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First published online 31 July 2007
doi: 10.1242/jcs.03480

Journal of Cell Science 120, 2851-2863 (2007)
Published by The Company of Biologists 2007
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Research Article

Reduced migration, altered matrix and enhanced TGFbeta1 signaling are signatures of mouse keratinocytes lacking Sdc1

Mary Ann Stepp1,2,*, Yueyuan Liu1, Sonali Pal-Ghosh1, Rosalyn A. Jurjus1, Gauri Tadvalkar1, Adith Sekaran1, Kristen LoSicco1, Li Jiang3, Melinda Larsen4,{ddagger}, Luowei Li5 and Stuart H. Yuspa5

1 Department of Anatomy and Cell Biology, George Washington University Medical School, Washington, DC 20037, USA
2 Department of Ophthalmology, George Washington University Medical School, Washington, DC 20037, USA
3 Institute for Biomedical Engineering, School of Engineering and Applied Science, George Washington University, Washington, DC 20037, USA
4 National Institute of Dental and Craniofacial Research/Laboratory of Cellular and Developmental Biology, National Institutes of Health, Bethesda, MD 20892, USA
5 National Cancer Institute/Laboratory of Cancer Biology and Genetics, National Institutes of Health, Bethesda, MD 20892, USA

* Author for correspondence (e-mail: mastepp{at}gwu.edu)

Accepted 12 June 2007


   Summary
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 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have reported previously that syndecan-1 (Sdc1)-null mice show delayed re-epithelialization after skin and corneal wounding. Here, we show that primary keratinocytes obtained from Sdc1-null mice and grown for 3-5 days in culture are more proliferative, more adherent and migrate more slowly than wt keratinocytes. However, the migration rates of Sdc1-null keratinocytes can be restored to wild-type levels by replating Sdc1-null keratinocytes onto tissue culture plates coated with fibronectin and collagen I, laminin (LN)-332 or onto the matrices produced by wild-type cells. Migration rates can also be restored by treating Sdc1-null keratinocytes with antibodies that block {alpha}6 or {alpha}v integrin function, or with TGFbeta1. Antagonizing either beta1 integrin function using a function-blocking antibody or TGFbeta1 using a neutralizing antibody reduced wild-type keratinocyte migration more than Sdc1-null keratinocyte migration. Cultures of Sdc1-null keratinocytes accumulated less collagen than wild-type cultures but their matrices contained the same amount of LN-332. The Sdc1-null keratinocytes expressed similar total amounts of eight different integrin subunits but showed increased surface expression of {alpha}vbeta6, {alpha}vbeta8, and {alpha}6beta4 integrins compared with wild-type keratinocytes. Whereas wild-type keratinocytes increased their surface expression of {alpha}2beta1, {alpha}vbeta6, {alpha}vbeta8, and {alpha}6beta4 after treatment with TGFbeta1, Sdc1-null keratinocytes did not. Additional data from a dual-reporter assay and quantification of phosphorylated Smad2 show that TGFbeta1 signaling is constitutively elevated in Sdc1-null keratinocytes. Thus, our results identify TGFbeta1 signaling and Sdc1 expression as important factors regulating integrin surface expression, activity and migration in keratinocyte and provide new insight into the functions regulated by Sdc1.

Key words: Syndecan-1, Keratinocytes, Integrins


   Introduction
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 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Syndecan-1 (Sdc1) is a single-pass, integral-membrane, heparan-sulfate proteoglycan that is abundantly expressed by epithelial keratinocytes early in development (Bernfield et al et al., 1999Go). Although the Sdc1-null mouse was found to be fertile and viable, when in vivo wound-healing studies were conducted, phenotypes began to emerge (Stepp et al., 2002Go; Gotte et al., 2002Go; Gotte et al., 2005Go). In cornea and skin of Sdc1-null mice, wound healing was found to be delayed due to slow re-epithelialization and increased inflammation. These mice also showed increased cardiac dysfunction after myocardial infarction associated with increased inflammation, matrix metalloproteinase (MMP) expression and function, and increased collagen disorganization (Vanhoutte et al., 2007Go). Paradoxically, overexpression of Sdc1 (Sdc/Sdc) in mice was also found to delay skin wound healing because of reduced cell proliferation, granulation tissue accumulation, and poor vascularization (Elenius et al., 2004Go). Together, these studies show that correct homeostasis of stratified squamous epithelial tissues demands that Sdc1 levels are precisely regulated.

Of the four known syndecans, Sdc1 is the most abundant on epithelial keratinocytes and can mediate cell-to-substrate adhesion by its ability to bind to laminin {alpha} chains (Salmivirta et al., 1994Go; Hoffman et al., 1998Go; Klass et al., 2000Go; Utani et al., 2001Go; Okamoto et al., 2003Go). In addition, the Sdc1 cytoplasmic domain has been shown to mediate cell spreading and migration (Chakravarti et al., 2005Go). whereas Sdc4 has been shown to interact with integrins at focal adhesions during wound healing (Alexopoulou et al., 2007Go), less is known about how Sdc1 mediates migration. Sdc1 has been shown to interact functionally with {alpha}v integrins in various cancer cell lines (Beauvais et al., 2004Go; Beauvais and Rapraeger, 2004Go; McQuade et al., 2006Go), and a recent report by Hayashida and colleagues has shown that expression of Sdc1 in epithelial keratinocytes is induced by TGFbeta1 through a protein kinase A (PKA)-dependent pathway (Hayashida et al., 2006Go). In response to injury in vivo, genes for TGFbeta1 and Sdc1 are upregulated in both epithelial and mesenchymal cells, and the Sdc1 ectodomain is shed at wound sites where the soluble molecule can regulate chemokine function (Gotte and Echtermeyer, 2003Go; Tkachenko et al., 2004Go) and modulate the activation of various MMPs (Kelly et al., 2000Go; Steffensen et al., 2001Go; Momota et al., 2005Go). The role Sdc1 plays in cancer is complex and tissue specific, but recent studies using Sdc1-null mice show that these mice are resistant to mammary carcinogenesis (Alexander et al., 2002; McDermott et al., 2007Go).

