induced collagenase-1 production by
keratinocytes. Primary human keratinocytes were cultured on type I
collagen (collagen alone) or heat-denatured collagen (gelatin) until
confluent. A, cells on collagen were treated with PD153035
(100 or 500 nM), EGFR blocking antibody 528 (0.1 or 1.0 µg/ml), or IL-1 RA (250 or 500 ng/ml). B and C,
keratinocytes on collagen were treated with TGF- (25 ng/ml),
hepatocyte growth factor (25 ng/ml), phorbol ester (20 ng/ml), or
IFN- (1000 units/ml) in the presence or absence of PD153035 (500 nM). Collagenase-1 protein in the conditioned medium after
72 h was quantified by ELISA, and values were normalized to total
protein content. Data shown are the means ± S.D. of triplicate
observations from the same cell preparation and are representative of
up to four separate experiments.
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We also found that other known stimulators of collagenase-1 expression
by keratinocytes required intermediate signaling through the EGFR.
Collagen-mediated induction of collagenase-1 expression was augmented
by transforming growth factor-
1 (TGF-
1), hepatocyte growth
factor/scatter factor, phorbol ester, and interferon-
(IFN-
).
However, in the presence of PD153035 (Fig. 2, B and
C), all such soluble factor-stimulated enzyme production was
inhibited. In contrast, IL-1 RA had no effect on collagenase-1
stimulation by each of these factors (data not shown). Therefore,
agents that stimulate collagenase-1 production by keratinocytes,
whether matrix or soluble, appear to require an obligatory intermediate
EGFR signaling step.
EGFR Blockade Inhibits Collagen-induced Collagenase-1 mRNA
Levels and Promoter Activity in Keratinocytes--
To gain an
understanding of the molecular level at which EGFR blockade inhibits
collagen-directed collagenase-1 production, total RNA was harvested
from keratinocytes grown on collagen alone, or on collagen and treated
with PD153035 or EGFR mAb 528 for 24 h. To detect collagenase-1
mRNA present in each sample, we used a semiquantitative RT-PCR
assay (9). Only collagenase-1-specific products are detected by
Southern hybridization or by ethidium bromide staining. Collagenase-1
mRNA was readily observed in keratinocytes cultured on collagen
alone (Fig. 3A). In contrast,
and paralleling the protein data, treatment of keratinocytes with
PD153035 (500 nM) or EGFR mAb 528 (1.0 µg/ml) inhibited
collagenase-1 mRNA expression by 96% and 92%, respectively,
indicating pretranslational regulation.

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Fig. 3.
Blockade of EGFR signaling inhibits
collagen-induced collagenase-1 mRNA levels and promoter activity in
keratinocytes. Primary human keratinocytes were cultured on type I
collagen. A, keratinocytes were treated with vehicle control
(collagen alone), PD153035 (100, 500 nM), or EGFR blocking
Ab 528 (0.1 or 1.0 µg/ml). Total RNA was harvested 24 h later
and processed for collagenase-1 or GAPDH RT-PCR as described under
"Experimental Procedures." A control sample was processed without
reverse transcriptase ( RT) to assure RNA purity.
B, keratinocytes cultured on type I collagen were
transfected at ~60% confluence with a human collagenase-1 promoter
construct containing the CAT reporter gene and a construct containing
the -galactosidase gene for normalization of transfection
efficiency. After 16 h, keratinocytes were washed and treated with
vehicle control (collagen alone), or with media containing PD153035 or
EGFR blocking Ab 528 at the indicated concentrations. CAT activity was
quantified by scintillation counting of non-acetylated and acetylated
chloramphenicol and shown as a percent conversion to the acetylated
forms normalized to -galactosidase activity.
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To determine if collagenase-1 transcription was affected following
inhibition of EGFR signaling, keratinocytes on collagen were
transfected with a CAT expression construct containing 2.2 kilobase
pairs of the human collagenase-1 promoter. As shown in previous
studies, changes in the activity of this promoter construct parallel
changes in the activity of the endogenous collagenase-1 gene (48), and
the activity of the promoter construct is about 5-fold greater in
keratinocytes plated on native collagen than in cells on heat-denatured
collagen (gelatin) (9). The normalized level of CAT activity detected
in keratinocytes plated on collagen was reduced in cells treated with
PD153035 or EGFR Ab 528 by 75% and 87.5%, respectively (Fig.
3B), indicating that inhibition of EGFR function blocks
collagenase-1 production by a transcriptional mechanism.
Keratinocyte Contact with Type I Collagen Induces EGFR
Phosphorylation--
Becuase keratinocyte contact with collagen is the
primary and requisite inductive event for collagenase-1 expression (9, 10) and because EGFR blockade inhibits enzyme production, we reasoned
that EGFR activation should follow initial matrix binding. To assess
this temporal relationship, we analyzed changes in keratinocyte steady-state mRNA levels of collagenase-1, EGF family members, and
the EGFR at different time points after plating on collagen (0-48 h)
(Fig. 4A). As we reported
previously (9), no collagenase-1 mRNA was detected in keratinocytes
prior to matrix contact (Fig. 4A, C'ase-1, 0-h).
