* Department of Medicine (Dermatology), Department of Cell Biology and Physiology, Washington University School of
Medicine, St. Louis, Missouri 63110; and § Department of Medicine, Harvard Medical School and Massachusetts General
Hospital, Boston, Massachusetts 02114
We have shown in a variety of human
wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary
keratinocytes by contact with native type I collagen and
not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the
catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea,
we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1
in promoting cell movement was demonstrated in four
distinct experiments. One, keratinocyte migration was
completely blocked by peptide hydroxymates, which
are potent inhibitors of the catalytic activity of MMPs.
Two, HaCaTs, a line of human keratinocytes that do
not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently
on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in
the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on
mutant type I collagen lacking the collagenase cleavage
site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely
blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti-MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against
the 2 integrin subunit but not by antibodies against the
1 or
3 subunits. We propose that interaction of the
2
1 integrin with dermal collagen mediates induction
of collagenase-1 in keratinocytes at the onset of healing
and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that
cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.
Normal cutaneous wound healing, as well as healing
in essentially all tissues, involves an orderly progression of events to reestablish the integrity of
the injured tissue. The initial injury starts a programmed
series of independent yet separate responses that includes
reepithelialization and epithelial proliferation, inflammation, angiogenesis, fibroplasia, matrix accumulation, and
eventually resolution. During each stage in this process, proteinases are needed to remove or remodel extracellular
matrix components in both the epithelial and interstitial
compartments, thereby accommodating cell migration and
tissue repair (Mignatti et al., 1996 Although extracellular matrix proteins can be degraded
by various proteinases, fibrillar type I collagen, the most
abundant protein in the dermis, is resistant to degradation
by most enzymes. Collagen degradation is initiated by the
catalytic activity of collagenases, a subgroup of the matrix
metalloproteinase (MMP)1 gene family, with the unique
ability to cleave fibrillar collagen types I, II, and III at a
specific locus in their triple-helical domain. At physiological temperature, cleaved collagen molecules denature into
gelatin and become susceptible to further digestion by
other proteinases. Of the three known human metallocollagenases, collagenase-1 (MMP-1) seems to be the enzyme
that is principally responsible for collagen turnover in
most tissues. In a variety of normal and disease-associated
tissue remodeling events, collagenase-1 may be expressed
by epithelial cells, fibroblasts, endothelial cells, chondrocytes, and macrophages (Saarialho-Kere et al., 1992 In a thorough examination of normally healing wounds
and a variety of chronic ulcers, we found that collagenase-1
is invariably expressed by basal keratinocytes at the leading edge of repairing tissue and that expression of this MMP
in the epidermis diminishes rapidly away from the wound
margin (Saarialho-Kere et al., 1992 Our in vivo findings suggest that altered cell-matrix interactions influence collagenase-1 expression during reepithelialization. Indeed, we have shown that native type I
collagen selectively supports primary human keratinocyte
attachment and induces collagenase-1, whereas denatured
collagen (gelatin), purified or combined basement membrane proteins, or other dermal-wound bed matrix components do not (Saarialho-Kere et al., 1993a The invariable expression of collagenase-1 in all forms
of wounds and the confinement of its expression to periods of active reepithelialization suggests that this enzyme
plays a critical role in keratinocyte migration during wound
repair. Beyond directly remodeling structural proteins,
MMPs are thought to break down extracellular matrix
barriers that impede cell migration. Clearly, this is a reasonable role for these proteinases in facilitating cell movement through a three-dimensional matrix, as is seen during blastocyst invasion (Librach et al., 1991 Cell Culture
Primary keratinocytes were isolated from full thickness skin obtained
from reductive mammoplasty or lateral abdominoplasty and grown in
DME (1.8 mM Ca2+) as described (Pentland and Needleman, 1986 In Situ Hybridization
Collagenase-1 mRNA was detected in formalin-fixed tissue samples and cultured cells by hybridization with 35S-labeled antisense RNA as described
(Prosser et al., 1989 Colloidal Gold Migration Assay
Keratinocytes were plated on chamber slides precoated with a mixture of
100 µg/ml type I collagen or gelatin or no matrix and colloidal gold particles in serum-containing DME with or without 30 ng/ml EGF (R&D Systems Inc., Minneapolis, MN). Colloidal gold-coated chamber slides were
prepared as described (Albrecht-Buehler, 1977 Colony Dispersion Migration Assay
Keratinocyte migration was also assessed in a colony dispersion assay by
plating 104 cells suspended in 150 µl DME within a siliconized cloning cylinder (6-mm internal diameter; Bellco Glass, Inc., Vineland, NJ) onto collagen- or gelatin-coated dishes. After a 24-h incubation period to allow
the cells to attach and become confluent, the cylinder was removed, and
cell colonies were allowed to migrate for 0, 24, 48, 96, or 120 h at 37°C in a
5% CO2 humidified incubator. Keratinocytes were fixed and 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.
Various reagents were added to promote or inhibit keratinocyte migration. In studies with HaCaT keratinocytes, collagenase-1 expression was
induced with 30 ng/ml EGF (R&D Systems, Inc.). HaCaT proliferation in
response to EGF was inhibited by treating cells with 100 mM hydroxyurea
(HU; Sigma Chemical Co., St. Louis, MO) 24 h before addition of EGF.
