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INTRODUCTION |
Heparan sulfate proteoglycans have been shown to have a regulatory
role in a number of cellular processes. This versatility is due to the
ability of glycosaminoglycan side chains to specifically bind proteins
such as growth factors, matrix components, or enzymes (1, 2). For
example, signaling by bFGF1
(3, 4) and wingless (5) is strongly augmented by the presence of cell
surface heparan sulfate (3, 4). This is possibly related to findings
that mutations in the members of glypican family of heparan sulfate
proteoglycans lead to delayed progression of cell cycle in Drosophila
(6), and to an overgrowth syndrome in man (7). Finally, the enzymatic
activity of proteases present in acute wound fluids is regulated by
heparan sulfate (8).
Syndecan-1 is an integral membrane proteoglycan that bears both heparan
and chondroitin sulfate side chains. In adult tissues, it is almost
exclusively found in simple and stratified epithelia. In simple
epithelia, it has been proposed to participate in the maintenance of
the epithelial phenotype (9, 10). In the epidermis, syndecan-1
expression is strongest in the suprabasal cell layers although
undetectable in the cornified layer (11). In cultured keratinocytes,
syndecan-1 mRNA levels increase upon differentiation of the cells
by exposure to high Ca2+ concentrations (12). Additional
regulation takes place at the posttranslational level: the
glycosaminoglycan chains of syndecan-1 are longer in stratified than in
undifferentiated keratinocytes (13). Remarkably, both syndecan-1
protein and mRNA levels are strongly induced in the wound edge
keratinocytes during wound healing (14). This elevated expression in
keratinocytes appears to be specific for syndecan-1, as no changes have
been detected for the other syndecans, although a strong induction of
syndecan-4 occurs in the dermis (15). The opposite situation is found
during the malignant transformation of keratinocytes. Syndecan-1 is
lost in invasive carcinomas and reduced in poorly differentiated
epidermal dysplasias (12, 16). Likewise, syndecan-1 levels are very low
in a cell line derived from a carcinoma induced by chemical carcinogenesis but normal in epithelial cells, such as immortal MCA-3D
keratinocytes (17).
In an effort to understand both the induction of syndecan-1 during
development and repair processes and, on the other hand, the
suppression of its expression in malignancies, we have cloned the
syndecan-1 gene (18) and analyzed the key regulatory regions for the
basal and induced expression (19, 20). Finally, we have found that an
enhancer for syndecan-1 gene (named FiRE for FGF-inducible response
element) is activated in vivo in wound edge keratinocytes
and that in cultured keratinocytes, FiRE can be activated by KGF and
EGF (21, 22). Interestingly, this enhancer element is not sufficient
for the default expression in suprabasal cells: the only epidermal
expression detected in the FiRE-LacZ mice occurs at the wound edge
(22). In this study, we addressed whether different matrix molecules
can modulate the syndecan-1 levels. We were interested in that aspect
of the regulation of syndecan-1 gene for the following reasons. First,
the activation of the syndecan-1 in the migrating epidermal sheet of
the wound edge is not uniform, as the merging frontiers of the two
sheets are syndecan-1-negative (14). Second, the extracellular matrix is a strong candidate to modify the gene expression of wound
keratinocytes that both encounter a new matrix and produce and remodel
it, as shown by the collagen-induced changes in the expression of the interstitial collagenase (23). In this paper, we demonstrate that
fibrillar collagen but not fibronectin suppresses the activation of
syndecan-1 by KGF. EGF and TGF-
, on the other hand, were able to
activate FiRE regardless the matrix. This regulation takes place at
transcriptional level and establishes FiRE as a target for both ECM and
growth factor receptor derived signals.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Growth Factor Treatments--
MCA-3D is a
keratinocyte cell line derived from carcinogen-treated mouse skin (24).
