(Received for publication, March 4, 1997, and in revised form, May 8, 1997)
From the Collagenase-1 is invariantly expressed by
migrating basal keratinocytes in all forms of human skin wounds, and
its expression is induced by contact with native type I collagen.
However, net differences in enzyme production between acute and chronic
wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a
dose-dependent manner. However, KGF was >100-fold more
effective than bFGF at inhibiting collagenase-1 expression, suggesting
that this differential signaling is transduced via an FGF receptor that
binds these ligands with different affinities. Reverse
transcriptase-polymerase chain reaction analysis of human keratinocyte
mRNA for fibroblast growth factor receptors (FGFRs) revealed
expression of only FGFR-2 IIIb, the KGF-specific receptor, which also
binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF
or KGF. FGFRs that bind bFGF with high affinity were not detected. Our
results suggest that bFGF and KGF inhibit collagenase-1 expression
through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF
induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix
metalloproteinases in a tissue-specific manner.
Wound repair is a highly organized process that requires a series
of spatially and temporally regulated events to heal a tissue defect.
Among these, effective proteolytic degradation of extracellular matrix
(ECM)1 macromolecules by various proteases
is necessary to remodel the damaged tissue, promote neovascularization,
and facilitate efficient migration of cells during re-epithelialization
(1). Yet, in chronic ulcers, the overproduction of matrix-degrading
proteases and/or the lack of production of their natural inhibitors
probably contributes to the underlying pathogenesis of the non-healing state by interfering with normal repair processes and by perpetuating matrix destruction.
Matrix metalloproteinases (MMPs) constitute a family of
zinc-dependent enzymes that collectively have the capacity
to degrade virtually all components of the ECM (2). While most members of this family possess overlapping substrate specificities, the metallocollagenases, a subgroup of the MMP gene family, have the unique
ability to initiate cleavage of fibrillar collagens I, II, and III at a
specific locus in their triple helical domain. At physiologic
temperature, cleaved collagen molecules denature into gelatin and
become susceptible to further digestion by other proteases. Of the
three known human collagenases, collagenase-1 (MMP-1) is the enzyme
principally responsible for collagen turnover in most tissues and, in
particular, the skin.
Previous studies from our laboratories and others have shown that basal
keratinocytes at the leading edge of migration in both normally healing
wounds and chronic ulcers invariantly express collagenase-1 (3-5).
Signal for collagenase-1 is confined to the basal layer of epidermis,
diminishes progressively away from the wound edge, and is absent in
intact skin. Furthermore, collagenase-1 expression is rapidly induced
in wound edge keratinocytes after injury, persists during the healing
phase, and ceases following wound closure (6). In chronic, non-healing
wounds expression of this MMP is prominent and excessive, whereas in
normally healing wounds its expression is transient and localized
precisely to areas of active re-epithelialization (3, 7). We have
demonstrated that collagenase-1 expression by basal keratinocytes is
induced following contact with native type I
collagen,2 and the activity of this enzyme
is required for cell migration (9). Thus, expression of
matrix-degrading enzymes by keratinocytes during cutaneous wound repair
is a normal and programmed response to injury, and altered cell-matrix
interactions may play a critical role in regulating this response.
In addition to cell-matrix interactions, soluble mediators present in
the ECM during wound repair may influence collagenase-1 expression.
Keratinocyte collagenase-1 production is stimulated by several growth
factors including transforming growth factor- Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth
factor (KGF, FGF-9) belong to a family of heparin-binding growth
factors that exert a variety of effects on multiple cell types (16).
bFGF is widely expressed in vivo, is a potent angiogenic factor, and induces collagenase-1 production by cultured fibroblasts (17, 18), endothelial cells (19, 20), and osteoblasts (21). In
addition, bFGF stimulates growth and proliferation of human
keratinocytes (22, 23). In contrast, KGF is expressed exclusively by
cells of mesenchymal origin, such as fibroblasts (24) and microvascular
endothelial cells (25), yet it specifically influences epithelial cells
by a paracrine signaling mechanism (24, 26, 27). Both bFGF and KGF are
expressed during epidermal wound repair (28, 29), and topical
application of bFGF to wounds accelerates healing (30). Likewise,
inhibition of KGF signaling in basal keratinocytes of epidermis
following injury impairs re-epithelialization, presumably by inhibiting
keratinocyte proliferation (31).