Peptide hydroxymate compounds SC44463 and SC44201 were used at
25 µM and were provided by Monsanto-Searle, Inc. (St. Louis, MO). Collagenase-1 antibody was affinity-purified and characterized as described
(Saarialho-Kere et al., 1993a Thiopeptolide Assay
Inhibition of MMP degradation of the thiopeptolide substrate Ac-ProLeu-Gly-thioester-Leu-Leu-Gly-Oet (BACHEM Bioscience, King of Prussia, PA) (Shipley et al., 1996 Collagenase-1 Is Expressed by Migrating Keratinocytes
In Vivo and In Vitro
The epidermis of neonatal human skin grafted onto SCID
mice heals by 4 d after wounding, and reepithelialization is
associated with the expression of identical integrins and
basement membrane proteins produced in actual human
wounds (Juhasz et al., 1993
When grown in DME medium, which contains 1.8 mM
CaCl2, cultured keratinocytes form subpopulations of migrating, proliferating, and differentiating cells (Pentland
and Needleman, 1986
Collagenase-1-expressing Keratinocytes Migrate on
Type I Collagen
To assure that collagenase-1-positive keratinocytes were
migrating, we plated cells on chamber slides coated with a
colloidal gold-type I collagen mixture and fixed the cultures 20 h later. Cells were treated with 30 ng/ml EGF to
augment collagenase-1 production over the relatively short
experimental period. Phagokinetic tracks created were
identified as areas lacking gold particles and were often
seen associated with single cells migrating along the collagen-coated surface (Fig. 2 B). Migration tracks were also
associated with single cells and doublets proximal to keratinocyte foci and were apparently moving away from them
(Fig. 2 C, arrows). In addition, gold particles were removed at the borders of cell foci (Fig. 2 C), suggesting
zones of collagenolysis. The dark clumps in keratinocytes
seen under phase (Fig. 2 C To verify that gold particle clearance was dependent on
metalloproteinase activity, we treated keratinocytes with a
peptide hydroxymate inhibitor. This compound is a substrate-based inhibitor containing a hydroxamic acid moiety that chelates the active site zinc cation and renders
MMPs catalytically inactive. SC44463 has a Ki of about 1 nM
for pure collagenase-1 in solution (Moore and Spilburg,
1986 Collagenase-1-deficient HaCaT Keratinocytes Migrate
on Gelatin but Not on Native Type I Collagen
To begin to assess if keratinocyte migration specifically requires the activity of collagenase-1, we assessed the ability of HaCaT cells, which do not express collagenase-1 in response to native type I collagen (see Fig. 4 A), to migrate
on native or denatured collagen. HaCaT cells were plated
within cloning cylinders on dishes coated with type I collagen or gelatin (heat-denatured type I collagen). The cylinders were removed 24 h later, and the cells were allowed
to migrate for 48 h. HaCaT cells cultured on native type I
collagen did not migrate from the colony border, whereas
cells grown on gelatin-coated dishes migrated efficiently from the cell colony (Fig. 3). MMP inhibitors did not affect
HaCaT migration on gelatin (data not shown). Because
the primary cleavage products of collagenase-1 denature
at 37°C but are not further degraded, we hypothesized that
collagenase-1-mediated cleavage of collagen followed by
denaturation to gelatin is required for effective migration.
Induction of Collagenase-1 Expression by HaCaT
Keratinocytes Induces Migration on Type I Collagen
High levels of collagenase-1 production were induced in
HaCaT keratinocytes after exposure to 30 ng/ml EGF (Fig.
4 A). As we saw in the primary keratinocyte cultures (Fig.
2 A), HaCaT cells formed tightly packed colonies, and collagenase-1 mRNA was expressed only by cells at the periphery of the foci (Fig. 4 B). In contrast, HaCaT cells
within the cell colony had no detectable collagenase-1
mRNA. Although EGF potently induced collagenase-1 expression, enzyme production was confined only to migrating cells, or at least to cells with the potential to migrate.
Supporting the idea that collagenase-1 is required for
keratinocyte motility on dermal matrix, EGF-treated cells
migrated progressively on a type I collagen-coated surface
over a 4-d period (Fig. 4 D). At 96 h, EGF-stimulated cells
covered an area four times greater than that of untreated
HaCaT cells, which did not produce collagenase-1 (Fig. 4 A)
and which had not progressed far from the border of the
cloning cylinder (Fig. 4, C and D). The small increase in
colony area evident at 72 and 96 h in control HaCaT cells
was likely due to cell proliferation.
HaCaT Migration on Type I Collagen Requires
MMP Activity
Induction of HaCaT cell migration on type I collagen by
EGF suggests that the activity of collagenase-1 facilitates
cell movement on this matrix. Indeed, treatment of HaCaT
cells with SC44463 inhibited EGF-mediated cell migration
~50% in the colony dispersion assay (Fig. 4 E, Because the colony dispersion assay requires treatment
with EGF for 96 h to see appreciable migration, we cannot
conclude that cell movement is due specifically and solely
to collagenase-1 activity. To address this concern, we used
the colloidal gold assay to assess keratinocyte migration
over a relatively shorter period of time (20 h). Duplicating
the findings of the colony dispersion assay, EGF-treated
HaCaTs migrated efficiently as single cells on a colloidal
gold-collagen substratum, and this activity was blocked by
SC44463 (Fig. 4 F). Migration was also completely inhibited by treatment with affinity-purified anti-collagenase-1 antibody, which blocks the catalytic activity of this MMP.