It is not, however, tumorigenic in nude mice (17). MCA-3D cells were
grown in Ham's F-12 medium supplemented with 10% fetal calf serum,
L-glutamine, streptomycin, and penicillin. The effects of
various growth factors were studied in cells starved for 24 or 48 h in Ham's medium without serum or in Ham's medium supplemented in
1% carboxymethyl-Sephadex-eluted fetal calf serum. Growth factors were
purchased from Peprotech and used in a concentration of 10 ng/ml. To
study the effects of ECM molecules, tissue culture plates were coated
overnight at +4 °C with 50 µg/ml fibrillar collagen (Vitrogen-100,
Collagen Corp.), fibronectin (Sigma), laminin-1 (EHS-laminin), or
native bovine serum albumin (Sigma). Cell proliferation was determined
as the incorporation of [125I]dUTP (Amersham Pharmacia
Biotech). To this end, the cells were treated with growth factors for
12 h following by pulse labeling with 0.25 µCi/ml
[125I]dUTP for additional 4 h. The cells were washed
two times with phosphate-buffered saline and solubilized in 1 N NaOH. Incorporated radioactivity was measured in a gamma
counter (LKB Wallac).
Analysis of Syndecan-1 Levels--
For the quantitation of
syndecan-1 in the conditioned medium of the MCA-3D keratinocytes,
starved cells were treated with growth factors for 24 h. The
medium was replaced with fresh medium, and cells were incubated for
another 4 h. This medium was then collected, and the cell number
was estimated by crystal violet staining as described in Ref. 25.
Syndecan-1 levels were measured in slot-blotted aliquots of the medium
by monoclonal antibody 281-2 (26) using enhanced chemiluminescence
detection (Amersham Pharmacia Biotech) and was normalized against cell
number for comparison among different growth factor treatments.
RNA Isolation and Northern Blotting--
Keratinocytes were
grown on coated 10-cm tissue culture dishes as described above. Total
RNA was isolated as described (27). Ten-µg aliquots of RNA were
electrophoresed in a 1% agarose-formaldehyde gel, blotted onto nylon
membranes (Hybond N, Amersham Pharmacia Biotech), and sequentially
probed with 32P-labeled DNA fragments for mouse syndecan-1
(28) and rat glyceraldehyde-3-phosphate dehydrogenase (29) using
hybridization conditions recommended by Amersham Pharmacia Biotech.
Reporter Gene Constructs, Transfections, and CAT Assays--
The
construction of the syndecan-1 enhancer-CAT constructs has been
reported earlier (20). The reporter gene construct p-271-FiRE (20)
contains the 280-base pair enhancer and a minimal 98-base pair promoter
of the syndecan-1 gene; the construct pSynProm-FiRE has a longer,
1.1-kilobase pair promoter with the same enhancer; and the construct
pSynProm carries the long promoter only, without the enhancer. The
creation of stably transfected cell populations of MCA-3D keratinocytes
was performed as in Ref. 19. The multiclonal cell lines selected for
for CAT assays were pools of independent cell clones. The rationale for
this was to avoid position effect in individual insertion events. The
cells were selected in 750 µg/ml G418 and maintained in 200 µg/ml
G418. In this study, cells up to sixth passage after pooling were used.
Cells were cultured and treated with growth factors as described above.
Chloramphenicol acetylatransferase activity was measured in cell
extracts as described (19). The values were corrected for the total
protein content of the extract (30). Anisomycin, an activator of JNK,
was used over a range of 30-40 µM to determine the role
of Jun amino-terminal kinase in enhancer activation.
Cell Extracts and Gel Mobility Shift Assays--
Whole cell
extracts were prepared from growth factor-treated and control cells
(20). The double-stranded oligonucleotides corresponding to the
transcription factor binding sites in the enhancer for syndecan-1 gene
and the conditions used in the binding reactions have been previously
described in Refs. 19 and 20. The specificities of the complexes were
determined by using a 100-fold excess of either specific or nonspecific
unlabeled competitors in the binding assays. Polyclonal antibodies from
Santa Cruz Biotechnology (indicated in the legends for figures 6 and 7)
were used to characterize the transcription factors present in the
specific complexes as described (20).