Low levels of collagenase-1 mRNA were observed as early as 2 h
after contact with collagen, and expression increased markedly and
progressively over the next 8 h. Collagenase-1 mRNA dropped to
lower levels between 24 and 48 h after plating, coincident with
the keratinocytes reaching confluence. Expression of the mRNAs for
EGF, TGF-
, and amphiregulin paralleled that of matrix-stimulated collagenase-1 expression (Fig. 4A).

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Fig. 4.
Collagenase-1, EGF, TGF- , amphiregulin, HB-EGF,
EGFR expression, and EGFR activation in keratinocytes cultured on type
I collagen. Normal human skin was processed for keratinocyte isolation
as described under "Experimental Procedures." As the
trypsin-dispersed cell suspension was prepared, some cells were
collected for RNA isolation (0 h). The remaining
keratinocytes were plated on collagen-coated dishes. A,
total RNA was isolated over a time course (0-48 h) and the mRNAs
for collagenase-1, EGF, TGF- , amphiregulin, HB-EGF, EGFR, and GAPDH
were amplified by RT-PCR and analyzed by Southern hybridization with
product-specific cDNA (c'ase-1 and GAPDH) or
oligonucleotide probes (all others). Duplicate samples were processed
without reverse transcriptase ( RT) to assure RNA purity.
B, total proteins were isolated from keratinocytes grown on
collagen (1 mg/ml) at the indicated time points and processed for
immunoprecipitation of tyrosine-phosphorylated species followed by
Western immunodetection of the EGFR as described under "Experimental
Procedures." Results presented in A and B are
representative of data obtained from at least three individual skin
donors.
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In contrast, mRNAs for HB-EGF and EGFR were constitutively
expressed at high levels in keratinocytes prior to collagen binding (Fig. 4A). EGFR mRNA became undetectable following
2 h of contact with collagen (Fig. 4A,
EGFR), but then increased progressively from 4 to 24 h,
diminishing slightly at 48 h. HB-EGF mRNA was expressed
at near-constant levels throughout the entire time course, except
for a single drop at 4 h.
We next determined whether collagen binding induced EGFR activation.
Primary keratinocytes were plated on collagen, and total cell lysates
were harvested over the next 120 min (Fig. 4B).
Phosphorylated species were immunoprecipitated with an
anti-phosphotyrosine monoclonal antibody, and phosphorylated EGFR was
detected by Western immunoblotting. EGFR was not phosphorylated in
freshly isolated keratinocytes from normal skin (Fig. 4B,
0 min). In contrast, marked phosphorylation of the EGFR was
observed as early as 10 min following contact with collagen and
persisted up to 120 min (Fig. 4B). Therefore, keratinocyte
contact with native type I collagen rapidly induces phosphorylation of
the EGFR. Furthermore, despite EGFR mRNA down-regulation at 2 h following matrix contact (Fig. 4A), the receptor remains present and functional on the cell surface as evidenced by continued phosphorylation (Fig. 4B).
EGFR Activity Mediates Sustained, but Not Early Collagen-directed
Keratinocyte Collagenase-1 Expression--
In the experiments shown in
Figs. 1-3, inhibitors of EGFR function blocked collagen-mediated
collagenase-1 expression as measured by enzyme protein and mRNA
levels at 24 h. We next performed a time course of collagenase-1
expression in the presence of EGFR inhibitors to determine if the
initial induction of collagenase-1 by collagen was mediated by EGFR
signaling. Freshly isolated keratinocytes from normal skin were
incubated with vehicle control or PD153035 (500 nM) for
2 h prior to plating on collagen. After plating, total RNA was
isolated over a time course (0-24 h), and collagenase-1 mRNA was
assessed by RT-PCR. Surprisingly, the initial matrix-induced expression
of collagenase-1 at 4 h was unaffected by inhibition of EGFR
signaling (Fig. 5, A and
B, +PD153035). In contrast, collagenase-1 mRNA was slightly diminished at 6 h and almost completely
absent at 24 h in cells treated with PD153035 when compared with
vehicle controls (Fig. 5, A and B). To ensure
that PD153035 inhibited EGFR activation, keratinocytes were plated on
collagen and treated with vehicle, 30 ng/ml EGF, or EGF + PD153035 (500 nM). Stimulation of collagenase-1 expression by EGF was
inhibited by PD153035 (data not shown). Therefore, the sustained
expression of collagenase-1 mRNA requires EGFR signaling, whereas
matrix contact alone is sufficient to induce enzyme expression.

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Fig. 5.
Sustained keratinocyte collagenase-1
expression is mediated via EGFR signaling. Keratinocytes were
isolated from normal human skin as described under "Experimental
Procedures" and pretreated for 2 h with vehicle control or
PD153035 (500 nM). After pretreatment some keratinocytes
were collected for RNA isolation (0 h). The remaining
keratinocytes were plated on collagen-coated dishes in the continued
presence of vehicle control or PD153035 and total RNA was isolated over
a time course (0-24 h). A, collagenase-1 mRNA was
amplified by RT-PCR analysis of DNase-treated RNA and visualized by
Southern hybridization with a radiolabeled cDNA probe. Duplicate
samples were processed without reverse transcriptase ( RT)
to assure RNA purity. B, collagenase-1 mRNA
hybridization bands were quantified by scanning digitized densitometry
and values were plotted over the time course.
|
|
Heparin-binding Epidermal Growth Factor Is the Ligand That Mediates
EGFR-dependent Sustained Collagenase-1
Expression--
Members of the EGF family, namely EGF and TGF-
,
up-regulate matrix metalloproteinase expression in several cell types,
including keratinocytes (49, 50). Furthermore, collagen-directed
collagenase-1 expression in keratinocytes is enhanced by treatment with
EGF (9, 10). Recent evidence has shown that autocrine production of
HB-EGF is likely responsible for potentiation and transmission of the
healing response during epidermal wound repair (51), yet effects of
this cytokine on collagenase-1 expression have not been studied.