In this report, we demonstrate that bFGF and KGF down-regulate
collagenase-1 expression by keratinocytes in a cell type-specific manner. Additionally, we show that KGF is >100-fold more potent than
bFGF in suppressing collagenase-1 production and that keratinocytes express only two fibroblast growth factor receptors (FGFRs): FGFR-3 IIIb, which does not bind bFGF or KGF, and FGFR-2 IIIb, which binds KGF
with high affinity, but poorly to bFGF. Thus, bFGF and KGF inhibition
of keratinocyte collagenase-1 expression probably occurs exclusively
through the KGF (FGFR-2 IIIb) receptor.
Recombinant human bFGF, recombinant human KGF,
and a polyclonal neutralizing antiserum to bFGF were obtained from
R & D Systems (Minneapolis, MN). Bovine type I collagen
(Vitrogen-100) was purchased from Celltrix Laboratories (Palo Alto,
CA).
Human
keratinocytes were harvested from healthy adult skin from reduction
mammoplasties or abdominoplasties as described previously (15, 32).
Briefly, the subcutaneous fat and deep dermis were removed, and the
remaining tissue was incubated in 0.25% trypsin in phosphate-buffered
saline. After 16 h, the epidermis was separated from the dermis
with forceps, and the keratinocytes were scraped into Dulbecco's
modified Eagle's medium. The keratinocyte suspension was added to
fresh Dulbecco's modified Eagle's medium supplemented with 5% fetal
calf serum and 0.1% penicillin/streptomycin. A specified amount of
keratinocyte suspension was then plated onto tissue culture dishes
coated with 1 mg/ml Vitrogen. Under these culture conditions, the
keratinocytes proliferate, migrate, differentiate, and cornify similar
to cells in vivo. Growth on native type I collagen is
necessary for induction of collagenase-1 and keratinocyte adhesion (5,
8, 15).
The amount of
collagenase-1 accumulated in keratinocyte-conditioned medium was
measured by indirect competitive ELISA (33). This ELISA is completely
specific for collagenase-1, has nanogram sensitivity, and detects both
active and zymogen enzyme forms, as well as collagenase-1 bound to
tissue inhibitor of metalloproteases (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.
Postconfluent keratinocytes plated on
type I collagen were cultured for 24 h in the presence of
Dulbecco's modified Eagle's medium/fetal calf serum containing
control or experimental solutions. The culture wells were then washed
and replaced with methionine-free Dulbecco's modified Eagle's medium
containing 5% dialyzed fetal calf serum (to remove free amino
acids), 1 mM sodium pyruvate, 2 mM
L-glutamine, 0.1 mM each of nonessential amino
acids, 50 µCi/ml [35S]methionine (ICN Radiochemicals,
Irvine CA), and the identical concentrations of experimental reagents.
Conditioned medium was collected after 24 h and stored at
Specific
polyclonal antisera to collagenase-1 (11), stromelysin-1 (34), 92-kDa
gelatinase (35), or TIMP-1 (36) were used to immunoprecipitate the
35S-labeled metalloproteinases from
keratinocyte-conditioned medium as described (37). Samples were
precleared with protein A-Sepharose (Zymed, San Francisco, CA), and
supernatants were incubated with antibody for 1 h at 37 °C and
then overnight at 4 °C. Immune complexes were precipitated with
protein A-Sepharose and washed extensively. Radiolabeled proteins were
resolved by polyacrylamide gel electrophoresis and visualized by
fluorography. Total incorporated radioactivity was determined from the
same conditioned medium by trichloroacetic acid precipitation.
Total RNA was
isolated from cultured keratinocytes by phenol-chloroform extraction
(38). RNA (5 µg) was denatured and resolved by electrophoresis
through a 1% formaldehyde-agarose gel, transferred overnight to Hybond
N+ (Amersham Corp.), and hybridized with radiolabeled collagenase-1
(39) and GAPDH cDNA probes. The cDNA probes were labeled by
random priming (Boehringer Mannheim, Mannheim, Germany) with
[ To determine which FGFRs
were expressed by both human keratinocytes and fibroblasts, total RNA
was harvested as above. RNA was treated with RQ1 RNase-free DNase
(Promega, Madison, WI) to remove any contaminating DNA as described
(40). DNase-treated RNA (5 µg) was reverse transcribed with random
hexamers using kit reagents and under the manufacturer's recommended
conditions (GeneAmp RNA PCR kit, Perkin-Elmer, Norwalk, CT). For each
sample, a parallel reaction was run without reverse transcriptase as a control.