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RESULTS |
KGF Fails to Activate FiRE in Cells Plated on Fibrillar
Collagen--
We had previously seen that the enhancer for syndecan-1
gene (the FiRE) is activated in wound edge keratinocytes in
vivo and that the FiRE can drive reporter gene expression after
KGF or EGF treatment (21, 22). At the wound edge, syndecan-1 induction is not uniform, however, as the leading front of migrating
keratinocytes remain syndecan-1-negative (14). In a search for possible
modulators of syndecan-1 activation in the cellular microenvironment at
wounds, we investigated whether the activation of FiRE is controlled by combined effects of growth factors and extracellular matrix.
We plated polyclonal MCA-3D cell lines harboring stably integrated
FiRE-CAT constructs onto either collagen, laminin, or fibronectin matrices and measured CAT activities in response to growth factor treatment. A consistent induction in the CAT activity was observed in
the cell lines harboring the constructs with the FiRE enhancer when
cells were grown on fibronectin and stimulated by KGF for 6-8 h (Fig.
1, A-C). The enhancer was
placed with the minimal promoter of syndecan-1 gene (p271-FiRE) or a
longer, 1.1-kilobase pair portion of syndecan promoter (pSynProm-FiRE).
The effect of the enhancer on the response to KGF was similar with both
of these promoters. No induction could be seen with the construct having the promoter alone (pSynProm).

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Fig. 1.
Activation of the enhancer for syndecan-1
gene (FiRE) by KGF. Stably transfected polyclonal MCA-3D cell
lines carrying syndecan-1 enhancer/promoter-CAT constructs were plated
on fibronectin, laminin, collagen, or native bovine serum albumin
(BSA), serum-starved, and treated for 6 h or overnight
with 10 ng/ml KGF. A, KGF induces CAT activity in 6 h
from the pPst-FiRE construct with the enhancer and a 0.1-kilobase pair
promoter in cells on fibronectin (FN, gray columns), laminin
(striped columns), BSA (white columns) but not in
cells on fibrillar collagen (COL, black columns).
B, CAT activities from the pPst-FiRE construct accumulated
in 6 or 12 h in cells on fibronectin (open symbols) or
collagen (filled symbols) indicate a delayed response to KGF
on collagen. C, CAT activity in a polyclonal cell line
carrying a construct in which the FiRE enhancer is in the context of a
longer (1.1 kilobase pair) promoter. D, CAT activity of the
1.1-kilobase pair promoter only construct. The results shown represent
mean and range (error bars)of two replicate wells in a
representative experiment out of at least three independent ones.
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Contrary to results with fibronectin, FiRE transfected cells grown on
collagen displayed no induction of CAT activity after the application
of KGF (Fig. 1, A-C). This suppression was targeted to the
FiRE only as the basal CAT activity of the promoter constructs was
similar on either matrix. When the KGF treatment was prolonged to 12 or
24 h, a modest induction could be see on collagen-coated plates,
as well (Fig. 1B). However, the measured CAT activities in
cells on collagen were always remarkably lower than in cells grown on
fibronectin coating. This suppression of the response to KGF was
detected in several serial passages of the cells, was independent on
the plating density of the cells, and was seen in both transient and
stable transfections of the reporter gene constructs (data not shown).
Induction by KGF in cells growing on laminin-1 was over 3-fold. Thus,
laminin-1 could not inhibit the activation of FiRE similarly to
collagen but acted in a manner similar to fibronectin (Fig. 1). The
activation of the enhancer by EGF was independent on the matrix (Fig.
2). Equal CAT activity was seen on all
the matrix molecules after EGF (or TGF-
) treatment. Similarly to the
KGF treatment, the promoter lacking an enhancer was not activated by EGF on any of the matrix molecules (Fig. 2).

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Fig. 2.
Induction of the FiRE activity by EGF.
The same cell lines as in Fig. 3 were plated on the indicated matrix
molecules, serum-starved, and treated with 10 ng/ml of EGF for 6 h. For all the plasmids, control values without the growth factor
represent 1.0 after the correction for total protein levels.