This study was undertaken to investigate the consequences of the loss of Sdc1 on keratinocyte function in vitro, and to identify alterations relevant to the delayed wound-healing phenotype observed in cornea and skin in vivo. Our results show that the loss of Sdc1 on activated keratinocytes enhances the overall amount of several integrins at the keratinocyte surface, increases adhesion, delays migration and causes an increase in the constitutive level of TGFbeta1-mediated gene expression. Furthermore, the response of Sdc1-null keratinocytes to TGFbeta1 is enhanced compared with that of wild-type (wt) keratinocytes. We also found that the reduced migration of Sdc1-null keratinocytes can be overcome by replating the keratinocytes on permissive substrates such as fibronectin–collagen-I (FNCNI), LN-332, matrix made from wt keratinocytes, or by treating Sdc1-null keratinocytes with TGFbeta1 or antibodies that block the function of {alpha}6 or {alpha}v integrins. Our results identify TGFbeta1 signaling and Sdc1 expression as important factors in activation and migration of keratinocytes in vitro and in vivo.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Sdc1-null keratinocytes reach high-saturation density and retain epithelial morphology and expression of keratins
In this study, primary Sdc1-null keratinocytes grew well in culture (Fig. 1A), and growth curves show that the Sdc1-null keratinocytes had equivalent plating efficiencies and reached confluence at the same number of days after seeding (Fig. 1B). However, the Sdc1-null keratinocytes achieved a higher population density than wt keratinocytes due to increased keratinocyte proliferation (Fig. 1C) and increased packing of cells in and around colonies (compare wt and Sdc1-null keratinocytes in Fig. 1A). Since studies using antisense methods (Kato et al., 1993Go) and tumor cell lines (Bayer-Garner et al., 2001; Kurokawa et al., 2006Go) have shown that loss of Sdc1 can cause epithelial cells to adopt a mesenchymal phenotype, wt and Sdc1-null keratinocytes were assessed for their localization of epithelial keratins (K-1, K-5, K-6, K-10, and K-14), E-cadherin, vimentin, and F-actin. Shown in Fig. 1D are keratinocytes stained simultaneously with an anti-K-14 antibody, phalloidin (for F-actin) and with the nuclear marker DAPI. The Sdc1-null keratinocytes retained epithelial morphology and continued to express K-14. They also expressed E-cadherin and other epithelial keratins and remained vimentin-negative (data not shown). However, differences in cytoskeletal organization can be seen; Sdc1-null keratinocytes showed thicker cortical actin bundles at cell peripheries compared with wt keratinocytes.


Figure 1
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  		Fig. 1. Although loss of Sdc1 alters keratinocyte growth characteristics, Sdc1-null keratinocytes retain their epithelial morphology and keratin expression. (A,B) Equivalent numbers of primary wt (+/+) and Sdc1-null (–/–) keratinocytes were plated out and grown for the times indicated; in A keratinocytes are viewed with phase-contrast optics at 3 days after being placed in culture, and in B the numbers of adherent primary +/+ and –/– keratinocytes were counted at the indicated days. Data are plotted as the mean ± s.e.m.; *, significantly more Sdc1-null keratinocytes per dish than wt keratinocytes at days 3 and 10. Bar in A, 10 µm. (C) Studies using [3H]thymidine showed that at day 7, Sdc1-null keratinocytes proliferated more than wt keratinocytes, even after controlling for differences in keratinocyte density. (D) Triple-labeling using FITC-labeled phalloidin (green) for F-actin, K14 (red) for intermediate filament protein keratin 14, and DAPI (blue) for nuclei in +/+ keratinocytes (a-d) and on –/– keratinocytes (e-h). The localization of K14 appears similar in wt and Sdc1-null keratinocytes. Sdc1-null keratinocytes show thicker cortical actin filament bundles that localized prominently at keratinocyte peripheries compared with wt keratinocytes. *, regions shown magnified in d and h to better emphasize the actin cortical filaments. Bar in D, 4 µm (a-c and e-g) and 1.3 µm (d and h).

 
Altered keratinocyte adhesion and delayed migration accompany the loss of Sdc1 in keratinocytes
In vivo, re-epithelialization of wounds has been shown to be delayed by 6-8 hours in Sdc1-null mice (Stepp et al., 2002Go); in the current study, adhesion and migration studies were performed to determine whether reduced migration rates after wounding in vivo correlated with altered properties of the keratinocytes in vitro. After 3 days in culture, equal numbers of wt and Sdc1-null keratinocytes were harvested and allowed to adhere to ECM-coated substrates for 60 minutes (Fig. 2A). Adhesion to all the matrices tested – fibronectin (FN), vitronectin (VN), laminin-111 (LN-111), collagen I (CNI) and collagen IV (CNIV) – was enhanced in the Sdc1-null keratinocytes compared with the wt keratinocytes; increased adhesion ranged from 3.3-fold higher for adhesion to VN to 1.5- to 2.0-fold higher for adhesion to the other matrix ligands. Because adhesion assays measure both cell attachment and spreading, the increased adhesion of the Sdc1-null keratinocytes on purified matrices could be owing to a combination of increased attachment and spreading of the Sdc1-null keratinocytes. When spreading studies were performed at 60 minutes after plating, there were no significant differences in keratinocyte spreading (data not shown). Since the adhesion assays presented in Fig. 2A were performed on keratinocytes 60 minutes after plating, the increased adhesion of the Sdc1-null keratinocytes seen is probably due to increased keratinocyte attachment.


Figure 2
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  		Fig. 2. Keratinocyte adhesion and time-lapse migration studies show that Sdc1-null keratinocytes adhere better and migrate poorly compared with wt keratinocytes, but only when migrating on matrix they produce themselves. (A) Cell adhesion studies were performed on equal numbers of wt and Sdc1-null keratinocytes, which were allowed to adhere on wells coated with fibronectin (FN), vitronectin (VN), laminin-111 (LN-111), collagen I (CN-I) and collagen IV (CN-IV). Note that the Sdc1-null keratinocytes are significantly more adherent than the wt keratinocytes to all ECM molecules tested. (B,C) Cell migration was assessed in time-lapse experiments in wt and Sdc1-null keratinocytes 5 days after initial plating. For details on the quantification, see Materials and Methods. Velocity measurements for wt and Sdc1-null keratinocytes are presented in B. *P<0.05. Typical tracks (red) of 15 keratinocytes are shown superimposed over final relief-contrast images of wt and Sdc1-null keratinocytes in C. Bar, 10 µm. (D) Velocities of wt and Sdc1-null keratinocytes were compared after replating onto FNCNI matrix and onto LN-332 as well as onto matrices deposited by each keratinocyte genotype. Data show that after replating, Sdc1-null keratinocytes migrated more slowly than wt keratinocytes but only when replated on the Sdc1-null keratinocyte matrix.

 

The attachment differences seen between the wt and Sdc1-null keratinocytes could impact the migratory behavior of Sdc1-null keratinocytes. To assess this, keratinocytes were seeded and grown for 5 days, and random movement of keratinocytes within wells in 24-well plates was assessed using time-lapse microscopy. At day 5, the Sdc1-null keratinocytes were found to migrate significantly slower than wt keratinocytes, as seen in the average velocities presented in Fig. 2B and the representative tracks presented in Fig. 2C. When data were evaluated for differences in persistence indices (net migration per total migration), no differences between wt and Sdc1-null keratinocytes emerged.

Altering the composition of the matrix the Sdc1-null keratinocytes are seeded upon can restore their migration rate to that of wt keratinocytes
The slower velocity of the Sdc1-null keratocytes could be caused by altered cytoskeletal dynamics or by their increased attachment to their substrate. To test this we evaluated cell migration after replating keratinocytes onto other matrices; data are presented in Fig. 2D. Keratinocytes were grown for 3 days and replated on purified FN-CNI or the LN-332-rich matrix secreted by 804G cells; as controls, migration rates of keratinocytes were also obtained for keratinocytes at day 4 after initial seeding. Day 3 keratinocytes were also replated onto wells that contained matrix (prepared as described in Materials and Methods) that had been produced and deposited by either wt or Sdc1-null keratinocytes. Triplicate wells were then used to track keratinocyte migration in time-lapse experiments. As expected, the day-4 Sdc1-null keratinocytes migrated more slowly than wt keratinocytes. When Sdc1-null keratinocytes were replated onto FN-CNI, LN-332 or onto the matrix deposited by wt keratinocytes, their migration rate was restored to that of wt keratinocytes; however, when replated onto their own matrix, they continued to migrate more slowly. Wild-type keratinocytes migrated at rates faster than those of control keratinocytes when replated onto day-3 Sdc1-null keratinocyte matrix. Therefore, these data show that the Sdc1-null keratinocyte matrix can support optimal cell migration when keratinocytes express Sdc1 and indicate that there is something distinct about the way the Sdc1-null keratinocytes interact with their matrix that reduces their ability to migrate quickly.