Expression of FGFRs 1-4 in human keratinocytes was detected by
polymerase chain amplification of cDNA using a single primer pair
to amplify conserved sequences in the tyrosine kinase domain of
all FGFRs (41). The primer sequences used for PCR were DO156 (5 To determine the expression of FGFR-2 isoforms (IIIb and IIIc) by human
keratinocytes and fibroblasts, we amplified random primed cDNA with
specific primers as described (42). Briefly, the cDNA was amplified
for FGFR-2 IIIb using the 5 To determine the expression of FGFR-3 isoforms (IIIb and IIIc) by human
keratinocytes and fibroblasts, we amplified random primed cDNA with
specific primers as described (45). The 5 Previous reports have documented the
capacity of bFGF to stimulate collagenase-1 production in cells of
mesenchymal origin (18, 19, 46, 47). Consistent with these studies, we
found that bFGF increased collagenase-1 production by human dermal
fibroblasts in a dose-dependent manner (Fig.
1A). At 1.0 ng/ml bFGF, collagenase-1 production was augmented 5-fold over control levels. To assess if bFGF
modulates keratinocyte collagenase-1 production, cells were exposed to
increasing concentrations of growth factor for 72 h, and
collagenase-1 accumulation in the medium was quantified by ELISA. In
contrast to other cell types, bFGF potently inhibited keratinocyte
collagenase-1 expression, with an ED50 of ~1.0 ng/ml (Fig. 1B). Preincubation with anti-bFGF neutralizing
antiserum abolished collagenase-1 down-regulation (Fig. 1C),
thus demonstrating that the effect was due to the growth factor itself
and not to a contaminant.
Metabolic labeling and immunoprecipitation experiments confirmed that
bFGF inhibited keratinocyte collagenase-1 production at the level of
new enzyme synthesis (Fig. 2A).
Immunoprecipitation of the same conditioned media for stromelysin-1
showed similarly reduced expression of this MMP (Fig. 2B),
whereas the synthesis of 92-kDa gelatinase and TIMP-1 was unchanged
(data not shown). Inhibition of collagenase-1 and stromelysin-1
expression was specific, since synthesis of total secreted proteins by
keratinocytes increased slightly following bFGF treatment (Table
I). The disparity in bFGF concentrations required to
effectively inhibit keratinocyte collagenase-1 production in Figs. 1
and 2 reflect the individual skin donors examined, whom we have found
to exhibit variable sensitivities to the growth factor.
Table I.
Total protein synthesis
Division of Dermatology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TGF-
)/epidermal
growth factor (10), hepatocyte growth factor/scatter factor (11),
transforming growth factor
1 (TGF-
1) (12, 13), and interferon-
(14). Furthermore, several of these growth factors (e.g.
epidermal growth factor and hepatocyte growth factor/scatter factor)
can augment ECM-directed collagenase-1 expression by keratinocytes (11,
15). In effect, while cell contact with specific matrices establishes
the primary "on and off" signals, soluble mediators may finely
control the net output of collagenase-1 by keratinocytes.
Materials
70 °C for analysis by immunoprecipitation.
-32P]dCTP (NEN Life Science Products). Following
hybridization, the membranes were washed and exposed to x-ray film for
an appropriate duration.
-TCNGAGATGGGAGRTGATGAA-3
) and DO158 (5
-CCAAGTCHGCDATCCTTCAT-3
), which produce a 341-bp product. PCR was for 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a final
extension step of 72 °C for 7 min. To determine which members of the
FGFR family were expressed, PCR products were analyzed by restriction
digestion analysis with PstI, BalI,
ScaI, or NarI. Digested fragments were separated
by nondenaturing polyacrylamide gel electrophoresis and visualized by
silver staining.
S primer corresponding to a region within
the FGFR-2 IIIb-specific exon K: 5
-CAATGCAGAAGTGCTGGCTCTGTTCAA-3
. FGFR-2 IIIc was amplified using the 5
S primer corresponding to a
region within the FGFR-2 IIIc specific exon B: 5
-GTTAACACCACGGACAA-3
. The 3
AS primer used in both PCR reactions was from nucleotides 2093-2112 of the cDNA coding for FGFR-2 IIIb. The same 3
AS primer was used for amplification of both FGFRs, since the nucleotide sequence
is identical for both isoforms in this region (27, 43, 44). PCR was for
40 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a final extension step of 72 °C for 7 min. The
predicted fragment size was 822 bp for the FGFR-2 IIIb and 830 for
FGFR-2 IIIc. The products were separated through a 2% agarose gel and
visualized by ethidium bromide staining. Further specificity was
determined by transfer to Hybond N+ followed by Southern hybridization
with a radiolabeled product-specific oligonucleotide probe. The probe
was labeled by terminal transferase (Boehringer Mannheim) with
[
-32P]dCTP. Following hybridization, the membranes
were washed and exposed to x-ray film for an appropriate duration.