Untreated HaCaT keratinocytes migrated efficiently on
gelatin, and cell movement on this substrate was not affected by the blocking antibody (Fig. 4 F). These findings
indicate that EGF-mediated migration of HaCaT keratinocytes on native collagen requires the activity of collagenase-1.
Migration of Primary Human Keratinocytes
In contrast to HaCaT cells, collagenase-1 is induced in primary human keratinocytes grown on native type I collagen
(Sudbeck et al., 1994
Table I.
Protein Synthesis
Table II.
Sustained Inhibitory Activity of SC44463
).
, 1993a;
Stricklin et al., 1993
; Wolfe et al., 1993
; Fisher et al., 1994
;
Galis et al., 1994
). Collagenase-2 (MMP-8) is found only in
neutrophils and chondrocytes (Hasty et al., 1990
; Chubinskaya et al., 1996
), and collagenase-3 (MMP-13), originally
cloned from a breast carcinoma line (Freije et al., 1994
), is
also expressed in articular cartilage (Mitchell et al., 1996
;
Reboul et al., 1996
) and developing bone (Gack et al.,
1995
).
, 1993a). In a time-course
study of reepithelialization in human skin, Inoue et al.
(1995)
reported that collagenase-1 expression is detectable
as early as 4 h after wounding, when migration is first apparent, and shuts off upon reformation of a contiguous epidermis. In chronic ulcers, collagenase-1 is expressed at
higher levels and over a greater length of epidermis compared to that seen in acute wounds (Saarialho-Kere et al.,
1993a
), suggesting that overexpression of this MMP may
contribute to the inability of certain lesions to heal. Importantly, this enzyme is always expressed by migrating keratinocytes that have moved off of an intact basement membrane and are in contact with dermal and provisional matrices (Saarialho-Kere et al., 1993a
). Conversely, wound
epidermal cells on an intact basement membrane, such as
seen in certain forms of blisters (Saarialho-Kere et al., 1995
),
and on nonwounded epidermis do not express this MMP.
; Sudbeck et al.,
1994
). Furthermore, we showed that contact with native
type I collagen induces collagenase-1 transcription and is
blocked by tyrosine kinase antagonists (Sudbeck et al.,
1994
). Thus, contact with dermal type I collagen is an important determinant in regulating the response of basal
keratinocytes to injury. Indeed, in this report, we show
that blocking antibodies to the
2 subunit of the
2
1 integrin inhibits collagen-mediated induction of collagenase-1
in keratinocytes.
), angiogenesis
(Fisher et al., 1994
), and extravasation and infiltration of
inflammatory cells (Shipley et al., 1996
). During normal
reepithelialization, however, keratinocytes migrate along
a path of least resistance, dissecting underneath the scab
while remaining superficial to the underlying dermis and
wound bed (Stenn and Malhotra, 1992
). Thus, epidermal
repair involves cell migration in a two-dimensional plane
rather than through a three-dimensional matrix-rich environment. As reported here, our findings indicate that keratinocytes use collagenase-1 to cleave collagen to gelatin,
thereby providing a substrate that is more conducive to
migration. Without collagenolytic activity, these cells do
not migrate on a collagen-containing matrix. We propose
that the ability of keratinocytes to bind tightly to collagen
and subsequently cleave the fibrils provides a mechanism that maintains the directionality of reepithelialization.
Materials and Methods
; Sudbeck et al., 1994
). Cells were plated on dishes or slides precoated with 1.0 mg/ml solution bovine type I monomeric collagen (Vitrogen; Collagen Corp., Palo Alto, CA) or with 1.0 µg/ml gelatin made by heating type I
collagen at 80°C for 10 min. The HaCaT human keratinocyte cell line
(Boukamp et al., 1988
) was provided by Dr. Norbert Fusenig (German
Cancer Research Center, Heidelberg, Germany) and was grown in DME
containing 10% heat-inactivated FCS, 1% Hepes, and antibiotics. Collagenase-1 accumulation in the medium was quantified by competitive ELISA and normalized to total cellular protein (Sudbeck et al., 1994
).
RNA isolation and detection of collagenase-1 mRNA by reverse transcriptase-PCR were done as described in detail (Sudbeck et al., 1997
). Total protein synthesis was determined in cultured keratinocytes as described (Sudbeck et al., 1994
).
; Saarialho-Kere et al., 1993b
). For in situ hybridization of acute wounds, we obtained sections of intact and 2-d wounded
neonatal human foreskin grafted onto severe combined immunodeficiency (SCID) mice from Drs. Horace DeLister and Steven Albelda (University of Pennsylvania, Philadelphia, PA). The creation of these grafted
mice and the incisional wounding and biopsy procedures have been described in detail (Juhasz et al., 1993
). Keratinocytes were grown on collagen-coated Lab-Tek chamber slides (Nunc, Naperville, IL) and were hybridized with 2.5 × 104 cpm/µl of 35S-labeled antisense or sense RNA
overnight at 57°C. After hybridization, slides were washed under stringent
conditions, including RNase A treatment, and were processed for autoradiography.