To determine whether there are differences in the organization of the wt and Sdc1-null matrices, we assessed the accumulation of collagen in keratinocyte cultures using a Sirius Red dye binding assay (Heng et al., 2006Go). Keratinocytes that had been used for tracking studies were immediately fixed and the collagen accumulation within the cultures was assessed. After data normalization for differences in keratinocyte numbers per well, we found that significantly less collagen was present within the wells of the Sdc1-null keratinocytes (Fig. 3A). To determine whether there were differences in the overall profile of proteins present in the matrices deposited by wt or Sdc1-null keratinocytes, we prepared matrices using methods identical to those used for the experiments shown in Fig. 2D, and ran extracts normalized for keratinocyte numbers onto 4-20% SDS-polyacrylamide gels that were then silver stained (Fig. 3B). Since one of the most abundant proteins present in these matrix preparations is LN-332, we also blotted these extracts for detection of LN-332 using the J18 antibody, which recognizes both unprocessed and processed LN-{alpha}3, beta3, and {gamma}2 chains (Fig. 3C). We did not see any difference in the overall amounts of high molecular weight molecules assembled into matrix at days 5 and 7 by wt or Sdc1-null keratinocytes. We found no difference in the amount of LN-332 present in these matrices; differences in LN-332 processing cannot be quantified without mouse LN-332-subunit-specific antibodies. Next, we looked directly at the LN-332 organization within matrices produced by the wt and Sdc1-null keratinocytes by performing immunolocalization experiments on matrix preparations using the J18 antibody (Fig. 3D). Data confirmed that the wt and Sdc1-null keratinocytes deposited similar amounts of LN-332-enriched matrix. However, Sdc1-null keratinocytes deposited LN-332 into more highly ordered (arrowhead) arrays oriented in the direction of keratinocyte migration, whereas LN-332 left behind by wt keratinocytes was organized into cloud-like aggregates (arrowheads, Fig. 3D).


Figure 3
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  		Fig. 3. Sdc1-null keratinocytes produce a matrix distinct from that made by wt keratinocytes. (A) Sirius Red dye binding assay to measure collagen accumulation in the wt and Sdc1-null cultures shows that the day after the keratinocytes had been tracked, Sdc1-null keratinocyte cultures had accumulated significantly less collagen per keratinocyte compared with wt keratinocytes. (B) Matrix preparations identical to those used in the experiments described in Fig. 2D were extracted as described, normalized and run on 4-20% gels that were silver-stained. High-molecular-mass proteins accumulated in the matrices, but there were no major differences between the amount of high-molecular-mass matrix deposited by the wt and Sdc1-null keratinocytes. The lower molecular mass bands are keratins, which stick non-specifically to the matrix after keratinocytes are lysed. The control extract shows those proteins deposited on wells which remain after ammonium hydroxide treatment of wells that had been coated with FN-CNI and fed serum-containing medium. (C) Immunoblots of the same matrix preparations shown in B normalized by cell count and probed for LN-332 using the J18 antibody showed similar patterns for LN-332 unprocessed (unp) and processed (prc) {alpha}3beta3{gamma}2 chains for matrices deposited by wt and Sdc1-null keratinocytes. (D) Immunofluorescence microscopy using the J18 antibody on matrix preparations from cultures of wt and Sdc1-null keratinocytes. (a,d) 20x images taken of wt and Sdc1-null keratinocytes; asterisks indicate elongated clear areas lacking LN-332 surrounded by areas positive for LN-332. In b and e, asterisks indicate regions shown at higher resolution. (c and f) Additional high-resolution images of wt and Sdc1-null keratinocyte matrices, respectively. Arrows in b,c,e,f indicate ordered streaks that are more prominent in Sdc1-null matrix than in wt matrix; arrowheads indicate amorphous cloud-like staining present in wt matrix but largely absent in Sdc1-null keratinocytes. Bar, 6 µm for a,d, 2 µm for b,c,e,f.

 

beta4 integrin regulates migration to a greater extent in Sdc1-null keratinocytes than in wt keratinocytes
Thus far, our data show that Sdc1-null keratinocytes migrated more slowly than wt keratinocytes owing to factors involving their ability to interact with the matrix they deposit. {alpha}6beta4 integrin is known to interact with LN-332 and has been implicated, together with {alpha}3beta1 integrin, in mediating keratinocyte migration (Belkin and Stepp, 2000Go). To test directly whether increased activity of integrins on the Sdc1-null keratinocytes contributes to their reduced migration rate, we repeated time-lapse experiments using integrin-function-blocking antibodies: GoH3 for {alpha}6, 9EG7 for beta1 integrins and RMV-7 for {alpha}v integrins. Data are presented in Fig. 4A as the fold change in velocity relative to untreated wt keratinocytes. Antagonizing the activity of either {alpha}6 or {alpha}v integrins fully restores velocity of Sdc1-null keratinocytes to the same or slightly higher rate than that of wt keratinocytes. Antagonizing beta1 integrins slows down migration rates of both wt and Sdc1-null keratinocytes but the affect is more profound in wt keratinocytes where migration rates were reduced by just over 60% of the rates seen in untreated or control-IgG-treated wt keratinocytes. For Sdc1-null keratinocytes, the beta1 antagonist decreased migration rates by ~30%, not significant compared with that of untreated Sdc1-null keratinocytes. Thus, Sdc1-null keratinocytes migrate at slower rates due to reduced beta1 integrin activity and/or increased {alpha}6beta4 and/or {alpha}v integrin activity.


Figure 4
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  		Fig. 4. The velocity of Sdc1-null keratinocytes can be restored to that of wt keratinocytes by addition of function-blocking antibodies against beta4 or {alpha}v integrins. (A) Time-lapse microscopy analyses were repeated on day 3 wt and Sdc1-null keratinocytes using integrin function-blocking antibodies at concentrations of 25 µg/ml added to serum-containing medium. Data are expressed as fold changes in velocity compared with untreated wt keratinocytes to facilitate comparisons between experiments. Control studies were performed using isotype specific antibodies. Note that the beta1 integrin antagonist (9EG7) inhibited wt keratinocyte migration rates significantly by ~60% whereas it inhibited Sdc1-null keratinocyte migration by only ~30% compared with untreated Sdc1-null keratinocytes, a difference which was not significant. By contrast, antagonizing either {alpha}6beta4 integrin using GoH3 or all {alpha}v integrins using RMV-7 allowed Sdc1-null keratinocytes to migrate at rates similar to those of wt keratinocytes. (B) beta4 integrin and LN-332 were simultaneously localized in day 3 wt and Sdc1-null keratinocytes. a,b and e,f show double-staining of actively migrating wt and Sdc1-null keratinocytes, respectively, whereas c,d and g,h show more stationary keratinocytes lacking LN-332 trails. Note the closer association between beta4 integrin and LN-332 at the edges of the migrating Sdc1-null keratinocytes in e and f (white arrows) compared with the wt keratinocytes. Bar, 5 µm.