Because HB-EGF was the only EGFR ligand constitutively expressed in
keratinocytes from intact skin (Fig. 4A), we hypothesized that sustained collagen-mediated production of keratinocyte
collagenase-1 is regulated via a HB-EGF/EGFR autocrine loop mechanism.
We treated primary keratinocytes with several EGF family members to
determine their effect on collagen-directed collagenase-1 expression
(Fig. 6A). Collagenase-1
production, as assessed by immunoprecipitation of metabolically labeled
proteins, was induced in keratinocytes cultured on collagen alone, and
this expression was slightly enhanced in cells treated with EGF (Fig.
6A). Collagenase-1 production was substantially increased in
keratinocytes treated with HB-EGF or TGF-
, whereas cells treated
with amphiregulin showed no stimulation above that induced by collagen.
Thus, of the EGF family members tested, HB-EGF and TGF-
were the
most potent modulators of matrix-mediated collagenase-1 production by
keratinocytes, making them likely candidates for autocrine activation
of the EGFR following contact with collagen.

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Fig. 6.
Sustained collagen-directed keratinocyte
collagenase-1 expression is dependent on HB-EGF activity. Primary
human keratinocytes were cultured on type I collagen until confluent.
A, keratinocytes were treated for 24 h with vehicle
control (collagen alone), HB-EGF (25 ng/ml), amphiregulin (25 ng/ml),
EGF (30 ng/ml), or TGF- (30 ng/ml). Proteins were metabolically
labeled in the presence of each solution for an additional 24 h,
and conditioned medium was analyzed by immunoprecipitation for
collagenase-1. Results from one cell population are shown and represent
data obtained from three separate skin donors. B,
keratinocytes were cultured on collagen over a time course (0-24 h)
and treated with vehicle control or with a HB-EGF neutralizing antibody
(10 µg/ml). Total RNA was harvested at the indicated time points and
processed for collagenase-1 or GAPDH RT-PCR as described under
"Experimental Procedures." A control sample was processed without
reverse transcriptase ( RT) to assure RNA sample purity.
C, keratinocytes were cultured on collagen and treated with
vehicle control or with a TGF- neutralizing antibody at the
indicated concentrations. Total RNA was harvested 24 h later and
processed for collagenase-1 RT-PCR as described under "Experimental
Procedures." A control sample was processed without reverse
transcriptase ( RT) to assure RNA sample purity. Results
presented in B and C are representative of data
obtained from at least two separate skin donors.
|
|
To address this issue, keratinocytes were isolated from normal human
skin and pre-incubated for 2 h with vehicle control or neutralizing antibodies to HB-EGF or TGF-
prior to plating on collagen. After plating, total RNA was harvested over the next 0-24 h,
and collagenase-1 mRNA was assessed by RT-PCR. The expression of
collagenase-1 by keratinocytes was inhibited by treatment with the
HB-EGF neutralizing antibody at both time points tested (Fig. 6B, 8 and 24 h). In contrast,
collagenase-1 expression at 24 h was unaltered by TGF-
neutralizing antiserum (Fig. 6C). Therefore, the sustained
production of keratinocyte collagenase-1 in the presence of collagen
requires an intermediate HB-EGF/EGFR autocrine signaling step.
Blockade of EGFR Signaling Inhibits ex Vivo Epidermal Collagenase-1
Expression and Prevents Re-epithelialization of Porcine Burn
Wounds--
We treated ex vivo wounded human skin explants
with inhibitors of EGFR tyrosine kinase activity to determine the
requirement, at the tissue level, of EGFR activation for keratinocyte
collagenase-1 expression. Punch biopsies of normal human skin were
cultured for 4 days with vehicle control or PD153035 (500 nM). As demonstrated by in situ hybridization of
the control specimens, collagenase-1 mRNA was expressed by
migrating keratinocytes at the leading edge of re-epithelialization
(Fig. 7, A and
A'). Collagenase-1 was also expressed by some dermal
fibroblasts. In marked contrast to control injured skin, no expression
of collagenase-1 mRNA was evident in wound-edge keratinocytes in
specimens treated with PD153035, whereas fibroblast production was
unaffected (Fig. 7, B and B').

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Fig. 7.