) with modifications for keratinocytes and the inclusion of matrix proteins (Woodley et al., 1988
; Kim
et al., 1994
). Primary or HaCaT keratinocytes (about 330 cells in 0.3 ml)
were added to each chamber, and 20 min later, nonadherent cells were removed and the medium was replaced. 20 h after plating, cultures were fixed in 1× Histochoice tissue fixative (Amresco, Solon, OH) for 1 min,
washed in PBS, and dehydrated through graded ethanols. Paths of cell migration (phagokinetic tracks) were identified by areas devoid of gold particles. A migration index was determined using image analysis software by
measuring the area of phagokinetic tracks associated with cells in randomly chosen fields under dark-field illumination at 100×. All cells in a
field were counted, and 20 cells were counted for each chamber. For each
experiment, all conditions were done in duplicate, and all experiments
were repeated at least four times with keratinocytes from different donors. The migration index of keratinocytes on colloidal gold with no matrix was essentially zero and was subtracted from experimental points.
). This polyclonal antibody recognizes both
the zymogen and activated forms of collagenase-1, free or bound to TIMP-1,
and fully inhibits the catalytic activity of collagenase-1. In an in vitro degradation assay, a 1:20 dilution of collagenase antibody completely inhibited the cleavage of type I collagen monomers by 5 µg/ml of purified human collagenase-1 (data not shown). Ammonium sulfate-precipitated nonimmune IgG (1:10 dilution) was used as a control in the antibodyblocking experiments. Recombinant human TIMP-1 (50 µg/ml), which has
full MMP-inhibitory activity, was provided as a gift from Dr. David Carmichael (Synergen Corporation, Boulder, CO). Wild-type and collagenase-1-resistant mouse type I collagens were used at 100 µg/ml. Generation of the mice and characterization of the mutant collagen were
reported earlier (Wu et al., 1990
; Liu et al., 1995
). Monoclonal blocking
antibodies to integrin subunits were purchased from Chemicon International Inc. (Temecula, CA) and were mAB1973 (anti-
1), mAB1950
(anti-
2), and mAB1952 (anti-
3). Integrin antibodies were added to a final concentration of 10 µg/ml. To prevent blocking of cell adhesion, keratinocytes were plated 2 h before addition of integrin-blocking antibodies.
) was used to determine the activity of
SC44463 in tissue culture. We obtained recombinant 92-kD gelatinase catalytic domain, which is spontaneously active, from Dr. J. Michael Shipley
(Washington University, St. Louis, MO) and used this protein as a source
of MMP activity to generate an initial cleavage rate. The inhibitory activity of fresh 25 µM SC44463 in DME was compared to that of SC44463 in
48- or 72-h keratinocyte-conditioned medium.
Results
). As demonstrated by in situ
hybridization, collagenase-1 was expressed by human
basal keratinocytes only at the leading edge of reepithelialization (Fig. 1, B and C), identical to the location of enzyme expression seen in a variety of human wounds and
ulcers (Saarialho-Kere et al., 1992
, 1993a, 1995). Collagenase-1 mRNA was not expressed in intact skin (Fig. 1 A).
The precise, spatially confined expression of collagenase-1
by migrating keratinocytes suggests that altered cell-matrix
interactions modulate enzyme expression, and indeed, collagenase-1-expressing keratinocytes were seen in intimate
contact with dermal fibrillar collagen (Fig. 1 C).
Fig. 1.
Collagenase-1 mRNA is expressed by
migrating keratinocytes in contact with dermal
collagen. (A) A section of uninjured neonatal foreskin grafted onto a SCID mouse was hybridized with a 35S-labeled antisense RNA probe specific for collagenase-1 mRNA. No detectable signal for collagenase-1 mRNA was seen in the
epidermis (E) or the dermis (D) of unwounded
skin. Mouse skin and the graft junctions are not
seen in this field. Under dark-field illumination, pigmented cells are iridescent and appear purplish. (B) In a section of a biopsy taken 2 d after
wounding grafted human neonatal skin, autoradiographic signal for collagenase-1 mRNA was
seen in basal keratinocytes at the leading edge of
reepithelialization (arrows). Signal was confined
to keratinocytes migrating into the wound area
(W), under the scab (S), and over the dermal
wound bed (D). No signal was detected in intact
epidermis (E) or in any cell in the dermis. (C) In
a section of pyogenic granuloma, which displays many features of an acute wound (SaarialhoKere et al., 1992), collagenase-1 mRNA was seen
in basal keratinocytes (arrows) along the dermal-epidermal junction (arrowheads) at the epidermal front. Collagenase-1-positive basal keratinocytes at the migrating front are in direct
contact with fibrillar collagen (arrowheads),
which appear as thick iridescent fibers under
Nomarski optics. (Inset) Under dark-field illumination, autoradiographic signal for collagenase-1 mRNA is seen in basal keratinocytes demarcated by arrows in the larger photomicrograph.
Autoradiographic exposure was 21 d for all sections. Bars: (A and B) 100 µm; (C) 25 µm; (Inset)
50 µm.
[View Larger Version of this Image (59K GIF file)]
). Differentiating keratinocytes are
seen as blurred foci when viewed from above and are surrounded by an apron of hyperproliferative cells (Fig. 2 A).
Bordering the hyperproliferative cells, and often detached from them, are migrating keratinocytes. Reflecting the phenotype of basal cells involved in reepithelialization in vivo,
collagenase-1 mRNA was expressed only in keratinocytes
migrating from the colonies of proliferating and differentiating cells (Fig. 2 A). No signal was seen in cultures hybridized with a 35S-labeled sense RNA probe (data not
shown). With time, proliferating cells and their migrating
daughter cells cover, or "heal," the tissue culture surface,
and collagenase-1 expression ceases (data not shown).