S primer used
(5
-GCACCGGCCCCATCCTGCAGGCGG-3
) corresponds to nucleotides 789-811 of
the human FGFR-3 gene, and the 3
AS primer used
(5
-TACACACTGCCCGCCTCGTCAGC-3
) corresponds to nucleotides 1135-1158
of the FGFR-3 gene, generating a product with a predicted size of 369 bp. PCR was for 30 cycles of 94 °C for 1 min, 68 °C for 1 min,
and 72 °C for 1 min followed by a final extension step of 72 °C
for 7 min. Following amplification, products were analyzed by
restriction digestion analysis with HaeII and
TaqI, allowing subsequent identification of IIIb and IIIc
isoforms. Products were separated by agarose gel electrophoresis, transferred to Hybond N+, and hybridized with a radiolabeled
product-specific oligonucleotide probe. The probe was labeled by
terminal transferase with
[32P]dCTP. Following
hybridization, the membranes were washed and exposed to x-ray film for
an appropriate duration.
bFGF Inhibits Collagenase-1 Production by Keratinocytes in a Cell
Type-specific Manner
Fig. 1.
bFGF inhibits keratinocyte collagenase-1
production in a cell type-specific and dose-dependent
manner. Human dermal fibroblasts (A) were cultured on
tissue culture plastic and keratinocytes (B and
C) on type I collagen until confluent. Increasing
concentrations of bFGF were added to the cell cultures, and
collagenase-1 protein accumulated in the conditioned media after
72 h of incubation was quantified by ELISA. In C, cells
were cultured on type I collagen alone (control), or in the presence of
bFGF neutralizing antiserum (10 µg/ml). bFGF (25 ng/ml) was
preincubated with antibody 2 h prior to addition to cultures. Cell
layers were analyzed for total protein content as described under
"Experimental Procedures." Data shown are the means of triplicate
observations from the same cell preparation.
[View Larger Version of this Image (16K GIF file)]
Fig. 2.
bFGF inhibits biosynthesis of keratinocyte
collagenase-1 and stromelysin-1. Human keratinocytes were cultured
on type I collagen until confluent. Cellular proteins were
metabolically labeled as described under "Experimental Procedures,"
and conditioned medium was analyzed for the presence of collagenase-1
(A) or stromelysin-1 (B) with specific antisera.
Cells were treated with 1.0 or 25 ng/ml of bFGF as indicated. Results
from one representative experiment of two different cell preparations
analyzed are shown.
[View Larger Version of this Image (52K GIF file)]
Condition
Protein synthesisa
Control
12,258 ± 1068
bFGF (1.0 ng/ml)
13,279 ± 2167
bFGF (25 ng/ml)
16,622 ± 956
KGF (1.0 ng/ml)
16,220 ± 3723
KGF (10 ng/ml)
15,237
± 676
a
Values for protein synthesis are in trichloracetic
acid-precipitable counts/min.
Total RNA was isolated from keratinocytes that
had been treated for 24 h in the absence or presence of bFGF (25 ng/ml) and was analyzed by Northern hybridization. bFGF inhibited
steady-state collagenase-1 mRNA levels, causing a 68% reduction
when compared with untreated controls (Fig.
3A). Identically treated cells were cultured
for 48 h, and collagenase-1 protein was quantified by ELISA (Fig.
3B). bFGF inhibited collagenase-1 protein expression (57%)
proportionally to the drop in mRNA levels (68%), indicating pretranslational regulation.