Blockade of EGFR signaling inhibits
collagenase-1 expression in ex vivo wounded
epidermis. Punch biopsies (2 mm) of human skin were treated in
culture for 4 days in the presence of vehicle control (A and
A') or PD153035 (500 nM) (B and
B'). The tissue was fixed, and sections were hybridized
with a 35S-labeled antisense RNA probe specific for
collagenase-1 mRNA. In the control section (A and
A'), prominent autoradiographic signal for collagenase-1
was detected in basal keratinocytes (arrows) at the
migrating front of the epidermis (E). Collagenase-1 mRNA
expression was also observed in several fibroblasts
(arrowheads) within the dermis (D). In contrast,
explants treated with PD153035 (B and B') showed
no detectable collagenase-1 mRNA signal in keratinocytes at the
wound margin (arrows), whereas expression was unchanged in
dermal fibroblasts (arrowheads) when compared with controls
(A and A').
|
|
Because EGFR signaling is required for sustained keratinocyte
collagenase-1 expression both in vitro and ex
vivo, and because the activity of this enzyme is essential for
cell migration across type I collagen (10), we next determined if EGFR
inhibition affected re-epithelialization of in vivo wounds.
Partial thickness burn wounds were created in pigs and were treated
with Silvadene® cream containing tyrphostin 1478 (20 µg/ml), a highly potent and specific EGFR tyrosine kinase inhibitor
(52), vehicle control (Silvadene® cream), or occlusive
dressing only. All wounds were covered with occlusive dressing. In
tyrphostin 1478-treated wounds, epithelial healing was significantly
impaired when compared with burns treated with vehicle control or
covered with occlusive dressing only (Fig. 8, A-D). Specifically, wounds
treated with tyrphostin 1478 had re-epithelialized only 22 ± 16%
(mean ± standard error) of the surface of the burn, whereas
wounds treated with vehicle control had closed an average of 90 ± 5% of the burn area (p = 0.008). Burns covered with
occlusive dressing only had re-epithelialized an average of 82% ± 12% of the wound surface (p = 0.016 versus 1478-treated wounds). There was no significant difference between epithelial healing of burns treated with vehicle control or covered with occlusive dressing (p = 0.88).

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Fig. 8.
Re-epithelialization of partial-thickness
porcine burn wounds is inhibited by blocking EGFR activity.
Partial thickness thermal burns were created on the dorsal skin of pigs
and treated topically with Silvadene® cream (A,
vehicle control), Silvadene® cream containing 20 µg/ml
tyrphostin 1478 (B) or occlusive dressing only
(C). All wounds were covered with occlusive dressing. The
two large arrowheads in each panel mark the original wounded
edges of the epidermal burn. The extent of epithelial healing for each
burn was assessed by measuring the distance between intact epithelial
edges divided by the total length of the wound and expressed as percent
re-epithelialization ± S.E. D, epithelial healing for
the three treatment groups was averaged and compared for statistical
significance by analysis of variance and Tukey's Honest Statistical
Difference post-test. E, primary human keratinocytes were
cultured on type I collagen until confluent. Cells were treated with
vehicle control (collagen alone), PD153035 (100 or 500 nM),
or tyrphostin 1478 (0.1-1.0 µM). Collagenase-1 protein
in 72-h conditioned medium was quantified by ELISA. Data shown are the
means ± S.D. from triplicate observations from the same cell
preparation and are representative of at least two separate skin
donors.
|
|
To verify that tyrphostin 1478 blocked collagenase-1 expression, human
keratinocytes plated on type I collagen were treated with PD153035
(100-500 nM) or tyrphostin 1478 (0.1-1.0
µM) for 72 h and collagenase-1 secreted into
conditioned medium was quantified by ELISA (Fig. 8E). Both
PD153035 and tyrphostin 1478 blocked collagen-induced collagenase-1
expression in a dose-dependent manner (Fig. 8E).
Taken together, these findings support the hypothesis that inhibition
of re-epithelialization in porcine burn wounds was due, at least in
part, to blocking collagenase-1 production.
 |
DISCUSSION |
Activation of the EGFR in basal keratinocytes following injury
provides several critical signals required for proper healing of the
tissue defect. For example, the wound-associated keratins 6 and 16 are
markedly up-regulated (53), and their promoters contain regulatory
elements that are responsive to EGFR activation (24). Continued
signaling through the EGFR promotes Bcl-X-L-mediated prevention of
keratinocyte apoptosis, thereby potentiating re-epithelialization by
maintaining survival of the migrating cells (54). We show here that the
sustained expression of collagenase-1 in two systems that mimic
epidermal healing (keratinocyte migration on type I collagen and
ex vivo skin explants) requires an EGFR autocrine loop
mechanism. In previous studies, we showed that collagenase-1 expression
is induced at the onset of re-epithelialization as keratinocytes
migrate onto dermis, and that collagenase-1 activity is required for
cell migration over a type I collagen substratum (10, 55). Thus, EGFR
activation is central to yet another important phase of the wound
repair process.