Fig. 2.
Collagenase-1 mRNA is expressed by migrating keratinocytes in culture. (A) Keratinocytes were plated on dishes precoated with native type I collagen and 24 h later were processed for in situ hybridization using a 35S-labeled collagenase-1-specific antisense RNA
probe. Signal for collagenase-1 mRNA was seen only in migrating keratinocytes (arrows), whereas no autoradiographic signal was detected in hyperproliferative or differentiating keratinocytes, which appear as blurred foci (f) when viewed from above. Autoradiographic exposure was 14 d. (B-D) Primary human keratinocytes were plated on culture slides coated with a mixture of type I collagen
and colloidal gold particles and were fixed 20 h later. With Nomarski optics (B) or under dark-field illumination (C), tracks of keratinocyte migration were seen as areas devoid of gold particles (arrows). (B) Often, single cells were seen migrating along large bundles of
collagen fibers. (C) Gold was also removed along the border of keratinocyte foci and in association with cells (arrows) that had apparently migrated from the keratinocyte islands. Under phase (C), internalized gold particles were seen as dense accumulations in keratinocytes. No internalized gold was evident in cells within foci (large arrows). (D and D
) No migration tracks or areas of lysis around cell
foci were seen in cultures treated with SC44463. Bars: (A) 50 µm; (B) 12 µm; (C-D
) 25 µm.
[View Larger Version of this Image (163K GIF file)]
) are phagocytized gold particles. Internalized gold particles were not present in keratinocytes within organized foci (Fig. 2 C
, large arrows), where collagenase-1 is not expressed (Fig. 2 A). Thus, the
gold-collagen substrate was removed only in areas that
corresponded to those where high levels of collagenase-1
expression were seen.
). Treatment with SC44463 completely blocked the formation of phagokinetic tracks or zones of lysis (Fig. 2,
D and D
). These observations demonstrate that collagenase-1-expressing keratinocytes are migrating and that
this process is inhibited by compounds that block the catalytic activity of this MMP.
Fig. 4.
HaCaT migration
on native type I collagen is
MMP dependent. (A)
HaCaT keratinocytes were
grown on type I collagen-
coated dishes and treated
with or without 30 ng/ml
EGF. Collagenase-1 accumulation in the medium was assessed 48 h later by ELISA
and normalized to total cellular protein. (B) HaCaT cells
were plated on collagencoated slides, stimulated with
EGF, and 24 h later were
processed for in situ hybridization with a collagenase-1 35S-labeled antisense RNA
probe. Only HaCaT cells at
the periphery of cell clusters
expressed collagenase-1
mRNA. Autoradiographic exposure was 14 d. (C-E)
HaCaT cells were plated
within cloning cylinders on
collagen-coated dishes. After 24 h, the cylinders were
removed, and the cells were
allowed to migrate on collagen alone or in the presence of EGF for 48, 72, or
96 h. Cells were stained, and
the area migrated was quantified by scanning densitometry. (E) During the initial
24-h culture period, some
HaCaTs were treated with
100 mM HU to inhibit EGFmediated proliferation. The
cylinders were removed, and
the cells were given fresh medium with or without 30 ng/
ml EGF or EGF plus 25 µM
peptide hydroxymate inhibitor SC44463. After 96 h, cultures were washed and
stained, and migration was
quantified by image analysis.
Migration data for HaCaT
cells pretreated with (+HU)
or without (HU) HU are
shown. The data in D and E
are the means ± SD or triplicate wells and are expressed in arbitrary units relative to
0-h controls. (F) HaCaT keratinocytes were plated on culture slides coated with a mixture of colloidal gold particles and type I collagen (Col) or gelatin (Gel).
Cells on collagen-coated chambers were treated with 30 ng/ml EGF. To inhibit collagenase-1 activity, cells were treated with (+) or without (
) collagenase-1 affinity-purified antibody or 25 µM SC44463 and were fixed 20 h later. Keratinocyte migration was quantified as described under Materials and Methods, and the data shown are the means ± SEM of duplicate samples from four experiments.
[View Larger Versions of these Images (38 + 136K GIF file)]
Fig. 3.
Collagenase-1-negative HaCaT keratinocytes migrate
on gelatin but not on native type I collagen. HaCaT cells were
grown within cloning cylinders on dishes precoated with type I
collagen (Col) or gelatin (Gel). The cylinders were removed 24 h
later, and cells were allowed to migrate for 48 h. The arrows designate the border of the culture when the cloning cylinder was removed. The micrographs shown are representative of three experiments.
[View Larger Version of this Image (73K GIF file)]
HU). The
incomplete inhibition of cell migration in SC44463-treated
cultures was likely due to EGF-stimulated cell proliferation, thereby increasing colony size independent of cell movement per se. To neutralize the proliferative effects of
EGF, cell colonies were pretreated for 24 h with HU, an
effective inhibitor of DNA synthesis and cell division. The
antiproliferative effect of HU pretreatment was dose dependent and persisted up to 96 h as determined by [3H]thymidine incorporation (data not shown). Under these conditions, EGF-stimulated cells migrated on collagen, but
they did not cover an area as large as EGF-treated HaCaT
cells not preexposed to HU (Fig. 4 E, +HU). Addition of
SC44463 completely blocked EGF-mediated cell migration
on type I collagen (Fig. 4 E, +HU). These results demonstrate that EGF-mediated HaCaT migration on type I collagen is an MMP-dependent process.