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Silencing of FiRE by Collagen Leads to Loss of Increase of
Syndecan-1 RNA and Core Protein--
To study whether the different
abilities of KGF and EGF to activate FiRE levels in cells on fibrillar
collagen are recapitulated at the mRNA level, we performed Northern
blotting analyses on combined effect of extracellular matrix and the
growth factor action. Cells grown on fibronectin showed increased
syndecan-1 mRNA levels after treatment with KGF for 4 h (Fig.
3A). Interestingly, the
KGF-dependent induction of syndecan-1 mRNA was
inhibited in cells grown on fibrillar collagen (Fig. 3A). In
the case of treatments with EGF, induction of syndecan-1 expression
appeared unaffected by the choice of matrix used (Fig. 3B).
The induction caused by EGF was usually higher than by KGF. Typically,
EGF increased syndecan-1 steady-state mRNA levels 6-8-fold,
whereas KGF resulted in an about 3-4-fold induction (Fig. 3,
A and B). Thus, a similar combined effect of KGF
and collagen was seen in mRNA levels as in reporter gene activation
by FiRE enhancer.

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Fig. 3.
Northern blot analysis of the syndecan-1
mRNA levels in keratinocytes grown on collagen or fibronectin.
Serum-starved cells on either collagen or fibronectin were treated with
10 ng/ml of KGF (A) or EGF (B) for 4 h.
Total RNA was isolated, and 10-µg aliquots were electrophoresed in
formaldehyde agarose gels and sequentially blotted for syndecan-1
(top) and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as a loading control (middle).
Quantitation of the hybridizations (bottom)
(syndecan/glyceraldehyde-3-phosphate dehydrogenase) was preformed with
an image analyzer.
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Syndecan-1 protein levels were measured from MCA-3D keratinocytes grown
on different matrix molecules or on native bovine serum albumin as a
nonspecific adhesive substrate. The concentration of shed syndecan-1
ectodomain in the conditioned medium of the keratinocytes was found to
be directly proportional to the level of the core protein synthesis
(not shown). Thus, the effect of the growth factors on the syndecan-1
protein levels were measured from conditioned medium collected during
the last 4 h of a 24-h growth factor treatment. At a concentration
of 10 ng/ml, KGF, EGF, and TGF-
were the most effective inducers of
the syndecan-1 (Fig. 4). Both EGF and
TGF-
resulted in a marked (over 10-fold) increase in the syndecan-1
ectodomain levels regardless of the matrix used as a substratum for
cell attachment (Fig. 4). Intriguingly, however, the production of
syndecan-1 protein in response to cell treatments with KGF appeared to
be dependent on the matrix. MCA-3D cells grown on fibronectin responded
to KGF by an increase in syndecan-1 production. On the other hand,
cells grown on fibrillar collagen failed to increase syndecan-1 levels
in response to KGF exposure (Fig. 4). Thus, the loss of activation seen
in the reporter gene assays and at the mRNA steady state levels was
finally reflected in the synthesis of syndecan-1 core protein.
Quantitation of syndecan production after growth factor treatment was
subsequently used to screen possible interactions of ECM signaling and
additional growth factors. A modest enhancement was seen in cells grown
on all matrix molecules and treated with either bFGF or acidic FGF (Fig. 4). No effects on the syndecan-1 levels were seen by TGF-
or
platelet-derived growth factor (data not shown). This result emphasizes
further the selectivity of KGF-response of FiRE enhancer as a target of
modulation by collagen.

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Fig. 4.
Syndecan-1 in the conditioned medium of
MCA-3D keratinocytes. The cells were plated on 24-well plates
coated with fibronectin or collagen or left uncoated. Next the cells
were starved in Ham's medium + 1% carboxymethyl-Sephadex-eluted fetal
calf serum for 48 h and treated for 24 h with 10 ng/ml of the
indicated growth factors. New medium with or without the growth factors
was changed for the last 4 h. Aliquots of these conditioned
mediums were slot blotted, and syndecan-1 levels were measured by monoclonal antibody 281-2 using enhanced chemiluminescence. Syndecan-1 levels were quantitated by
an image analyzer (Molecular Imaging) and normalized by the cell
number. Columns and bars represent the mean and
S.E. of the values from four replicate wells in a representative
experiment.