 
We went on to examine localization of {alpha}3 and beta4 integrin in the wt and Sdc1-null keratinocytes. Finding only subtle differences in {alpha}3beta1 localization (data not shown), we focused on colocalization studies of LN-332 and beta4 integrin in wt and Sdc1-null keratinocytes. Whereas there are a wide variety of keratinocyte morphologies and beta4 integrin and LN-332 localization profiles in primary keratinocyte cultures, differences in beta4 integrin localization and cell morphology emerged when we compared localization of these two proteins in actively migrating wt and Sdc1-null keratinocytes surrounded by prominent LN-332 trails (Fig. 4B). Migrating wt keratinocytes were smaller and less uniform in shape compared with Sdc1-null keratinocytes, and there was less beta4 integrin present at cell peripheries. LN-332 and beta4 integrin are present beneath the cell nucleus in both migrating and stationary wt and Sdc1-null keratinocytes. In migrating Sdc1-null keratinocytes, we were frequently able to see close association of beta4 integrin with LN-332 at cell peripheries, and Sdc1-null keratinocytes were less uniformly round in shape. For both the wt and Sdc1-null keratinocytes, those that were not actively moving, as evidenced by the absence of LN-332 trails, were more round with less beta4 integrin present at cell peripheries. beta4 integrin was present on the basal surface and surrounding the perinuclear region and, although it appeared more organized in the Sdc1-null keratinocytes, there was significant variability in this phenotype.

{alpha}v integrins, especially {alpha}vbeta6 and {alpha}vbeta8, are involved in mediating the activation of TGFbeta1 signaling in epithelial cells (Sheppard, 2005Go). Little is known about how the activity of {alpha}v integrins might affect keratinocyte migration. TGFbeta1 has long been known to alter the surface expression of integrins, including those containing the {alpha}v and beta1 subunits (Gailit et al., 1994Go; Decline et al., 2003Go). Furthermore, Sdc2 has recently been shown to facilitate binding and activity of TGFbeta1 on cell surfaces (Chen et al., 2004Go), and TGFbeta1 signaling has been shown to induce Sdc1 expression through a PKA-dependent pathway (Hayashida et al., 2006Go). To determine whether TGFbeta1 played a role in mediating the migration rates of the wt and Sdc1-null keratinocytes, we treated keratinocytes with a neutralizing antibody against TGFbeta1 (TGFbeta1NA) or with exogenous addition of TGFbeta1, and measured keratinocyte migration rates using time-lapse microscopy. Data are presented in Fig. 5A,B. Addition of the TGFbeta1NA to both wt and Sdc1-null keratinocytes reduced cell migration rates of both genotypes significantly; control IgGs at the same concentration had no affect on the rate of cell migration (data not shown). Despite the fact that TGFbeta1NA reduced the migration rates of both wt and Sdc1-null keratinocytes, it had a more profound affect on migration rates of wt keratinocyte because it reduced wt migration to ~55% that of untreated wt control keratinocytes and reduced migration of Sdc1-null cells by ~30% compared with untreated Sdc1-null keratinocytes (Fig. 5A). These data suggest that TGFbeta1 signaling plays a role in regulating the overall velocity of keratinocytes in primary culture, and further suggests that differences between wt and Sdc1-null keratinocytes in TGFbeta1 signaling exist. Consistent with these data, addition of 0.25 ng/ml TGFbeta1 to both wt and Sdc1-null keratinocytes increased their migration rates. Whereas wt keratinocytes were no longer able to increase their migration rates in response to higher doses (2.5 ng/ml) of TGFbeta1, Sdc1-null keratinocytes migrated faster than untreated keratinocytes in response to the higher dosage of growth factor (Fig. 5B).


Figure 5
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  		Fig. 5. Disruption and activation of TGFbeta1 signaling has distinct effects on the migration rates of wt and Sdc1-null keratinocytes. (A) wt and Sdc1-null keratinocytes were grown for 3 days, after which keratinocytes were treated with a TGFbeta1 neutralizing antibody overnight and then tracked by time-lapse microscopy the next day. Data show that neutralizing TGFbeta1 reduced wt keratinocyte migration rates by over 50% but inhibited Sdc1-null keratinocyte migration by less than 30% compared with untreated or control IgG treated Sdc1-null keratinocytes. (B) wt and Sdc1-null keratinocytes were grown for 3 days, after which keratinocytes were treated with 0.25 or 2.5 ng/ml TGFbeta1 overnight and then tracked the next day. The migration rates of the Sdc1-null keratinocytes were restored to those of wt keratinocytes after treatment with 0.25 ng/ml TGFbeta1. Further, the Sdc1-null keratinocytes migrated significantly faster than the wt keratinocytes when given 2.5 ng/ml TGFbeta1. Whereas lower concentration of TGFbeta1 stimulated wt keratinocyte migration rates, higher concentration did not. *P<0.05; grey line highlights values above untreated wt controls. (C) Localization of LN-332 and beta4 integrin in migrating wt and Sdc1-null keratinocytes 24 hours after treatment of keratinocytes with either TGFbeta1-neutralizing antibody (a-d) or with 0.25 ng/ml of TGFbeta1 (e-f). Bar, 5 µm.

 

To get a better idea about how TGFbeta1 affects migration of wt and Sdc1-null keratinocytes, we visualized beta4 integrin and LN-332 in wt and Sdc1-null keratinocytes that had been treated either with TGFbeta1NA or with 0.25 ng/ml TGFbeta1 (Fig. 5C). TGFbeta1NA-treated wt and Sdc1-null keratinocytes were similar in overall morphology and beta4 integrin localization compared with untreated keratinocytes (compare Fig. 5C with Fig. 4B) and both genotypes showed increased close association of LN-332 with beta4 integrin. The migrating TGFbeta1-treated wt keratinocytes (Fig. 5Ce,f) were more spread out than untreated wt keratinocytes (Fig. 4Bb), whereas the migrating TGFbeta1-treated Sdc1-null keratinocytes (Fig. 5Cg,h) were less well spread out compared with migrating untreated Sdc1-null keratinocytes (Fig. 4Be,f). Also, the close association of beta4 integrin and LN-332 at the keratinocyte peripheries seen in migrating untreated Sdc1-null cells was decreased. These data suggest that treatments that restore Sdc1-null keratinocyte migration rates to levels similar to those of wt keratinocytes reduce the close association of beta4 integrin with LN-332, whereas treatments that reduce wt and Sdc1-null keratinocyte migration enhance the close association of beta4 integrin with LN-332.

Cell-surface integrins are constitutively elevated in Sdc1-null keratinocytes and do not change in response to TGFbeta1 treatment
The increased attachment and reduced migration rates observed in the Sdc1-null keratinocytes, and the changes in migration that accompany activation of TGFbeta1 signaling by growth factor treatment could be due to altered expression of integrins in keratinocytes lacking Sdc1. To test this, we assessed total integrin expression in wt and Sdc1-null keratinocytes and found that, for each of the eight keratinocyte integrin subunits assessed by immunoblotting and after normalization for total protein and/or actin, there were no differences in expression in wt versus Sdc1-null keratinocytes (see supplementary material Fig. S1). Expression of {alpha}9 integrin in both wt and Sdc1-null keratinocytes was downregulated after keratinocytes were placed in culture and, therefore, could not be detected by immunoblotting.