). 120 h after plating, primary keratinocytes cultured on collagen alone migrated well beyond
the original cell colony border (Fig. 5), and addition of the
MMP inhibitor SC44463 completely blocked this response
(Fig. 5). The ability of SC44463 to inhibit cell migration was
dose dependent over a range of 0.1 to 25 µM, demonstrating an ~50% reduction in cell movement with 1.0 µM in both EGF-stimulated HaCaT keratinocyte and primary human keratinocyte cultures (data not shown). Compound
SC44201, a stereoisomer of 44463 differing only in the
plane of a single hydrogen atom, is a weak inhibitor of
MMP activity, with a relative Ki for all MMPs over 1,000fold greater than that of SC44463 (Moore and Spilburg, 1986
). Consistent with the idea that keratinocyte migration
on type I collagen is MMP dependent, SC44201 did not affect cell movement (Fig. 5). As determined by a [3H]leucine pulse and trichloroacetic acid precipitation, the hydroxymate compounds at concentrations up to 25 µM did
not affect the synthesis of total secreted protein (Table I).
Furthermore, as determined by an in vitro MMP activity
assay, the inhibitory activity of SC44463 was not diminished after 72 h in culture (Table II).
Fig. 5.
Keratinocyte migration on native type I collagen is MMP
dependent. Primary human keratinocyte migration was assessed
by the colony dispersion assay. Cells were cultured on collagen
with or without peptide hydroxymate compounds SC44463 or
SC44201. The data shown are the means ± SD of triplicate wells
and are expressed in arbitrary units relative to 0-h controls.
[View Larger Version of this Image (19K GIF file)]
Primary Keratinocytes Do Not Migrate on Collagenase-resistant Type I Collagen
Inhibition of keratinocyte migration on type I collagen by
the peptide hydroxymate compound demonstrated that
MMP activity was required for cell motility. This compound, like other hydroxymates, inhibits a broad spectrum
of metalloproteinases (Moore and Spilburg, 1986). Therefore, based on these data alone, we cannot conclude that
collagenase-1 is the specific MMP required for migration on collagen. Because primary keratinocytes have the potential to express other MMPs, namely 92-kD gelatinase
(Sudbeck et al., 1997
), stromelysin-1, and stromelysin-2
(Windsor et al., 1993
; Saarialho-Kere et al., 1994
), we used
more specific reagents to determine which enzyme is required for migration. Northern hybridization demonstrated
that human keratinocytes express only collagenase-1 and
do not produce collagenase-2 or -3 (data not shown).
To determine if the proteolytic activity of collagenase-1
is required for cell motility, we cultured primary human
keratinocytes on a collagenase-resistant mutant type I collagen. Human collagenase-1 cleaves fibrillar type I collagen
at Gly775-Ile776 in the 1(I) chains and at Gly775-Leu776 in
the
2(I) chain, and these sites are conserved among mammalian type I collagens. This cleavage renders the molecule thermally unstable and susceptible to further degradation by other proteinases. The mutant collagen used in this study contains a double substitution of Pro for Glu774
and Ala777 and Met for Ile776 in the region of the collagenase cleavage site of the
1(I) chain, rendering the molecule resistant to proteolysis by collagenase-1 (Wu et al.,
1990
; Liu et al., 1995
). Collagenase-1 expression was induced in primary keratinocytes grown on wild-type native
type I collagen, and cells migrated efficiently across this
matrix (Fig. 6, A and B). In contrast, keratinocytes cultured on collagenase-resistant mutant collagen did not migrate, although enzyme expression was induced (Fig. 6, A
and B). As an internal control, inhibition of all MMP activity with SC44463 completely inhibited the ability of
these cells to migrate on native type I collagen (Fig. 6 A).
Keratinocytes plated on mutant type I collagen adhered
equally to those on wild-type collagen and expressed collagenase-1, indicating that the cell-recognition site is distinct
from the enzyme cleavage site.
Migration of Primary Keratinocytes on Type I Collagen Is Blocked by Collagenase Antibodies
In both the colony dispersion and colloidal gold assays, anti-collagenase-1 antibodies inhibited keratinocyte migration in a dose-dependent fashion, whereas cell motility was not affected by nonimmune IgG (Fig. 7, A and B). The concentrations of collagenase-1 antibody that inhibited cell migration are the same as those that blocked activity of pure enzyme in solution (data not shown). In addition, human recombinant TIMP-1, a naturally occurring inhibitor of collagenase-1 and other MMPs, inhibited cell migration (Fig. 7 A).
In the colloidal gold assay, most keratinocytes showed evidence of migration on collagen (Fig. 7 C). Collagenase-1 antibody blocked keratinocyte migration on native type I collagen proportionately to the concentration of antibody added (Fig. 7 B). Similar to the inhibition seen with SC44463 (Fig. 7 B), essentially no migration was seen in cultures treated with the highest concentration of antibody (Fig. 7, B and D). In contrast, collagenase-1 antibody did not affect keratinocyte migration on gelatin (Fig. 7, B and E). Keratinocyte migration on gelatin was slightly reduced compared to that on collagen, likely because the cells did not adhere as efficiently to the heat-denatured substrate. These findings demonstrate that the proteolytic activity of collagenase-1, and not that of other MMPs, is required for keratinocyte migration on native type I collagen.