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KGF Is Able to Promote the Proliferation of MCA-3D Cells Plated on
Collagen--
We were interested to see whether collagenous matrix can
suppress all of the end points of KGF signaling in MCA-3D cells. The
ability of KGF to promote the proliferation of MCA-3D cells was assayed
by the means of iodo-deoxyuridine (Idu) incorporation into newly
synthesized DNA. A similar induction of DNA synthesis was seen in cells
on all ECM molecules (Fig. 5). Thus, all
aspects of KGF signaling are not compromised in the cells growing on
collagen. This is supported by Western blot analysis of FGFR-2, which
revealed similar amounts of the KGF-receptor in cells plated on either fibronectin or collagen (not shown).

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Fig. 5.
Proliferation of MCA-3D keratinocytes on
collagen and fibronectin. The cells were plated on coated 24-well
plates, serum-starved, and treated with 10 ng/ml KGF or EGF. DNA
synthesis was monitored by adding 0.25 µCi/ml
[125I]dUTP to the culture medium for 6 h. The cells
were solubilized in 1 N NaOH, and the incorporated
radioactivity was measured. Note that collagen coating does not
compromise the proliferative effect of KGF.
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Fibrillar Collagen Does Not Change the Binding of Transcription
Factors to FiRE--
Next, we investigated whether matrix composition
can cause changes in the pattern of transcription factors that bind
FiRE and enable syndecan-1 induction. We have previously characterized the enhancer by DNaseI footprinting and gel mobility shift assays (20,
21). An overview of the enhancer is presented in the Fig.
6A. In 3T3 fibroblasts, the
enhancer can be bound by both transcription factors that are induced by
bFGF and by factors constitutively present in the cells. The enhancer
harbors binding sites for two growth factor-inducible AP-1 complexes
and for one inducible, as yet uncharacterized transcription factor
(20). In MCA-3D keratinocytes, on the other hand, transcription factor binding is not affected by growth factors that activate FiRE (21). However, there remained a possibility that ECM can cause the induction or suppression of transcription factor binding and thus modulate the
growth factor effects on the cell.

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Fig. 6.
Transcription factors binding to the motifs 2 and 3 of FiRE in MCA-3D cells growing on fibronectin or collagen.
A, an overview of the structure of the 170-base pair FiRE
that is located 10 kilobases upstream from the syndecan-1 transcription
start sites. B, the E-box in motif 2 binds USF in MCA-3D
cells. An electrophoretic mobility shift with radiolabeled
oligonucleotide encompassing motif 2 with 5 µg of total cellular
extracts from MCA-3D cells on collagen or fibronectin treated with KGF
(lanes 1 and 3) or EGF (lane 4) or
left untreated (lanes 2 and 5-7). On the
right, a supershift with polyclonal antibodies indicates the
presence of USF in the shifted complex (lane 6). As a
control antibody, an equal amount of anti-Max was used (lane
7). C, motif 3 does not bind fibroblast growth
factor-inducible nuclear factor (FIN-1) in MCA-3D cells on fibronectin
or collagen (lanes 1-6). A binding reaction, shown in
lane 7, presented a positive control with an extract from
3T3 fibroblasts treated with 10 ng/ml bFGF. Additionally, competition
experiments with double-stranded unlabeled specific and nonspecific
binding site oligonucleotides were carried out (not shown; Ref. 20) to
demonstrate the specific complexes in the gels
(arrow).
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In order to investigate the effect of ECM on transcription factor
binding to FiRE, we used gel mobility shift assays. The most 3'-motif
(motif 1) of the enhancer binds an uncharacterized 46-kDa protein
constitutively expressed in 3T3 cells (21). The DNA binding activity of
this protein was equally present in treated and control MCA-3D cells
and was unchanged by extracellular matrix molecules (data not shown).