KGF Inhibits Keratinocyte Collagenase-1 Production
Although
bFGF consistently inhibited keratinocyte collagenase-1 expression, we
often had to use relatively high concentrations (10 ng/ml) of the
growth factor to observe this activity (Fig. 2 and other data not
shown). Because multiple FGFs bind to more than one FGFR with different
affinities (48-51), we postulated that other members of the FGF family
might be more potent inhibitors of collagenase-1 production. KGF, a
mesenchymal cell-derived cytokine that acts specifically on epithelial
cells (24), was chosen as a candidate because of its relevance to
epidermal wound repair. Paralleling the effects of bFGF, treatment of
cultured keratinocytes with increasing concentrations of KGF resulted
in a dose-dependent inhibition of collagenase-1 expression
(Fig. 4A). Futhermore, KGF was more potent,
consistently demonstrating an ED50 of ~0.01 ng/ml, at
least 100-fold lower than bFGF.
As demonstrated by metabolic labeling and immunoprecipitation, KGF inhibited collagenase-1 production (Fig. 4B). Again, KGF was effective at lower concentrations than bFGF (Fig. 4A versus Fig. 1B). As observed for bFGF, stromelysin-1 biosynthesis was also inhibited by KGF treatment, whereas 92-kDa gelatinase and TIMP-1 were unaffected (data not shown). Total protein synthesis was mildly increased by KGF (Table I), indicating the specificity of its collagenase-related activity.
KGF Inhibits Keratinocyte Collagenase-1 Expression PretranslationallyNorthern hybridization was performed to
determine if KGF inhibited collagenase-1 production in a manner similar
to bFGF. Keratinocytes treated with KGF (1.0 ng/ml) displayed a
dramatic reduction in collagenase-1 mRNA compared with untreated
cells (Fig. 5A). To compare KGF inhibition of
collagenase-1 mRNA with collagenase-1 protein, conditioned media
samples from the same skin donor were analyzed by ELISA. Quantitation
demonstrated that inhibition of secreted collagenase-1 protein closely
paralleled decreased mRNA levels (Fig. 5B; 69 versus 70%, respectively).
Inhibition of Keratinocyte Collagenase-1 Expression by bFGF and KGF Is Transduced through the KGF Receptor
FGFs activate a family of four receptor tyrosine kinases, which bind each member with different affinities (48, 49, 51, 52). Further specialization of these receptors occurs through alternative mRNA splicing, leading to unique ligand binding properties (27, 52, 53). We examined FGFRs present on keratinocytes to determine whether cell surface receptor expression could explain the differences in ED50 between bFGF and KGF required to obtain an equivalent inhibition of collagenase-1 expression by keratinocytes.
We used established reverse transcriptase-PCR methods to determine
which members of the FGFR family are expressed by human keratinocytes
(41, 42, 45). Total RNA was isolated from keratinocytes of two separate
skin donors, and a random-primed cDNA library was generated by
reverse transcription. Amplification of the cDNA using a single
primer pair to generate all FGFRs yielded the expected 341-bp fragment
(Fig. 6A). As seen by differences in band
intensities using EtBr staining, levels of FGFR expression varied among
the two populations of keratinocytes. To distinguish among keratinocyte
FGFRs 1-4, PCR products were analyzed by restriction digestion
analysis. Cultured keratinocytes expressed similar levels of FGFRs 2 and 3 but did not express FGFRs 1 and 4 (Fig. 6B). Extraneous bands of sizes different from that predicted were seen following silver staining. These bands probably resulted from low level
contamination by genomic DNA. Our assignment of receptor isotypes
remains unchanged, however, because nonspliced regions produce
fragments larger than those predicted by restriction digestion. The
expression of only FGFRs 2 and 3 was a consistent finding among several
skin donors (n = 5), but, as previously stated, expression levels varied among samples.
Alternative splicing of primary transcripts of FGFRs 1-3 generates cell surface receptors having unique sequences within the ligand-binding Ig-like domain III (52-54). These isoforms, designated IIIb and IIIc, have distinct ligand affinities that regulate FGF signaling (51). Because cultured keratinocytes expressed only FGFRs 2 and 3 (Fig. 6), we determined which isoform(s) (IIIb or IIIc) of each receptor were expressed.
Alternative splicing of FGFR-2 produces two distinct isoforms: FGFR-2
IIIb and FGFR-2 IIIc (27, 44). The IIIb isoform binds KGF with high
affinity but does not efficiently bind to bFGF. In contrast, FGFR-2
IIIc affinity for bFGF is high, whereas KGF does not bind (51). FGFR-2
IIIb, but not FGFR-2 IIIc, was expressed by primary keratinocytes as
demonstrated by EtBr staining (Fig. 7, A and
C). In contrast, human foreskin fibroblasts expressed only
FGFR-2 IIIc (Fig. 7, A and C). Specificity
was verified by Southern hybridization with a product-specific
oligonucleotide probe (Fig. 7, B and D). These
data agree with our findings that equivalent inhibition of keratinocyte
collagenase-1 expression required much higher concentrations of bFGF
than KGF and that keratinocytes and fibroblasts exhibited different
responses to bFGF.