Integrins are present within intracellular compartments as well as on the cell surface. To analyze integrin surface expression on wt and Sdc1-null keratinocytes, we initially used flow cytometry. Data are presented in Fig. 6A for beta1 and beta4 integrins and indicate 1.2 and 1.6 times more, respectively, of both integrins on the surface of untreated Sdc1-null keratinocytes compared with untreated wt keratinocytes. Since there are few antibodies available that detect extracellular epitopes of mouse integrins, we measured surface integrins by surface-labeling keratinocytes in suspension at 4°C using biotinylation, followed by immunoprecipitation (IP) with integrin antibodies and detection of biotin-labeled integrin heterodimers using horseradish peroxidase (HRP)-conjugated avidin. After demonstrating that the biotinylation efficiency of the Sdc1-null keratinocytes was similar to that of the wt keratinocytes – using a dot-blot technique and by assessing biotinylation of total protein extracts from both wt and Sdc1-null keratinocytes (Fig. 6B) – extracts were normalized based on equal amounts of total protein, and IP was performed. Although under the conditions used for the IPs both {alpha} and beta subunits were pulled down, biotin labels primary amine groups and the numbers of biotins added per integrin molecule vary between the different subunits. As a result, frequently only one of the two subunits was detected in the unreduced mini-gels as shown in Fig. 6B; data were quantified and are shown in Fig. 6C.


Figure 6
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  		Fig. 6. Sdc1-null keratinocytes have increased expression of several different integrins on their surface but, unlike wt keratinocytes, they do not increase their integrin surface expression in response to TGFbeta1. (A) Flow cytometry analysis on unfixed isolated wt and Sdc1-null keratinocytes revealed increased surface expression of beta1 and beta4 integrins, as determined using Student's t-test (P< 0.05). (B) Tests of biotinylation efficiency show that the amount of biotin incorporated per ng total protein for wt and Sdc1-null keratinocytes is similar, as is the overall profile of biotinylated proteins. (C,D) Biochemical analyses of surface integrins using biotinylation and immunoprecipitation reveals elevated levels of several integrins in untreated Sdc1-null keratinocytes, excluding {alpha}3 integrin; the increase seen for {alpha}2 integrin was not significant. Treating wt and Sdc1-null keratinocytes with 0.25 ng/ml TGFbeta1 for 24 hours significantly increased the expression in wt keratinocytes of all the integrins tested, excluding {alpha}3beta1, but had no significant effect on integrin surface expression by the Sdc1-null keratinocytes. Numbers in C represent fold increase in surface integrins in the untreated and TGFbeta1-treated Sdc1-null keratinocytes relative to wt keratinocytes.

 

Comparing integrin surface expression in wt and Sdc1-null keratinocytes, we show that Sdc1-null keratinocytes expressed significantly (1.3 to 1.5 times) more beta4, {alpha}v, beta6 and beta8 integrins than wt keratinocytes. Because the difference between the surface expression of integrins on the wt and Sdc1-null keratinocytes was less than twofold, these experiments were repeated seven times to determine statistical significance. The increase in {alpha}2 integrin was not significant and {alpha}3 integrin expression was not elevated in the Sdc1-null keratinocytes (Fig. 6C). In wt keratinocytes but not in Sdc1-null keratinocytes, TGFbeta1 significantly enhanced surface expression of several integrins including beta4, {alpha}2, {alpha}v, beta6 and beta8, with increases ranging from 1.5 times higher ({alpha}2) to over 2 times higher ({alpha}v). Only {alpha}3 integrin expression at the keratinocyte surface remained at similar levels on both control and TGFbeta1-treated wt and Sdc1-null keratinocytes. Adding 2.5 ng/ml TGFbeta1 to day-3 cultures of wt and Sdc1-null keratinocytes yielded differences in integrin expression similar to those seen for 0.25 ng/ml (data not shown). These data show that adding TGFbeta1, which increases migration in both wt and Sdc1-null keratinocytes, also increased integrin surface expression in wt keratinocytes. However, Sdc1-null keratinocytes showed elevated levels of several integrins before treatment with TGFbeta1, and those levels were not altered in response to TGFbeta1.

Sdc1-null keratinocytes have constitutively elevated TGFbeta1-mediated signaling and respond to TGFbeta1 over a wider range of concentrations than wt keratinocytes
All of the integrins whose surface expression in wt keratinocytes was altered by addition of 0.25 ng/ml TGFbeta1, namely {alpha}2, {alpha}v, beta4, beta6 and beta8 integrins, were also surface-elevated in the Sdc1-null keratinocytes prior to TGFbeta1 treatment. These data, together with the data showing different migratory responses to high concentrations of TGFbeta1, suggest the possibility that the Sdc1-null keratinocytes have alterations in TGFbeta1 signaling.

To assess the possibility of defective TGFbeta1 signaling, we investigated the ability of increasing TGFbeta1 concentrations (0.015 ng to 1 ng/ml) to inhibit DNA synthesis. Like wt keratinocytes, Sdc1-null keratinocytes ceased proliferation with a similar dose response when treated with increasing concentrations of TGFbeta1 (Fig. 7A). We then looked in more detail at TGFbeta1-induced gene expression by using a dual reporter assay, we assessed the ability of TGFbeta1 to induce transcription of Smad4-dependent promoters. Data are presented in Fig. 7B for keratinocytes assayed 20 hours after TGFbeta1 treatment and expressed as levels of TGFbeta1-induced gene expression after controlling for differences in transfection efficiency. Similar results were obtained 6 hours after TGFbeta1 treatment (data not shown). Both wt and Sdc1-null keratinocytes had detectable TGFbeta1-mediated gene expression prior to the addition of TGFbeta1; however, the constitutive level of TGFbeta1-mediated gene expression in the Sdc1-null keratinocytes (6.7-fold) was significantly greater than that in wt keratinocytes (3.9-fold). The increase in levels of TGFbeta1-induced gene expression measured after addition of 0.5 ng/ml TGFbeta1 was 47-fold in Sdc1-null keratinocytes compared with 30-fold in wt keratinocytes. Increasing the concentration of TGFbeta1 in the medium from 0.25 to 0.50 ng/ml increased TGFbeta1-induced gene expression in Sdc1-null keratinocytes but had no affect on wt keratinocytes.


Figure 7
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  		Fig. 7. TGFbeta1-mediated signaling is altered in Sdc1-null keratinocytes. (A) Addition of increasing concentrations of TGFbeta1 to wt and Sdc1-null keratinocytes inhibits keratinocyte proliferation with both genotypes showing identical responses. (B) A dual reporter assay was used to determine the fold induction of transcription of a TGFbeta1-induced promoter (Smad4) compared with a control promoter (transketolase) 20 hours after wt and Sdc1-null keratinocytes were treated with 2 ng TGFbeta1, as described in Materials and Methods. Note that the baseline of TGFbeta1-mediated gene transcription for untreated keratinocytes was significantly higher for Sdc1-null keratinocytes, and that Sdc1-null keratinocytes had higher TGFbeta1-induced gene expression at all concentrations of TGFbeta1 tested; at 20 hours, these increases were significant for 250 and 500 ng TGFbeta1. (C) Smad2 phosphorylation was measured directly in wt and Sdc1-null keratinocytes before and after TGFbeta1 addition. In response to TGFbeta1, wt keratinocytes showed an increase in P-Smad2, whereas Sdc1-null keratinocytes did not. (D) Amount of total TGFbeta1 secreted into conditioned media was assessed via ELISA assay at the times indicated and data expressed as picogram (pg) per 106 cells. (E) The amount of active TGFbeta1 secreted into conditioned medium as well as in cell extracts was assessed using a standard mink lung epithelial reporter cell assay as described (see Materials and Methods); data are expressed in relative luciferase units.