Motif 2 contains an E-box that is bound by USF-1 in both cell lines
(20, 21). Fig. 6B shows equal binding activity of USF in
both control and growth factor-treated cells on fibronectin and in
KGF-treated cells grown on collagen. An antibody against USF abolished
the bound specific complex, whereas antibodies against other bHLH
factors (anti-Max shown) did not affect the complex formation (Fig.
6B). Motif 3, which binds an bFGF-inducible unidentified
factor in 3T3 cells does not bind that protein in growth factor-treated
MCA-3D cells (21). Neither fibronectin nor collagen alone or in
combination with growth factors induced specific transcription factor
binding to this site (Fig. 6C). Motifs 4 and 5 are occupied
by the members of AP-1 (Fos/Jun) family (20, 21). The AP-1 binding
activity was unchanged by the extracellular matrix (Fig.
7A). We furthermore investigated whether the composition of AP-1 complexes is affected by
matrix. Again, the same complexes were formed in cells growing on
collagen as in those on fibronectin. Antibodies against c-Fos or JunD
abrogated or supershifted the complexes with motif 4 (Fig. 7B). The same complex was observed in cells growing on all
tested ECM molecules and treated with either KGF or EGF (data not
shown). In summary, changes in transcription factor binding activities did not explain the diminished activation in cells grown on collagen. This suggested that matrix-dependent regulation of
syndecan-1 induction during growth factor treatment occurred at the
posttranslational modifications of transcription factors.

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Fig. 7.
Transcription factors binding to the AP-1
sites of the FiRE in MCA-3D cells growing on fibronectin or
collagen. A, growth factors do not increase the amount
of AP-1 complexes binding to FiRE either on collagen or on fibronectin.
The radiolabeled motifs 4 (lanes 1-6) and 5 (lanes
7-12) were used to detect the AP-1-binding proteins from cells
treated with growth factors on either collagen or fibronectin.
B, supershifts demonstrate the presence of c-Fos and JunD in
the complexes. The gel was run for a longer time than other presented.
A competition with a 100-fold excess of unlabeled motif 4 oligonucleotide (lane 2) compared with a nonspecific
competitor (lane 3, 100-fold excess of an Sp1 binding site)
shows the specific binding. An antibody against c-Fos abolishes the
binding to motif 4 (lane 4). To asses the Fos family members
in the complexes, antibodies against FosB (lane 5), Fra-1
and Fra-2, and a pan-Fos antibody were used (not shown). In the Jun
family, the predominant protein participating the complexes is JunD
(lane 8; the supershift is marked by an asterisk), whereas
antibodies against c-Jun (lane 7) or JunB (not shown) did
not compromise the interaction. For both the Jun and Fos families, all
the extracts from different growth factor/matrix combinations resulted
in the similar results in the supershift assays (not shown).
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As AP-1 sites were necessary for the FiRE activation in
vitro (20, 21), we examined several pathways that converge AP-1 activation. Anisomycin, a known inducer of Jun amino-terminal kinase
and p38 stress-activated mitogen-activated protein kinase (JNK)
activity (31), caused a reduction in FiRE activation in cells growing
on collagen compared with fibronectin (Fig.
8A). Thus, it appears that the
activation of JNK could, in turn, activate FiRE in keratinocytes in a
manner that can be inhibited by the ECM. The activity of the pSynProm
construct without the enhancer sequences was unchanged by anisomycin
(Fig. 8B). This indicates that the induction seen with the
enhancer construct is not due to nonspecific effect of the anisomycin
on the translation or stability of the CAT protein. Interestingly, KGF
and anisomycin had a synergistic effect, as the activation of the
enhancer was higher when they were administered together (Fig.
8A). This synergistic activation was, as well, lower on
collagen than on fibronectin (Fig. 8A). The finding suggests
that parallel signaling pathways act in concert to regulate FiRE.