Integrin-mediated Induction of Collagenase-1
To assess which integrin-matrix interactions transduce the
collagen-mediated induction of collagenase-1, we treated
primary keratinocytes plated on native collagen with 1-,
2-, or
3-blocking antibodies. Each of these subunits is
expressed by basal keratinocytes in intact and wounded
skin and complex with the
1 subunit to form potential
collagen-binding receptors (Larjava et al., 1993
; Lange et al.,
1994
). Collagen-mediated induction of collagenase-1 mRNA
was potently inhibited by treatment with the
2 antibody but not by antibodies against the
1 or
3 subunits (Fig.
8). Blocking collagenase-1 expression with the
2
1 antibody inhibited keratinocyte migration on collagen by ~75%
relative to untreated controls, whereas cell movement was
unaffected by the
1 or
3 antibodies (Fig. 8). To prevent
potential disruption of cell attachment to collagen, we
added the integrin-blocking antibodies 2 h after keratinocytes were plated, and migration and collagenase-1
mRNA levels were assessed 20 h later. Although collagenase-1 mRNA levels were completely repressed by 20 h,
some migration was evident, which may be because of
some collagenase-1 produced during the 2-h attachment
period.
In this report, we demonstrate that the production and
activity of collagenase-1 is required for keratinocyte migration on native type I collagen. In response to cutaneous
injury, basal keratinocytes at the edge of the wound dislodge, dissolve, or loosen their cell-cell and cell-matrix
contacts, move from the basement membrane, and migrate in an organized front over the viable dermis and
wound bed. Initiation of keratinocyte migration is one of
the earliest responses of the epidermis to wounding and
precedes by hours stimulation of cell proliferation (Garlick and Taichman, 1994). As we have shown in several different types of human wounds (Saarialho-Kere et al., 1992
,
1993a, 1995), collagenase-1 is invariably expressed by
keratinocytes that have moved off of the basement membrane and onto the underlying matrix, and studies by Inoue
et al. (1995)
demonstrated that collagenase-1 is induced as
soon as 4 h after wounding. The confinement of collagenase-1 expression to a spatially defined subpopulation of
keratinocytes suggests that altered cell-matrix contacts
mediate induction of this MMP. Indeed, as we demonstrate here, contact with dermal type I collagen induces
collagenase-1 expression only by migrating keratinocytes
(Fig. 2 A). Other components of the dermal and provisional matrices, such as type III collagen, fibronectin, and
fibrin, do not induce or affect collagenase-1 production or
are not even recognized by keratinocytes (Sudbeck, B.D.,
B.K. Pilcher, H.G. Welgus, and W.C. Parks, unpublished
observations). Consistent with the idea that migration
from basement membrane onto a type I collagen-containing matrix is required for collagenase-1 expression, this
MMP is not induced in keratinocytes grown on basement
membrane proteins (Saarialho-Kere et al., 1993a
; Sudbeck
et al., 1994
). Thus, altered cell-matrix interactions, and in
particular, contact with dermal type I collagen, may initiate the keratinocyte's response to injury, which is characterized by the expression of collagenase-1 at the migrating
front.
We propose that collagenase-1 acting on its principal
substrate in the dermis, type I collagen, provides migrating
keratinocytes with a mechanism to maintain their course
and directionality in the wound environment during reepithelialization. In intact skin, basal keratinocytes constitutively express the type I collagen-binding integrin 2
1
along their basolateral surfaces (Hertle et al., 1991
; Symington et al., 1993
). In wounded epidermis, migrating keratinocytes continue to express their collagen-binding receptors (Cavani et al., 1993
; Hertle et al., 1992
; Juhasz et al.,
1993
), but
2
1 becomes redistributed and concentrated
at the frontobasal end of the cells (Guo et al., 1991
). This
redistribution places
2
1 where it would likely come into
intimate contact with dermal type I collagen. Although
basal keratinocytes also express
1
1 and
3
1, keratinocytes preferentially use
2
1 to bind to type I collagen
(Lange et al., 1994
). Indeed, we demonstrate that blocking the ability of this integrin to bind type I collagen inhibits collagenase-1 expression and, consequently, keratinocyte
migration, whereas blocking ligand interaction to
1
1 or
3
1 did not affect enzyme production or cell movement
(Fig. 8). In contrast, Zhang and Kramer (1996)
reported
that blocking antibodies to
3
1 inhibited keratinocyte
migration on collagen. However, in their assays, inhibition
of cell movement was seen at a concentration of collagen at which we have demonstrated to be a weak stimulator of
collagenase-1 expression (Saarialho-Kere et al., 1993a
). At
higher concentrations of collagen, cell migration was barely
inhibited (Zhang and Kramer, 1996
). Still, their data suggest that
3
1 plays a role in keratinocyte migration, but
our findings indicate that this integrin does not influence
collagenase-1 expression or collagenase-1-dependent motility.
2
1 binds native collagen with high affinity (Staatz et al.,
1989
), and thus, clustering of this integrin at the forward edge of keratinocytes may actually tether the cells to the
matrix, rendering them unable to migrate. Therefore, the
proteolytic activity of collagenase-1 may aid in dissociating
keratinocytes from these high-affinity attachments to a
collagen matrix. As stated, collagenase-1 does not degrade
fibrillar type I collagen but rather makes a single, site-specific cleavage through the triple helix about 3/4 the length
from the NH2 terminus. The resultant fragments, called
TCA and TCB fragments, are thermally unstable at body
temperature and may spontaneously denature into gelatin.