Similar to FGFR-2, alternative splicing of FGFR-3 primary transcripts
results in two distinct isoforms, FGFR-3 IIIb and FGFR-3 IIIc (53, 55,
56). FGFR-3 IIIb does not bind bFGF or KGF, whereas FGFR-3 IIIc binds
bFGF with high affinity but does not bind to KGF (51). Keratinocytes
expressed only FGFR-3 IIIb, as determined by restriction digestion
analysis and EtBr staining of PCR products (Fig.
8A). Specificity was verified by Southern hybridization with a product-specific oligonucleotide probe (Fig. 8B).
The precise regulation of MMP expression is critical for normal wound repair and for maintaining tissue homeostasis. Aberrant expression following tissue injury may lead to a failure of healing. Indeed, we have demonstrated increased expression of collagenase-1 and stromelysin-1 in certain ulcerative skin lesions when compared with normally healing wounds (34, 57, 58). Furthermore, inflammatory/proliferative diseases, such as rheumatoid arthritis (59), are associated with unregulated production of MMPs, leading to widespread matrix destruction. Therefore, precise control of MMP expression in multiple cell types is necessary to maintain proper tissue organization and to promote events essential to postinjury repair.
Previous reports from our laboratories and others have shown that
expression of collagenase-1 during cutaneous wound repair is restricted
to basal keratinocytes at the leading edge of re-epithelialization (3,
4, 57, 60). These cells are in contact with dermal ECM (5, 7), and
collagenase-1 production by keratinocytes in vitro is
primarily induced by contact with native type I collagen (7, 8),
facilitating cell migration on this matrix (9). Three human
interstitial collagenases have been reported to date. 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 (3, 4, 57, 61, 62). In contrast,
expression of collagenase-2 (MMP-8) is limited to neutrophils and
chondrocytes (63, 64), and collagenase-3 (MMP-13), originally cloned
from a breast carcinoma cell line (65), is expressed in articular
cartilage (66, 67) and developing bone (68). Recent studies by
Johansson et al. (69) have reported expression of
collagenase-3 by HaCaT keratinocytes following treatment with TGF-
and TGF-
. In contrast, however, primary human epidermal keratinocytes fail to express both collagenase-2 and -3, and our results confirm these observations (data not shown), thereby suggesting that collagenase-1 is the principal collagen-degrading enzyme produced
by keratinocytes during repair.
In addition to cell-matrix interactions, soluble factors present within the extracellular environment may also play an important role in regulating the expression of MMPs by keratinocytes (10, 11, 13, 14, 70). Indeed, in this report we demonstrate that members of the FGF family inhibit the production of collagenase-1 by keratinocytes. Perhaps more interesting, however, are the findings that inhibition by these growth factors is cell type-specific and that ligand signaling most likely occurs through the KGF binding isoform (IIIb) of FGFR-2.
The molecular mechanisms responsible for cell type-specific regulation
of MMP expression may be numerous and distinct. For example,
intranuclear events mediate TGF-1 inhibition of collagenase-1 production in fibroblasts and its induction in keratinocytes (13). Mauviel et al. (13) demonstrated that distinct
jun trans-activating factors result in the differential
regulation of collagenase-1 transcription in these two cell types.
Although not reported to date, other cell-specific post-receptor signal
transduction pathways could also mediate the different responses of
cell types to a soluble factor. Furthermore, responses of distinct cell
types to extracellular cation concentrations also regulate MMP
production. Indeed, increased intracellular Ca2+ induces
collagenase-1 in fibroblasts (71), whereas its secretion is inhibited
in keratinocytes (72). Finally, the binding of a single cytokine or
growth factor to distinct cell-surface receptors provides yet another
potential pathway for cell-specific MMP regulation. Many studies had
previously shown bFGF to induce the expression of MMPs in various cell
types, including fibroblasts, smooth muscle cells, osteoblasts, and
endothelial cells (17-21). Indeed, bFGF has been regarded as a
prototypic MMP-inducing agent. Our findings represent the first report
demonstrating the inhibition of MMP expression by any member of the FGF
family and also the first report of cell-specific responses in MMP
expression mediated by a single ligand's binding to different
receptors on two distinct cell types.