 
The data from the dual-reporter assay show that, (1) Sdc1-null keratinocytes have increased constitutive signaling through the TGFbeta1 pathway and, (2) Sdc1-null keratinocytes respond to levels of TGFbeta1 above those that elicit a transcriptional or migratory response in wt keratinocytes. We confirmed the data regarding increased constitutive signaling by assessing Smad2 phosphorylation in wt and Sdc1-null keratinocytes. Prior to the addition of exogenous TGFbeta1, Sdc1-null keratinocytes showed elevated levels of phosphorylated Smad2 (P-Smad2) compared with those in wt keratinocytes (Fig. 7C). Addition of TGFbeta1 induced a reproducible increase in P-Smad2 in wt keratinocytes within 15 minutes, which was sustained until at least 60 minutes later. By contrast, the Sdc1-null keratinocytes showed no change in P-Smad2 levels in response to the addition of TGFbeta1.

Next, we performed semi-quantitative RT-PCR to assay the levels of mRNAs known to be upregulated by TGFbeta1 in wt keratinocytes using untreated day-3 wt and Sdc1-null keratinocytes. Data are presented numerically in Table 1; supplementary material Fig. S2 shows the gels. After normalizing against the mRNA levels in wt keratinocytes, we saw increased levels of several TGFbeta1-inducible mRNAs, including proteoglycans-like biglycan (1.8 times) and lumican (1.8 times), as well as matrix molecules including collagens {alpha}1-I (1.8 times) and {alpha}2-VI (1.9 times) despite the fact that we had not given the keratinocytes TGFbeta1. Thrombospondin and Mmp9 mRNAs also showed a modest (1.5 times and 1.3 times, respectively) increase in Sdc1-null keratinocytes. These mRNA studies support the conclusion that the Sdc1-null keratinocytes were engaged in an elevated level of constitutive TGFbeta1 signaling.


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  		Table 1. Semi-quantitative RT-PCR analyses of expression of TGFbeta1-induced mRNAs

 

We next considered whether the overall production of TGFbeta1 by Sdc1-null keratinocytes was greater than that of wt keratinocytes. Evaluating total TGFbeta1 accumulation in conditioned media we observed, at the earliest time point detectable, that Sdc1-null keratinocytes secreted ~1.3 times more total TGFbeta1 per cell than wt keratinocytes. By day 15, the Sdc1-null keratinocytes had secreted about two times more total TGFbeta1 than had the wt keratinocytes (Fig. 7D). When the amount of active TGFbeta1 in the conditioned medium was also assessed by using a standard cell assay with luciferase-tagged transfected mink lung epithelial cells (TMLCs) (Fig. 7E), 1.6 times more active TGFbeta1 per 106 cells was seen in the medium of Sdc1-null keratinocytes than in medium of wt keratinocytes. In addition, we also assessed the amount of active TGFbeta1 present in keratinocyte extracts obtained from the wt and Sdc1-null keratinocytes (Fig. 7E). At day 14, the extracts obtained from the Sdc1-null keratinocytes showed amounts of active TGFbeta1 similar to those from wt keratinocytes.


   Discussion
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 Summary
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 Results
 Discussion
Materials and Methods
 References
 
In this study, we show that cultured Sdc1-null keratinocytes migrate more slowly than wt keratinocytes, seemingly because of factors related to their interaction with and the assembly of their extracellular matrix. We further show that Sdc1-null keratinocytes have elevated constitutive TGFbeta1 signaling and also respond to concentrations of TGFbeta1 above those that elicit responses in wt keratinocytes. By 3 days in culture, the Sdc1-null keratinocytes expressed higher levels of integrins on their surface than wt keratinocytes, despite the fact that both wt and Sdc1-null keratinocytes isolated directly from wt and Sdc1-null mouse skin showed similar levels of integrins on their surfaces. When we evaluated the migratory phenotypes of wt and Sdc1-null cells after treatment of cells with antibodies that block {alpha}6, beta1 and {alpha}v integrins, we were able to show involvement of {alpha}v-family integrins in mediating {alpha}6beta4 activity.

Association of Sdc1 with the {alpha}v-integrin family in wt cells promotes {alpha}6beta4-integrin-mediated migration over cell adhesion
When we inhibited beta1 integrin function the rate of migration was reduced for both the wt and Sdc1-null keratinocytes, but the difference between wt and Sdc1-null keratinocyte velocities after blocking beta1-family integrin function was still significant: Sdc1-null keratinocytes still migrated slower than wt cells when they were forced to use {alpha}v-family integrins and {alpha}6beta4 as their primary cell-to-substrate adhesions. This result implicates either {alpha}v-family integrins or {alpha}6beta4 as causative in the delayed migration rates of Sdc1-null keratinocytes. Blocking {alpha}6 function restored migration rates of Sdc1-null keratinocytes to the same levels as in wt cells; thus, when Sdc1-null keratinocytes are forced to use {alpha}v-family and beta1-family integrins, they no longer experience any delay in their migration rate. For {alpha}6beta4 to reduce Sdc1-null keratinocyte migration, the activity of {alpha}v family integrins is needed. Blocking {alpha}v function restored keratinocyte migration rates in Sdc1-null cells but, unlike {alpha}6 integrin, the {alpha}-integrin antagonist reversed the phenotype so that the Sdc1-null keratinocytes were migrating faster than wt keratinocytes. Thus, when Sdc1-null keratinocytes depend on {alpha}6beta4 and beta1-family integrins for their migration, they migrate faster than similarly treated wt cells. Taken together, these data implicate {alpha}v-family integrins as positive regulators of the adhesive functions of {alpha}6beta4. In wt keratinocytes, Sdc1 cooperates with {alpha}v integrins to decrease the adhesion and increase the migratory activity of {alpha}6beta4.


Figure 8
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  		Fig. 8. Cartoon highlighting results from the studies using integrin-function-blocking antibody. PM, plasma membrane; N,nucleus; {alpha}vbeta, integrin heterodimers that contain the {alpha}v subunit, are expressed in keratinocytes and include integrins {alpha}vbeta5, {alpha}vbeta6 and {alpha}vbeta8; {alpha}beta1, integrin heterodimers that contain the beta1 subunit, are expressed in keratinocytes and include integrins {alpha}2beta1, {alpha}3beta1 and {alpha}5beta1 (see supplementary material Fig. S1 for expression profiles of total keratinocyte integrins and Fig. 6B for surface integrins).

 
Our data show that Sdc1 on keratinocytes modulates the function of {alpha}6beta4 integrin by facilitating detachment from LN-332 matrices during migration and it does so by indirectly interacting with {alpha}v-family integrins. A model summarizing these results is presented in Fig. 8. Incorporated into the model is our knowledge that in normal and migrating epithelial tissues in skin and cornea, Sdc1 is not localized at the basal membrane of the basal keratinocyte surface where {alpha}6beta4 is found but, rather, is present on basolateral and apical membranes and in endosomal compartments (Stepp et al., 2002Go). Although during wound healing the localization of {alpha}6beta4 extends to the basolateral membrane surfaces, it remains enhanced at the basal surface where Sdc1 is absent. Thus, any effect that Sdc1 has in mediating keratinocyte adhesion and migration would have to be a regulatory one.