Our findings that both matrix- and soluble factor-induced collagenase-1
expression in keratinocytes require autocrine signaling through the
EGFR (Fig. 2) draw interesting parallels to reports in human
fibroblasts showing that all up-regulators of collagenase-1 production
operate via an IL-1
/IL-1
receptor autocrine loop. Fini and
colleagues (46, 47) have demonstrated that only IL-1
directly
stimulates collagenase-1 gene transcription in fibroblasts. All other
inducers, ranging from cytokines such as TNF-
to phorbol esters to
agents causing cytoskeletal rearrangement, must first stimulate IL-1
gene transcription and subsequent release of IL-1
protein. This
cytokine then binds to the cell-surface IL-1
receptor, resulting in
triggering of collagenase-1 expression. Consequently, the addition of
an IL-1
receptor antagonist to fibroblast cultures blocks
collagenase-1 gene expression by any stimulating agent. Our findings
suggest an analogous role for HB-EGF/EGFR in keratinocytes and offer
the possibility that autocrine signaling pathways may represent a
general biologic mechanism for inducing the expression of
collagenase-1, and perhaps even other MMPs, in different cell types.
Our results implicate HB-EGF as the key ligand that mediates autocrine
signaling through the EGFR, resulting in sustained keratinocyte
production of collagenase-1. Evidence demonstrating multiple effects of
HB-EGF on keratinocytes has accumulated through studies of its role in
epidermal wound repair. This newer member of the EGF family is
synthesized by multiple cell types, including vascular endothelium,
smooth muscle, and keratinocytes (56). Exogenously added HB-EGF,
TGF-
, or EGF promotes autoinduction of HB-EGF mRNA, suggesting
that this protein is an autocrine growth factor for these cells (56).
The requirement for activation of the EGFR to sustain matrix-induced
collagenase-1 production is an example of wound keratinocyte phenotype
modulation by HB-EGF. Although HB-EGF mRNA is expressed in freshly
isolated keratinocytes from normal skin, the membrane-bound form of
this protein may not be processed and released in a soluble form until
later in the wound healing response. In fact, other groups have shown
that soluble HB-EGF does not appear in conditioned medium from
excisional wounds or human skin explants until 24 h after injury
(51, 57). These findings correlate with our observations that complete
inhibition of collagenase-1 expression by blocking EGFR or HB-EGF
activity does not occur until 24 h following keratinocyte contact
with collagen.
Our findings indicate that matrix-induced keratinocyte collagenase-1
expression involves two distinct pathways, an initial response not
requiring EGFR activation and a sustained response obligatory to EGFR
activation. Both of these responses are integrin-mediated, but early
collagenase-1 expression is EGFR-independent. Our data demonstrate that
2
1 integrin activation alone is
sufficient to induce the early (0-8 h) expression of collagenase-1 in
keratinocytes following contact with type I collagen. However, the
sustained production (
8 h) of this MMP requires signaling through the
EGFR in addition to
2
1 binding (Figs. 5
and 6B). Indeed, blocking
2
1
activity blocks all matrix-induced collagenase-1 production (10),
whereas only the sustained expression is inhibited when EGFR activity
is blocked (this report) (Fig. 5). Stimulation by both integrin
adhesion and cell binding of a soluble factor are required for a
variety of cellular responses during tissue morphogenesis. Unique to
our system, however, is that both integrin and EGFR signaling are
required only after prolonged exposure to type I collagen (the inducing stimulus).
Similar to our findings, EGFR autophosphorylation is induced by contact
of glomerular epithelial cells with type I collagen (58). Our data
demonstrate that EGFR (and HB-EGF) mRNA is expressed in
keratinocytes freshly isolated from intact skin and that the receptor
is rapidly autophosphorylated following contact with type I collagen.
These findings represent further examples of how keratinocytes in
unwounded skin are "primed" to respond to injury. Indeed,
keratinocytes in intact skin express endogenous
2
1 integrin (59). Interestingly, although
the EGFR is autophosphorylated rapidly following keratinocyte binding
to collagen, collagenase-1 expression does not require EGFR signaling
until later in the healing response. We speculate, however, that other
early functions of the wound keratinocyte, such as non-enzymatic
migration-related events or cell proliferation may require early EGFR
signaling, although this remains to be determined.
In addition to our in vitro data, we also show a role for
EGFR signaling in the re-epithelialization of porcine burn wounds in vivo. Blocking receptor activity with a specific
inhibitor of the EGFR tyrosine kinase (tyrphostin 1478) inhibited
keratinocyte collagenase-1 production and markedly delayed
re-epithelialization when compared with normal controls. Similarly,
treatment of ex vivo human skin punch biopsies with PD153035
inhibited collagenase-1 production in keratinocytes at the wound edge.
Taken together, these data suggest that blocking EGFR activity inhibits
keratinocyte migration across the dermal matrix and that this is due,
at least in part, to inhibition of collagenase-1 expression. Although
blocking EGFR signaling may inhibit other biological events necessary
for the complete wound repair (e.g. cell proliferation), we
suggest that EGFR-dependent collagenase-1 expression is
critical for sustained keratinocyte migration and normal
re-epithelialization.
Our findings in this report add substantially to understanding the
mechanisms that regulate collagenase-1 expression during wound repair.