Members of the FGF family have the capacity to activate up to four receptor tyrosine kinases (48, 49, 52). In addition, FGFRs 1-3 undergo alternative mRNA splicing, generating IIIb and IIIc isoforms (27, 52, 53). Thus, regulation of cell signaling results from different ligand binding affinities of each receptor variant (51). Because KGF is produced in high quantities by dermal fibroblasts underlying the edges of the wound bed (29), we examined its capacity to regulate collagenase-1 expression by keratinocytes in vitro. We found that KGF inhibited keratinocyte collagenase-1 production and that it was effective at >100-fold lower concentrations than bFGF. We next observed that human keratinocytes in vitro expressed only FGFR isoforms that bind KGF with high affinity (FGFR-2 IIIb) and bind bFGF very weakly (FGFR-2 IIIb and FGFR-3 IIIb). Of the isoforms that bind weakly to bFGF, FGFR-2 IIIb does so with slightly higher affinity than FGFR-3 IIIb (51). We therefore propose that in keratinocytes, bFGF and KGF signal through FGFR-2 IIIb (the KGF receptor), accounting for the requirement of increased concentrations of bFGF when compared with KGF to obtain an equivalent level of collagenase-1 inhibition. Furthermore, human fibroblasts expressed FGFR-2 IIIc, which displays high binding affinity for bFGF but fails to complex KGF, and presumably mediates collagenase-1 up-regulation in these cells.
Expression of different FGFR isoforms in distinct compartments of the skin may contribute to spatially localized expression of MMPs. Regulation of FGFR isoforms is cell type-specific, with exon b expression limited to epithelial cells and exon c expression limited to cells of mesenchymal origin (56, 73, 74). Thus, expression of c isoforms by dermal fibroblasts and endothelial cells following tissue injury permits responsiveness to FGFs within the ECM and would promote collagenase-1 production, which is essential to ECM remodeling and angiogenesis. In contrast, production of b isoforms in keratinocytes behind the migrating front of epithelium would inhibit collagenase-1 expression and allow cell proliferation and differentiation.
Recent studies have begun to delineate a role for KGF production during wound repair. Following tissue injury, the expression of KGF is markedly up-regulated by fibroblasts within the damaged dermis and acts in a paracrine manner to stimulate the overlying epithelium (29, 75). Additionally, KGF applied to full-thickness wounds results in increased re-epithelialization associated with epidermal thickening (76), and the targeted overexpression of this growth factor to keratinocytes leads to marked acanthosis (8). In contrast, expression of a dominant negative FGFR-2 IIIb driven by the K-14 promoter in transgenic mice resulted in epidermal atrophy, abnormal hair follicles, and impaired re-epithelialization (31). Taken together, these data suggest that the primary influence of KGF following injury is to promote proliferation and differentiation of basal keratinocytes.
Our data suggest that KGF may restrict keratinocyte MMP expression after wounding, thereby preventing the excessive degradation of the ECM. Interestingly, KGF receptors are expressed throughout the full thickness of intact skin. Upon wounding, receptor expression is dramatically decreased in migrating keratinocytes, and this pattern persists throughout the healing phase. However, KGF receptors are still prominently expressed by proliferating basal cells just behind the migrating front and in noninvolved areas of epidermis (75). When injury results in the production of KGF by underlying dermal fibroblasts, keratinocytes at the edge of tissue damage augment their basal proliferating phenotype, supplying new cells for the migrating front. Inhibition of MMP expression by KGF in these proliferating wound edge keratinocytes may be needed to prevent the degradation of reforming basement membrane or the aberrant destruction of underlying ECM. Because migrating wound keratinocytes have markedly down-regulated KGF receptor (i.e. FGFR-2 IIIb) expression, KGF would not affect these cells, allowing collagenase-1 production to facilitate migration. Thus, KGF may play a dual role in wound repair, as a factor that stimulates cell proliferation and differentiation at the wound edge but also restricts MMP production to just the actively migrating cells.
We thank Dr. David M. Ornitz and Donald G. McEwen (Washington University, St. Louis, MO), for technical help with reverse transcriptase-PCR assays and critical evaluation of the manuscript; Dr. Alice Pentland (Washington University), for help with obtaining skin for keratinocyte culture; and Dr. Gregory Goldberg (Washington University) for the collagenase-1 cDNA.