Evidence supporting multipathway regulation came from our recent
experiments with FiRE-LacZ, in which anisomycin and a growth factor
mixture were needed together to activate FiRE in an organ culture of
unwounded skin (22). Additionally, a wound healing assay performed on
the FiRE-LacZ mice determined that PD 098059, an inhibitor of
extracellular matrix regulated kinases 1 and 2, can inhibit reporter
gene activity (22). Likewise, we found that inhibition of extracellular
matrix regulated kinase activity by PD098059 inhibits the reporter gene activity in cells growing on fibronectin (data not shown), which is
further evidence for an involvement of several parallel signaling pathways in the regulation of FiRE activity.

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Fig. 8.
Anisomycin induces a higher CAT activity in
cells growing on fibronectin than on collagen. A, the
stably transfected cells harboring p-271-FiRE CAT-construct were
treated with 30 µg/ml Anisomycin for 30 min before the addition of 10 ng/ml KGF or EGF for overnight or treated with anisomycin alone
(labeled A). Columns and bars
represent the combined mean and S.E. from three independent
experiments. Note that the difference in the EGF treatment between
cells on collagen or fibronectin is not statistically significant.
B, anisomycin treatment of the pSynProm-CAT cells does not
cause any changes in the CAT activity compared with untreated
controls.
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DISCUSSION |
In adult tissues, syndecan-1 is predominantly expressed by
stratified and simple epithelia. This expression in simple polarized epithelia is constitutive and is probably regulated by the short Sp1-activated basal promoter of the gene (19). On the other hand,
syndecan-1 expression in epidermal keratinocytes is inducible, requiring events associated with differentiation or repair of epidermal
injuries (14). We have shown that a treatment of mouse MCA-3D
keratinocytes by KGF or members of the EGF family results in the
activation of an enhancer for syndecan-1 gene, an increase in
syndecan-1 steady state mRNA levels, and a rise in the
concentration of syndecan-1 protein in the conditioned medium of the
cells (Ref. 21 and this study). Taken together with our previous
findings that syndecan-1 levels are induced severalfold in
keratinocytes at the leading edge of the wound (14) and that FiRE
directs reporter gene expression to the wound edge in transgenic mice (22), these data suggest that certain growth factors can be the key
activators of syndecan-1 gene in keratinocytes.
KGF is a known to increase re-epithelialization in experimental wound
healing studies (32, 33). Accordingly, the over-expression of a
dominant-negative form of the KGF-receptor variant of the FGFR-2
retards healing in transgenic mice (34), although wounds in KGF
knock-out mice appear to heal normally (35). Our study establishes that
KGF signaling to FiRE is regulated by ECM. Most notably, KGF failed to
rapidly activate syndecan-1 expression in cells plated on fibrillar
collagen, whereas there was a severalfold induction in cells growing on
fibronectin, nonspecific substrata, or the ambient matrix laid on
uncoated plastic dishes. This indicates that extracellular matrix is
selectively able to modulate the regulation of syndecan-1 expression.
Fibronectin is a major component of the provisional matrix, the first
extracellular matrix that is deposited in the wound space. It supports
keratinocyte adhesion and migration (36, 37) and, by acting through
different integrin receptors, modulates gene expression (38, 39). In
this study, fibronectin supported an induction of syndecan-1 expression
by KGF. Collagen as well promotes keratinocyte adhesion and migration
(38). It is present in the provisional matrix but is deposited in the
granulation tissue later than fibronectin (40). Thus, the local
composition of the matrix and the availability of growth factors may
determine the activation status of the syndecan-1 gene in
keratinocytes. Hence, it is possible that interactions with the
provisional matrix could mediate the differential expression of
syndecan-1 in wound keratinocytes. The role of fibrillar collagen has
previously been implicated in the regulation of other events critical
to wound healing, such as the induction of interstitial collagenase in migrating keratinocytes (23). In this case, the role is inductive, as
the collagenase expression is detected only in primary keratinocytes growing on collagen but not on gelatin (23). This, in turn, corresponds
to the in vivo findings that collagenase expression is
detected in keratinocytes that during wound healing encounter the
dermal matrix.
