Extracellular Matrix-dependent Activation of Syndecan-1 Expression in Keratinocyte Growth Factor-treated Keratinocytes*

Syndecan-1 is a major heparan sulfate proteoglycan of the epidermis. Its expression is strongly induced in migrating and proliferating keratinocytes during wound healing and, on the other hand, diminished or lost in invasive squamous cell carcinoma. We have recently found in the syndecan-1 gene an enhancer (fibroblast growth factor-inducible response element (FiRE)) that activates gene expression in wound edge keratinocytes (Jaakkola, P., Kontusaari, S., Kauppi, T., Määttä, A., and Jalkanen, M. (1998)FASEB J. 12, 959–969). Now, we demonstrate that the activation of this enhancer by keratinocyte growth factor (KGF) is modulated by the components of the extracellular matrix (ECM). MCA-3D mouse immortal keratinocytes growing on fibrillar collagen failed to activate FiRE and subsequently to induce syndecan-1 in response to KGF. The same cells growing on fibronectin or laminin, however, increased FiRE-dependent reporter gene expression upon KGF treatment. The inhibition of the KGF induction by collagen appears to be specific for signaling to FiRE, as the increase in cell proliferation by KGF was not affected. The effect was selective to KGF, as EGF-induction was independent on ECM composition. Changes in the transcription factor binding were not involved in the differential activation of FiRE, as the levels and composition of the AP-1 complexes were unchanged. However, application of anisomycin, an activator of Jun amino-terminal kinase, resulted in a lower response in cells growing on collagen compared with fibronectin. These results indicate that the composition of ECM and availability of growth factors can play a role in the epidermal regulation of syndecan-1 expression and that FiRE is a novel target for gene regulation by the extracellular matrix.

Syndecan-1 is a major heparan sulfate proteoglycan of the epidermis. Its expression is strongly induced in migrating and proliferating keratinocytes during wound healing and, on the other hand, diminished or lost in invasive squamous cell carcinoma. We have recently found in the syndecan-1 gene an enhancer (fibroblast growth factor-inducible response element (FiRE)) that activates gene expression in wound edge keratinocytes (Jaakkola, P., Kontusaari, S., Kauppi, T., Mä ä ttä , A., and Jalkanen, M. (1998) FASEB J. 12, 959 -969). Now, we demonstrate that the activation of this enhancer by keratinocyte growth factor (KGF) is modulated by the components of the extracellular matrix (ECM). MCA-3D mouse immortal keratinocytes growing on fibrillar collagen failed to activate FiRE and subsequently to induce syndecan-1 in response to KGF. The same cells growing on fibronectin or laminin, however, increased FiREdependent reporter gene expression upon KGF treatment. The inhibition of the KGF induction by collagen appears to be specific for signaling to FiRE, as the increase in cell proliferation by KGF was not affected. The effect was selective to KGF, as EGF-induction was independent on ECM composition. Changes in the transcription factor binding were not involved in the differential activation of FiRE, as the levels and composition of the AP-1 complexes were unchanged. However, application of anisomycin, an activator of Jun amino-terminal kinase, resulted in a lower response in cells growing on collagen compared with fibronectin. These results indicate that the composition of ECM and availability of growth factors can play a role in the epidermal regulation of syndecan-1 expression and that FiRE is a novel target for gene regulation by the extracellular matrix.
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 bFGF 1 (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 Ca 2ϩ 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.

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 ϩ4°C with 50 g/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 [ 125 I]dUTP (Amersham Pharmacia Biotech). To this end, the cells were treated with growth factors for 12 h following by pulse labeling with 0.25 Ci/ml [ 125 I]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-g aliquots of RNA were electrophoresed in a 1% agarose-formaldehyde gel, blotted onto nylon membranes (Hybond N, Amersham Pharmacia Biotech), and sequentially probed with 32 P-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 pSyn-Prom 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 g/ml G418 and maintained in 200 g/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 M 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).

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).
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).
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 KGFdependent 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.
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, how-  Fig. 3 were plated on the indicated matrix molecules, serumstarved, 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. ever, 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.
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 iododeoxyuridine (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).
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.
In order to investigate the effect of ECM on transcription factor binding to FiRE, we used gel mobility shift assays. The most 3Ј-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 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-g aliquots were electrophoresed in formaldehyde agarose gels and sequentially blotted for syndecan-1 (top) and glyceraldehyde-3phosphate dehydrogenase (GAPDH) as a loading control (middle). Quantitation of the hybridizations (bottom) (syndecan/ glyceraldehyde-3-phosphate dehydrogenase) was preformed with an image analyzer.
regulation of syndecan-1 induction during growth factor treatment occurred at the posttranslational modifications of transcription factors.
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. 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. The cells were plated on coated 24-well plates, serumstarved, and treated with 10 ng/ml KGF or EGF. DNA synthesis was monitored by adding 0.25 Ci/ml [ 125 I]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.

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 overexpression 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 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 g/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. 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.