Previously, we found that collagenase-1 gene transcription is induced
following injury when keratinocytes move off their underlying basement
membrane and contact type I collagen of the dermal matrix (3, 7, 5). We
identified the cell-surface recognition integrin as
2
1 and showed that collagenase-1 activity was requisite for keratinocyte migration over a type I collagen substratum (10). Our present data indicate that intact skin is primed
and ready to respond to injury with high endogenous levels of
keratinocyte EGFR and HB-EGF mRNA. Upon keratinocyte
2
1 binding to type I collagen, EGFR
phosphorylation occurs within minutes. Collagenase-1 mRNA levels
are induced within 2 h, however, by a mechanism that is
EGFR-independent. Nevertheless, as collagenase-1 expression continues,
the sustained high levels of enzyme production from
8 h following
contact with type I collagen are dependent on an EGFR/HB-EGF autocrine
signaling loop. Since re-epithelialization of even minor wounds takes
days, this sustained collagenase-1 expression is likely essential for
most keratinocyte migration and for complete re-epithelialization.
Important unanswered questions raised by our findings include: 1) what
signaling pathway does
2
1 use to transmit
the rapid initial, EGFR-independent induction of collagenase-1?; and 2)
upon completion of re-epithelialization, does the cessation of
collagenase-1 production involve the dismantling of the EGFR/HB-EGF
autocrine loop? Studies to address these important questions are
currently in progress.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Steven Frisch, La Jolla Cancer
Research Foundation, La Jolla, CA, for the human collagenase-1 promoter
construct and Dr. Robert Panek, Parke-Davis Warner Lambert, Ann Arbor,
MI for the PD153035 EGFR antagonist. We also thank Jennifer
Gaither-Ganim, Molly R. Cofman, and Theresa J. Burke for technical
assistance, and Jill Roby (Barnes-Jewish Hospital) for assistance with
in situ hybridization studies.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health and the Dermatology Foundation.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.
§
Recipient of a National Institutes of Health K-01 Mentored Research
Fellowship and a Research Career Development Award from the Dermatology
Foundation. To whom correspondence should be addressed: Dermatology,
BJH North, Washington University School of Medicine, 216 S. Kingshighway, St. Louis, MO 63110. Tel.: 314-454-8290; Fax:
314-454-8293; E-mail: bpilcher{at}im.wustl.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
ECM, extracellular
matrix;
HB, heparin-binding;
EGF, epidermal growth factor;
EGFR, epidermal growth factor receptor;
IFN, interferon;
TGF, transforming
growth factor;
IL, interleukin;
RA, receptor antagonist;
MMP, matrix
metalloproteinase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
ELISA, enzyme-linked immunosorbent assay;
PBS, phosphate-buffered
saline;
DMEM, Dulbecco's modified Eagle's medium;
Ab, antibody;
mAb, monoclonal antibody;
RT-PCR, reverse transcription-polymerase chain
reaction;
CAT, chloramphenicol acetyltransferase.
 |
REFERENCES |
-
Mignatti, P.,
Rifkin, D. B.,
Welgus, H. G.,
and Parks, W. C.
(1996)
in
The Molecular and Cellular Biology of Wound Repair (Clark, R. A. F., ed), 2nd Ed., pp. 427-474, Plenum Press, New York
-
Parks, W., and Mecham, R.
(eds)
(1998)
Matrix Metalloproteinases, 1st Ed., Academic Press, New York
-
Saarialho-Kere, U. K.,
Chang, E. S.,
Welgus, H. G.,
and Parks, W. C.
(1992)
J. Clin. Invest.
90,
1952-1957[Medline]
[Order article via Infotrieve]
-
Stricklin, G.,
and Nanney, L.
(1994)
J. Invest. Dermatol.
103,
488-492[Abstract]
-
Saarialho-Kere, U. K.,
Vaalamo, M.,
Airola, K.,
Niemi, K.-M.,
Oikarinen, A. I.,
and Parks, W. C.
(1995)
J. Invest. Dermatol.
104,
982-988[Abstract]
-
Inoue, M.,
Kratz, G.,
Haegerstrand, A.,
and Ståhle-Bäckdahl, M.
(1995)
J. Invest. Dermatol.
104,
479-483[Abstract]
-
Saarialho-Kere, U. K.,
Kovacs, S. O.,
Pentland, A. P.,
Olerud, J.,
Welgus, H. G.,
and Parks, W. C.
(1993)
J. Clin. Invest.
92,
2858-2866[Medline]
[Order article via Infotrieve]
-
Inoue, M.,
Kratz, G.,
Haegerstrand, A.,
and Ståhle-Bäckdahl, M.
(1995)
J. Invest. Dermatol.
104,
479-483[Abstract]
-
Sudbeck, B. D.,
Pilcher, B. K.,
Welgus, H. G.,
and Parks, W. C.
(1997)
J. Biol. Chem.
272,
22103-22110[Abstract/Free Full Text]
-
Pilcher, B. K.,
Sudbeck, B. D.,
Dumin, J.,
Krane, S. M.,
Welgus, H. G.,
and Parks, W. C.
(1997)
J. Cell Biol.
137,
1445-1457[Abstract/Free Full Text]
-
Sudbeck, B. D.,
Parks, W. C.,
Welgus, H. G.,
and Pentland, A. P.
(1994)
J. Biol. Chem.
269,
30022-30029[Abstract/Free Full Text]
-
van der Geer, P.,
Hunter, T.,
and Lindberg, R.
(1994)
Annu. Rev. Cell Biol.
10,
251-337[CrossRef]
-
Miettinen, P.,
Berger, J.,
Meneses, J.,
Phung, Y.,
Pederson, R.,
Werb, Z.,
and Derynck, R.