Besides being highly susceptible to complete degradation
by different proteinases, gelatin binds
2
1 with a much
lower affinity than does native collagen (Staatz et al., 1989
).
Thus, we speculate that by cleaving type I collagen, which
then denatures into gelatin, collagenase-1 effectively mediates the loosening of the tight contacts keratinocytes
may establish with the dermal matrix. This function is distinct from the often suggested idea that migrating cells use
metalloproteinases to remove matrix barriers that may
physically impede movement.
Although keratinocytes may actually migrate on other
molecules found in the provisional wound matrix, such as
fibronectin and vitronectin, the proteolytic activity of collagenase-1 is still needed to initiate and maintain this process. Basal HaCaT cells did not express collagenase-1 (Fig.
4 A) and did not migrate on a collagen matrix, even
though serum adhesive proteins were present (Figs. 3 and
4). These cells did, however, migrate easily on a gelatin
matrix (Fig. 3), and stimulation of collagenase-1 expression
by exogenous EGF induced migration of HaCaT cells on type I collagen (Fig. 4). Furthermore, we were able to
block migration of primary human keratinocytes by any
method that perturbed the ability of collagenase-1 to
cleave collagen, be it addition of hydroxymate inhibitors,
collagenase-1-blocking antibodies, or TIMP-1 or plating
cells on collagenase-resistant collagen (Figs. 5, 6 and 7). In
addition, blocking the induction of collagenase-1 expression in primary keratinocytes by treatment with 2 antibody inhibited keratinocyte migration. We do not interpret these data to indicate that
2
1 binding to collagen is
directly required for keratinocyte migration, but rather
that this cell-matrix interaction mediates induction of collagenase-1, which, in turn, is essential for cell movement.
As stated, we propose that collagenase-1 facilitates keratinocyte migration by affecting the conformation of type I collagen and, consequently, the avidity with which cells interact with it. One may argue that this is an inherently inefficient mechanism. If activated keratinocytes migrate over the viable dermis interacting with provisional matrix proteins, then why do they need to cleave type I collagen? Why would they adhere to the dermis with high affinity if their objective is to close the wound as quickly as possible? The answer, we believe, is that the process of interacting with and then cleaving type I collagen provides keratinocytes with a mechanism to determine and maintain their directionality during reepithelialization.
An important observation relevant to our directionality
hypothesis is that collagenase-1 production is induced in
keratinocytes by native type I collagen but not by denatured forms of the molecule (see Fig. 6 B and Sudbeck et
al., 1994). Thus, collagenase-1 acting on collagen creates a
mediator that does not support or maintain its own production. The conversion of collagen to gelatin would replace the inductive stimulus with a neutral substrate (gelatin), and in stationary cells, collagenase-1 expression would
decline. Indeed, collagenase-1 expression is rapidly turned
off at the completion of reepithelialization (Inoue et al.,
1995
). Although cell-cell contacts may be involved in this
process, the initial expression of collagenase-1 may mediate cleavage of the collagen substrate, thereby neutralizing
the inductive effect of the underlying matrix. If keratinocytes
continue to interact with type I collagen, presumably by migrating, then they would continue to express collagenase-1.
During wound healing in vivo, collagenase-1 cleavage of
collagen would leave a trail of denatured collagen (gelatin) that would not attract keratinocyte attachment. Using
high-affinity interactions with native type I collagen as a
"molecular compass," keratinocytes could then bind to
components of the provisional matrix to support motility.
Because gelatin does not induce or maintain collagenase-1
expression, keratinocytes would not have the proteinase
needed to invade into the dermis if they would begin to
stray. The activity of collagenase-1 would allow keratinocytes to break away from collagen while it continually extends and interacts with new native collagen molecules
present in the superficial plane of the viable dermis. In a
stratified epithelium, cell migration is thought to proceed
in a leap-frog fashion, whereby the cell at the front extends
along and attaches to the matrix and is replaced by cells
coming from behind and above (see Stenn and Malhotra, 1992). Thus, these interrelated collagenolytic and migratory processes may occur within a limited microenvironment.
Received for publication 28 October 1996 and in revised form 27 November 1996.
1. Abbreviations used in this paper: HU, hydroxyurea; MMP, metalloproteinase; SCID, severe combined immunodeficiency; TIMP-1, tissue inhibitor of metalloproteinases-1.We thank Dr. Dan Getman and his associates at Monsanto-Searle for the synthetic metalloproteinase inhibitors, Dr. Alice Pentland (University of Rochester, Rochester, NY) and her associates for their assistance in obtaining tissue for keratinocyte cultures, Drs. Horace DeLister and Steven Albelda for wound samples grafted onto SCID mice, Dr. Norbert Fusenig for the HaCat keratinocytes, Dr. David Carmichael for the recombinant human TIMP-1, Dr. J. Michael Shipley for 92-kD gelatinase catalytic domain, Dr. Ulpu Saarialho-Kere and Ms. Jill Roby (Barnes Jewish Hospital) for assistance with in situ hybridization studies, and Catherine Fliszar (Barnes-Jewish Hospital) for assistance with enzyme activity assays.
This work was supported by grants from the National Institutes of Health (NIH) and by a Monsanto-Searle/Washington University Research Award. Dr. Pilcher is the recipient of an NIH Individual National Research Service Award.
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