Although other studies have suggested that {alpha}6beta4 integrin affects migration by altering LN-332 matrix assembly (Rabinovitz et al., 2001Go; Sehgal et al., 2006Go), our study is the first to implicate TGFbeta1 and Sdc1 in this process. Conversion of {alpha}6beta4 integrin from a state that mediates migration to one that mediates firm adhesion has been shown to be regulated by phosphorylation of the integrin beta4 subunit by the EGF receptor (Mainiero et al., 1996Go; Mariotti et al., 2001Go). Sdc1 is known to associate with LN {alpha} chains (Hoffman et al., 1998Go), but recently a study by Ogawa and colleagues has shown that Sdc1 also associates with a fragment derived from the LN-332 {gamma}2 chain and this complex inhibits phosphorylation of {alpha}6beta4 integrin (Ogawa et al., 2007Go). Interaction between Sdc1 and the LN {gamma}2 fragment causes integrin {alpha}6beta4 to promote adhesion over migration. These data are consistent with previous data showing that proteolytic processing of LN-332 favors stable adhesion, whereas unprocessed forms of the molecule favor migration (Goldfinger et al., 1999Go). Ogawa and colleagues have also indicated that the interaction between Sdc1 and integrin {alpha}6beta4 is indirect (Ogawa et al., 2007Go). Realizing that integrin {alpha}6beta4 and Sdc1 generally exist in separate domains on the plasma membrane, we propose that Sdc1 interacts with {alpha}v-family integrins on basolateral and apical membranes such that both molecules are sequestered from {alpha}6beta4 leaving it available to exist in its migratory state, shown by others to be phosphorylated by the EGF receptor. In Sdc1-null keratinocytes, {alpha}v-family integrins do not associate with Sdc1, are not sequestered apart from {alpha}6beta4 and their presence at the basal surface of the cell may block phosphorylation of {alpha}6beta4 integrin.

Loss of Sdc1 affects matrix assembly
Studies have shown that deposition of LN-332 is polarized, and that {alpha}2beta1 and {alpha}3beta1 integrins regulate persistent migration in keratinocytes (Nguyen et al., 2000Go; Frank and Carter, 2004Go), a process that also involves formation of Rac1 gradients towards the forward- or leading-edge during directed cell migration (Pankov et al., 2005Go; Choma et al., 2004Go; Sehgal et al., 2006Go). Whereas Sdc1-null cells show differences in migration rate, we found no differences in their persistence indices on any of the substrates tested.

In a model for the study of coronary infarcts, Sdc1-null mice showed reduced deposition and increased disorder of collagens after wounding, which was reversed when Sdc1-null mice were infected with an adenovirus expressing Sdc1 before wounding. Disorganized collagen contributed to cardiac dilatation and reduced coronary function after infarcts in Sdc1-null mice (Vanhoutte et al., 2007Go). Here, we show that keratinocytes isolated from Sdc1-null mice also show reduced matrix assembly. These are the same cultures at the same time point that we also showed were synthesizing more of several different collagen mRNAs. Defective matrix assembly in Sdc1-null keratinocytes could be due to several factors; Vanhoutte and colleagues (Vanhoutte et al., 2007Go) have suggested in their heart model that elevated levels of MMP9 secreted by inflammatory cells contribute to collagen degradation before newly synthesized collagens had assembled into mature fibers. Sdc1 has been shown to bind to and sequester proteases after wounding in the skin (Bernfield et al., 1999Go) and, therefore, defective proteolytic balance could contribute to matrix destruction in vivo. Our data in vitro in the absence of inflammatory cells suggests that the matrix assembly defect is inherent in epithelial cells derived from the Sdc1-null mouse. If shed Sdc1 extracellular domain serves to protect nascent collagen molecules from destruction, its lack could lead to denaturation and unfolding, which could alter the ability of the matrix to support keratinocyte adhesion and migration.

The fact that the LN-332 tracks are better organized in the Sdc1-null keratinocytes may result from the enhanced adhesion-promoting activity of {alpha}6beta4 integrin in these cells. Whereas the LN-332 deposited by Sdc1-null cells was better organized, the Sdc1-null cell matrix itself contained similar amounts of LN-332 and was able to support robust migration when wt keratinocytes were plated on it. TGFbeta1 treatment of wt keratinocytes increased cell-surface expression of integrins but induced only modest increases in keratinocyte migration; similar treatment of Sdc1-null keratinocytes had a marked effect on migration rate, enhancing it significantly without altering the overall levels of integrins on the Sdc1-null keratinocyte surfaces. The mechanism whereby the Sdc1-null keratinocytes increase their migration rate above those of untreated wt cells after TGFbeta1 treatment remains a subject of ongoing investigation. We have shown here that the Sdc1-null keratinocytes cease proliferating after TGFbeta1 treatment with the same dose-response as wt control keratinocytes but we do not know whether or how long the wt and Sdc1-null cells remain viable after they cease proliferating.

TGFbeta1 can have differing affects on cell migration, depending upon cell or tissue studied and integrins expressed
Previous studies have shown conflicting results relating to the affect of TGFbeta1 signaling on epithelial cell migration. In human keratinocytes, TGFbeta1 increased migration rates (Decline et al., 2003Go). In vivo skin-wound-healing experiments using Smad3-null mice have shown accelerated wound healing, suggesting that endogenous TGFbeta1 signaling impedes re-epithelialization after wounding (Ashcroft et al., 1999Go). Transgenic mice overexpressing Smad2 have shown delayed wound healing, again supporting the idea that elevated TGFbeta1 signaling delays healing in vivo (Hosokawa et al., 2005Go). Other data have shown that neutralizing TGFbeta1 in a wound-healing model that allows keratinocytes to migrate as sheets after injury, accelerates sheet movement (Neurohr et al., 2006Go). Experimental models for the study of epithelial cell migration in vitro via sheet movement are limited, but it appears that the differing mechanisms used by {alpha}vbeta6 and {alpha}vbeta8 integrins to activate TGFbeta1 at the cell surface play important roles in relaying TGFbeta1 signals from outside to inside keratinocytes during sheet movement (Sheppard, 2005Go).

By 3 days in culture, Sdc1-null keratinocytes have elevated levels of {alpha}vbeta6, {alpha}vbeta8 and {alpha}6beta4 on their surfaces, the same integrins whose surface expression is enhanced when wt cells are treated with TGFbeta1. How epithelial integrins accumulate at the surface of Sdc1-null keratinocytes is unclear. They could accumulate as a result of the elevated constitutive TGFbeta1 signaling, which acts to enhance integrin surface expression on keratinocytes. Sdc-2 has recently been shown to mediate TGFbeta-induced fibrosis in a kidney cell culture model via a mechanism that involved binding of Sdc2 with betaglycan, one of the TGFbeta receptors present on kidney cell surfaces (Chen et al., 2004Go). The molecular mechanisms underlying syndecan-induced TGFbeta affects are likely to vary in cell-type specific ways, especially in epithelial and mesenchymal cells. Several studies have implicated the Sdc1 cytoplasmic domain in mediating endocytosis of cytokine receptors (Fuki et al., 2000Go; Chen et al., 2004Go; Zimmermann et al., 2005Go), making it possible that the lack of Sdc1 alters integrin and growth-factor-receptor-mediated endocytosis (Caswell and Noman, 2006Go). Such differences could account for aspects of both the cell migration defects and enhanced responsiveness of the Sdc1-null keratinocytes to TGFbeta1.