(1995)
Nature
376,
337-341[CrossRef][Medline]
[Order article via Infotrieve]
-
Theadgill, D.,
Dlugosz, A.,
Hansen, L.,
Tennenbaum, T.,
Lichti, U.,
Yee, D.,
LaManti, C.,
Mourton, T.,
Herrup, K.,
Harris, R.,
Barnard, J.,
Yuspa, S.,
Coffee, R.,
and Magnuson, T.
(1995)
Science
269,
230-234[Medline]
[Order article via Infotrieve]
-
Sibilia, M.,
and Wagner, E.
(1995)
Science
269,
234-238[Medline]
[Order article via Infotrieve]
-
Chen, J.,
Kim, J.,
Zhang, K.,
Sarret, Y.,
Wynn, K.,
Kramer, R.,
and Woodley, D.
(1993)
Exp. Cell. Res.
209,
216-223[CrossRef][Medline]
[Order article via Infotrieve]
-
Nanney, L.,
and King, J. L. E.
(1996)
in
The Molecular and Cellular Biology of Wound Repair (Clark, R., ed), 2nd Ed., pp. 171-194, Plenum Press, New York
-
Stoscheck, C.,
Nanney, L.,
and King, J. L.E.
(1992)
J. Invest. Dermatol.
99,
645-649[Abstract]
-
Ju, W.,
Schiller, J.,
Kazempour, M.,
and Lowy, D.
(1993)
J. Invest. Dermatol.
100,
628-632[Abstract]
-
Chen, Y. Q.,
Kähäri, V.-M.,
Bashir, M. M.,
Rosenbloom, J.,
and Uitto, J.
(1992)
J. Invest. Dermatol.
98,
615 (Abstr.)
-
Cha, D.,
O'Brien, P.,
O'Toole, E.,
Woodley, D.,
and Hudson, L.
(1996)
J. Invest. Dermatol.
106,
590-597[Abstract]
-
Brown, G.,
Nanney, L.,
Griffin, J.,
Cramer, A.,
Yancey, J.,
Curtsinger, L.,
Holtzin, L.,
Schultz, G.,
Jurkiewicz, M.,
and Lynch, J.
(1987)
N. Engl. J. Med.
321,
76-79[Abstract]
-
Schultz, G.,
White, M.,
Mitchell, R.,
Brown, G.,
Lynch, J.,
Twardzick, D.,
and Todaro, G.
(1987)
Science
235,
350-352[Medline]
[Order article via Infotrieve]
-
Jiang, C.-K.,
Magnaldo, T.,
Ohtsuki, M.,
Freedberg, I.,
Bernerd, F.,
and Blumenberg, M.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
6786-6790[Abstract]
-
McCawley, L.,
O'Brien, P.,
and Hudson, L.
(1997)
Endocrinology
138,
121-127[Abstract/Free Full Text]
-
Wilcox, B.,
Dumin, J.,
and Jeffrey, J.
(1994)
J. Biol. Chem.
269,
29658-29664[Abstract/Free Full Text]
-
Sorvillo, J. M.,
McCormack, E. S.,
Yanez, L.,
Valenzuela, D.,
and Reynolds, J., F. H.
(1990)
Oncogene
5,
377-386[Medline]
[Order article via Infotrieve]
-
Kawamoto, T.,
Sato, J.,
Le, A.,
Polikoff, J.,
Sato, G.,
and Mendelsohn, J.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
1337-1341[Abstract]
-
Pentland, A. P.,
and Needleman, P.
(1986)
J. Clin. Invest.
77,
246-251[Medline]
[Order article via Infotrieve]
-
Prosser, I. W.,
Stenmark, K. R.,
Suthar, M.,
Crouch, E. C.,
Mecham, R. P.,
and Parks, W. C.
(1989)
Am. J. Pathol.
135,
1073-1088[Abstract]
-
Saarialho-Kere, U. K.,
Welgus, H. G.,
and Parks, W. C.
(1993)
J. Biol. Chem.
268,
17354-17361[Abstract/Free Full Text]
-
Cooper, T. W.,
Bauer, E. A.,
and Eisen, A. Z.
(1982)
Collagen Relat. Res.
3,
205-211
-
Stricklin, G. P.,
Eisen, A. Z.,
Bauer, E. A.,
and Jeffrey, J. J.
(1978)
Biochemistry
17,
2331-2337[Medline]
[Order article via Infotrieve]
-
Welgus, H. G.,
Campbell, E. J.,
Bar-Shavit, Z.,
Senior, R. M.,
and Teitelbaum, S. L.
(1985)
J. Clin. Invest.
76,
219-224[Medline]
[Order article via Infotrieve]
-
Gullick, W. J.,
Downward, J.,
Parker, P. J.,
Whittle, N.,
Kris, R.,
Schlessinger, J.,
Ullrich, A.,
and Waterfield, M. D.
(1985)
Proc. R. Soc. Lond. B
226,
127-134[Medline]
[Order article via Infotrieve]
-
Pilcher, B.,
Gaither-Ganim, J.,
Parks, W.,
and Welgus, H.