It appears that the default signaling pathway activated by KGF leads to
syndecan-1 expression in keratinocytes and that this regulation is
specifically blocked by a collagenous matrix. The KGF-induced pathway
is likely to converge with EGF signaling, because the FiRE enhancer
binds the same transcription factors upon the treatment by either
growth factor.
Interestingly, there was no difference between the cells grown on
either fibronectin or collagen in the binding activities of the
transcription factors recognizing the enhancer element, suggesting that
both the effect of growth factors and the modulation by collagenous
matrix occur at the level of the modification of pre-existing factors.
Alternatively, levels of modulator(s) of the transcriptional activity
that mediate the interaction of enhancer-bound complexes to the basal
transcriptional machinery or function as co-activators are regulated in
these cells. For example, JAB1, a co-activator of c-Jun and JunD, does
not affect the association rate of AP-1 to its binding site but
increases the stability and transcriptional activity of the complexes
(41).
The potential ability of the matrix to regulate the capacity for
transcriptional activity of the factors binding to the enhancer is
suggested by the results obtained with anisomycin, an activator of JNK
(stress-activated mitogen-activated protein kinase) (31, 42). JNK can
phosphorylate the amino-terminal activation domain of transcription
factor c-Jun (42). Anisomycin elicited a stronger response in CAT
activity of in cells growing on fibronectin than on collagen. This
response was specific for the enhancer constructs, whereas the CAT
activity with the promoter alone remained unaffected. This might
indicate that FiRE can be activated via the JNK pathway. Moreover,
regulation of JNK kinase activity could be a target for matrix induced
signals. Interestingly, anisomycin had a synergistic effect on reporter
gene activation when administered together with growth factors.
Additionally, an inhibitor to extracellular matrix regulated kinase
activity, PD098059 was able to block the LacZ expression in wounds of
transgenic mice (22) and largely to suppress increase in CAT activity
in the case of both growth factors regardless of the matrix. Thus,
there is evidence for existence of parallel signaling pathways that
activate FiRE in keratinocytes. It is likely that during wound healing,
several independent signals are needed for high level syndecan-1
expression. Furthermore, it is possible that anisomycin activates a
different signaling cascade to KGF but that they both are independently controlled by collagenous matrix.
Several studies indicate how changes in the expression of proteoglycans
can have profound biological consequences. In the case of the syndecan
family of cell surface proteoglycans, the studies have focused on the
effect of syndecan-1 on the regulation and maintenance of the simple
polarized epithelial cell phenotype (9, 10) or on the ability of the
cells to bind and respond to bFGF (43, 44). As a forced expression of
syndecan-1 in fibroblasts leads to a loss of response to bFGF, it has
been postulated that syndecans, unlike some other proteoglycans, such
as perlecan (45), could be inhibitory for the FGF signaling (44). In
wound fluids, syndecan-1 ectodomain acts as a dual regulator of bFGF activity. The intact ectodomain inhibits growth factor action, but
degradation of the glycosaminoglycan chains by platelet heparanase liberates heparan sulfate fragments that activate bFGF (46). So far it
remains to be studied how elevated syndecan-1 levels participate in
re-epithelialization of wounds. We have recently started transgenic
approaches to look for syndecan-1 functions in mice. We found that
targeting of syndecan-1 into basal keratinocytes by the means of
keratin K14 promoter effects epidermal differentiation, resulting in an
increase in cell layers of the
epidermis.2
In general, a convergence of signaling by cell-matrix interactions and
growth factor receptor activation is a well documented phenomenon. For
example, cell adhesion modulates signaling from platelet-derived growth
factor or EGF receptors at the level of receptor phosphorylation (47,
48). Moreover, integrin signaling can modify events that occur
downstream at the levels of Raf or MEK activation (49). In this study,
we found that KGF signaling is modulated by the extracellular matrix.
This co-ordinated regulation is likely to play a role in the specific
spatial and temporal expression of syndecan-1 during wound healing.
Further studies are needed to characterize the integrins and signaling
pathways involved. One point, perhaps worth of further investigation,
is what events mediate KGF regulation of proliferation distinctly from
FiRE activation.