Our data on Sdc1-null keratinocytes grown in vitro are consistent with the hypothesis that the delayed corneal and skin wound healing we reported previously in vivo (Stepp et al., 2002Go) results from (1) the migrating Sdc1-null keratinocytes being more adherent to their underlying matrix due to increased adhesion promoting activity mediated by {alpha}6beta4 integrin and, (2) altered responsiveness of the activated Sdc1-null keratinocytes to TGFbeta1 in their environment, which increases surface expression of integrins and alters matrix synthesis and deposition. The Sdc1-null keratinocytes produce and secrete more active TGFbeta1 in vitro. At the site of a wound, TGFbeta1 can be released by keratinocytes, mesenchymal cells and by the inflammatory cells that are known to be present in elevated numbers after wounding in Sdc1-null mice (Stepp et al., 2002Go: Gotte and Echtermeyer, 2003Go; Neurohr et al., 2006Go). Altered responsiveness of keratinocytes to TGFbeta1 could affect Sdc1-null keratinocyte migration rates by altering matrix remodeling and reassembly. Additional studies on the effects of the depletion of Sdc1 on signal transduction networks in vivo in skin and cornea will shed light on the mechanisms underlying the wound-healing defects induced by loss of this important proteoglycan and, in doing so, provide insight into the roles played by Sdc1 in forming and maintaining epithelial tissues in health and disease.


   Materials and Methods
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 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Antibodies
For immunoblots and immunoprecipitations, we used the following antibodies against: actin (Chemicon International, Temecula, CA; MAB1501R), {alpha}v integrin (Chemicon; AB1930), beta5 integrin (Chemicon; AB1926), beta6 integrin (Chemicon; MAB2076Z), beta8 integrin (Santa Cruz Biotechnology, Santa Cruz, CA; sc-10817), {alpha}2 integrin (Chemicon; AB1936), {alpha}5 integrin [BD Pharmingen, Franklin Lakes, NJ; 5H10-27 (MFR5)], and LN-332 (Jonathan Jones, Northwestern University, Chicago, IL). The beta1, beta4, {alpha}3 and {alpha}9 integrin antibodies were rabbit polyclonals against cytoplasmic domain peptides (Sta Iglesia et al., 2000Go). For function blocking of integrins, 25 µg/ml each of the rat anti-mouse monoclonal antibodies 9EG7 for beta1 integrin, GoH3 for {alpha}6 integrin and RMV-7 for {alpha}v integrins were used. These, along with an isotype-specific control IgGs were obtained from BD Pharmingen. For immunofluorescence microscopy localization of integrins, the same antibodies used for biochemical analyses listed above were used. For F-actin localization, we used Alexa-Fluor-488-labeled phalloidin (Molecular Probes/Invitrogen, Carlesbad, CA; A-12379), and for keratin-14, we used a rabbit polyclonal against mouse keratin-14 (Covance Research Products, Princeton, NJ; PRB-155P). For flow-cytometry analysis, we used the following antibodies: {alpha}6-FITC (BioLegend, San Diego, CA; 313605), beta1-phycoerythrin (PE) (BioLegend; 102207), and beta4-PE (Santa Cruz; sc-18883). For TGFbeta1 neutralization studies, the antibody was obtained from R&D Systems (Minneapolis, MN; AB-101-NA) and was used at 1 µg/ml; a chicken polyclonal anti-vimentin antibody (Novus Biologicals, Littleton, CO 80120) was used at the same dilution as a control.

Primary mouse keratinocyte cell culture
Wild-type (wt) mice (Balb/C) were obtained from NCI-Frederick (Frederick, MD). Tissue culture media, stocks, and buffers were obtained from Gibco/Invitrogen (Carlesbad, CA) unless otherwise indicated. Construction of Sdc1-deficient mice has been described previously (Stepp et al., 2002Go); mice have been backcrossed into a Balb/C genetic background (McDermott et al., 2007Go; Alexander et al., 2000Go). Primary mouse keratinocytes were isolated from skin of newborn Balb/C or Sdc1-null mice as described (Dlugosz et al., 1995Go), resuspended in freezing media (S-MEM with 8% fetal calf serum (FCS), 1.4 mM calcium, 10% dimethylsulfoxide, 10 mM Hepes, pH 7.3) and stored in liquid nitrogen until use. For each experiment, primary keratinocytes were grown in regular low-Ca2+ media (S-MEM and 8% FCS with calcium concentration of 0.05 mM) for the times indicated. Tissue culture plates were routinely coated with a mixture of human plasma fibronectin and collagen I (FNCNI; 10 µg/ml human plasma FN (BD Pharmingen, San Jose, CA); 1% Vitrogen (v:v) and 100 µg/ml bovine serum albumen (BSA) in S-MEM) for 15 minutes at 37°C prior to plating keratinocytes. For studies involving growth curves, data are presented for adherent keratinocytes only.

Immunoblotting, flow cytometry and surface labeling using biotinylation
Wt or Sdc1-null keratinocytes were cultured for 4 days. Medium was removed and the keratinocytes were washed three times with PBS. Then, 250 µl M-Per protein extraction reagent (Pierce Chemical Company, Rockland, IL; 78503) with proteinase inhibitor (1:100 dilution) (Pierce Chemical Co.; inhibitor cocktail, 78415) was added to each of the 10-cm cell culture dishes, and the keratinocytes were harvested by scraping. A total of 10 µg protein from each extract was loaded to the 4-20% gel (Invitrogen, EC6025BOX) and SDS-PAGE electrophoresis was performed at 140 V. Proteins were transferred to PVDF membrane (Millipore, Billerica, MA; IPVH15150) at 300 mA for 1.5 hours, and the blot was then blocked in blocking solution [Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and 10% milk] overnight at 4°C. Blots were subjected to enhanced chemiluminescence (ECL) reaction (Amersham/GE Healthcare Services, Piscataway NJ; RPN2132), and chemiluminescence was detected using X-ray film. When appropriate, data were quantified using NIH ImageJ software, v1.345 (available as a free download at http://rsb.info.nih.gov/ij/).

For-flow cytometry, keratinocytes were trypsinized and resuspended in serum-containing media, and concentrations were adjusted to normalize the cell counts for wt and Sdc1-null keratinocytes. Per antibody tested, 200,000 keratinocytes were spun down and resuspended in blocking buffer [PBS supplemented with 3% BSA containing 1 µl Fc-receptor (AbD Serotec, Raleigh, NC; BUF041A)]. Antibodies used were conjugated directly with phycoerythrin (PE) and controls included keratinocytes incubated with isotype-matched PE-conjugated antibodies, as well as keratinocytes incubated in blocking buffer alone. For quantitation, FloJo software (Windows Version 7.1.2, Tree Star, Inc., Ashland, OR) was used; median values for fluorescence intensity were obta