(1997)
J. Biol. Chem.
272,
18147-18154[Abstract/Free Full Text]
-
Dunsmore, S. E.,
Rubin, J. S.,
Kovacs, S. O.,
Chedid, M.,
Parks, W. C.,
and Welgus, H. G.
(1996)
J. Biol. Chem.
271,
24576-24582[Abstract/Free Full Text]
-
Imai, T.,
Kurachi, H.,
Adachi, K.,
Adachi, H.,
Yoshimoto, Y.,
Homma, H.,
Tadokoro, C.,
Takeda, S.,
Yamaguchi, M.,
Sakata, M.,
Sakoyama, Y.,
and Miyake, A.
(1995)
Biol. Reprod.
52,
928-938[Abstract]
-
Forsyth, I. A.,
Taylor, J. A.,
Keable, S.,
Turvey, A.,
and Lennard, S.
(1997)
Mol. Cell. Endocrinol.
126,
41-48[CrossRef][Medline]
[Order article via Infotrieve]
-
Fen, Z.,
Dhadly, M. S.,
Yoshizumi, M.,
Hilkert, R. J.,
Quertermous, T.,
Eddy, R. L.,
Shows, T. B.,
and Lee, M.
(1993)
Biochemistry
32,
7932-7938[Medline]
[Order article via Infotrieve]
-
Chomczynski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[CrossRef][Medline]
[Order article via Infotrieve]
-
Swee, M. H.,
Parks, W. C.,
and Pierce, R. A.
(1995)
J. Biol. Chem.
270,
14899-14906[Abstract/Free Full Text]
-
Brown, G. L.,
Curtsinger, I., L.,
Brightwell, J. R.,
Ackerman, D. M.,
Tobin, G. R.,
Polk, J., H. C.,
George-Nascimento, C.,
Valenzuela, P.,
and Schultz, G. S.
(1986)
J. Exp. Med.
163,
1319-1324[Medline]
[Order article via Infotrieve]
-
Levitzki, A.,
and Gazit, A.
(1995)
Science
267,
1782-1788[Medline]
[Order article via Infotrieve]
-
Fry, D.,
Kraker, A.,
McMichael, A.,
Ambroso, L.,
Nelson, J.,
Leopold, W.,
Connors, R.,
and Bridges, A.
(1994)
Science
265,
1093-1095[Medline]
[Order article via Infotrieve]
-
Fini, M.,
Strissel, K. J.,
Girard, M. T.,
Mays, J. W.,
and Rinehart, W. B.
(1994)
J. Biol. Chem.
269,
11291-11298[Abstract/Free Full Text]
-
West-Mays, J.,
Strissel, K. J.,
Sadow, P. M.,
and Fini, M. E.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
6768-6772[Abstract]
-
Doyle, G. A. R.,
Pierce, R. A.,
and Parks, W. C.
(1997)
J. Biol. Chem.
272,
11840-11849[Abstract/Free Full Text]
-
Saarialho-Kere, U. K.,
Chang, E. S.,
Welgus, H. G.,
and Parks, W. C.
(1993)
J. Invest. Dermatol.
100,
335-342[Abstract]
-
Woodley, D. T.,
Kalebec, T.,
Baines, A. J.,
Link, W.,
Prunieras, M.,
and Liotta, L.
(1986)
J. Invest. Dermatol.
4,
418-423
-
Stoll, S.,
Garner, W.,
and Elder, J.
(1997)
J. Clin. Invest.
100,
1271-1281[Abstract/Free Full Text]
-
Levitski, A.,
and Gazit, A.
(1995)
Science
267,
1782-1788[Medline]
[Order article via Infotrieve]
-
Paladini, R.,
Tkakahashi, K.,
Bravo, N.,
and Coulombe, P.
(1995)
J. Cell Biol.
132,
381-397[Abstract]
-
Stoll, S.,
Benedict, M.,
Mitra, R.,
Hiniker, A.,
Elder, J.,
and Nunez, G.
(1998)
Oncogene
16,
1493-1499[CrossRef][Medline]
[Order article via Infotrieve]
-
Saarialho-Kere, U. K.,
Vaalamo, M.,
Airola, K.,
Niemi, K.-M.,
Oikarinen, A. I.,
and Parks, W. C.
(1995)
J. Invest. Dermatol.
105,
982-988
-
Hashimoto, K.,
Higashimaya, S.,
Asada, H.,
Hashimura, E.,
Kobayashi, T.,
Sudo, K.,
Nakagawa, T.,
Damm, D.,
Yoshikawa, K.,
and Taniguchi, N.
(1994)
J. Biol. Chem.
269,
20060-20066[Abstract/Free Full Text]
-
Marikovsky, M.,
Breuing, K.,
Liu, P. Y.,
Eriksson, E.,
Higashiyama, S.,
Farber, P.,
and Abraham, J. M. K.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
3889-3893[Abstract]
-
Cybulsky, A.,
McTavish, A.,
and Cyr, M.-D.
(1994)
J. Clin. Invest.
94,
68-78[Medline]
[Order article via Infotrieve]
-
Larjava, H.,
Salo, T.,
Haapasalmi, K.,
Kramer, R. H.,
and Heino, J.
(1993)
J. Clin. Invest.
92,
1425-1435[Medline]
[Order article via Infotrieve]
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