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* This work was supported in part by National Institute of Health, NICHD Grant HD-37490 and grants from the Cutaneous Biology Research Center through the MGH/Shiseido Company (to P. F. G.) and the Dermatology Foundation Research (to F. D.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This paper is dedicated to the memory of Merton Bernfield who pioneered the field of syndecan biology. ‡ Supported by National Institutes of Health Postdoctoral Fellowship F32 HD41235.
The cell-surface heparan sulfate proteoglycan syndecan-4 acts in conjunction with the α5β1 integrin to promote the formation of actin stress fibers and focal adhesions in fibronectin (FN)-adherent cells. Fibroblasts seeded onto the cell-binding domain (CBD) fragment of FN attach but do not fully spread or form focal adhesions. Activation of Rho, with lysophosphatidic acid (LPA), or protein kinase C, using the phorbol ester phorbol 12-myristate 13-acetate, or clustering of syndecan-4 with antibodies directed against its extracellular domain will stimulate formation of focal adhesions and stress fibers in CBD-adherent fibroblasts. The distinct morphological differences between the cells adherent to the CBD and to full-length FN suggest that syndecan-4 may influence the organization of the focal adhesion or the activation state of the proteins that comprise it. FN-null fibroblasts (which express syndecan-4) exhibit reduced phosphorylation of focal adhesion kinase (FAK) tyrosine 397 (Tyr397) when adherent to CBD compared with FN-adherent cells. Treating the CBD-adherent fibroblasts with LPA, to activate Rho, or the tyrosine phosphatase inhibitor sodium vanadate increased the level of phosphorylation of Tyr397 to match that of cells plated on FN. Treatment of the fibroblasts with PMA did not elicit such an effect. To confirm that this regulatory pathway includes syndecan-4 specifically, we examined fibroblasts derived from syndecan-4-null mice. The phosphorylation levels of FAK Tyr397 were lower in FN-adherent syndecan-4-null fibroblasts compared with syndecan-4-wild type and these levels were rescued by the addition of LPA or re-expression of syndecan-4. These data indicate that syndecan-4 ligation regulates the phosphorylation of FAK Tyr397 and that this mechanism is dependent on Rho but not protein kinase C activation. In addition, the data suggest that this pathway includes the negative regulation of a protein-tyrosine phosphatase. Our results implicate syndecan-4 activation in a direct role in focal adhesion regulation.
protein kinase C
focal adhesion kinase
phorbol 12-myristate 13-acetate
Syndecan-4 is a member of a family of transmembrane heparan sulfate proteoglycans (syndecans 1–4) that are characterized by divergent extracellular domains and short cytoplasmic domains that contain two constant regions separated by a variable region that is unique to each family member (reviewed in Refs.
). Comparison of the localization of syndecan-4 with the focal adhesion marker protein vinculin suggests that syndecan-4 does not localize to newly formed contacts but with more established adhesion sites (
). Focal adhesions are composed of transmembrane receptors (primarily syndecan-4 and members of the integrin superfamily), structural molecules (such as actin, talin, tensin, vinculin, and α-actinin), and signaling molecules (i.e. focal adhesion kinase (FAK), PKC, and Src). Focal adhesions, therefore, serve not only as structural supports but also as signaling conduits between the actin cytoskeleton and the surrounding environment of the cell.
The generation of focal adhesions in fibronectin (FN)-adherent cells is dependent on the ligation of two different transmembrane receptors: integrins and syndecan-4. Fibroblasts seeded on the cell-binding domain (CBD) of FN (which contains only the integrin-binding RGD sequence) will attach but not form focal adhesions or actin stress fibers (
The generation of syndecan-4-null mice demonstrated no initial obvious phenotype and showed, surprisingly, that cells seeded onto FN will form stress fibers and focal adhesions in the absence of syndecan-4 (
). These data point to another cell-surface heparan sulfate proteoglycan that can compensate for the absence of syndecan-4. Treatment of CBD-adherent syndecan-4-null fibroblasts with antibodies to syndecan-4 do not form focal adhesions or stress fibers although wild type fibroblasts do, suggesting that the syndecan-4 signaling pathway can be selectively activated and does not function in the syndecan-4-null cells (
). Interestingly, further studies have documented that syndecan-4-null mice do not respond to physiological insults as well as their wild type counterparts implying that syndecan-4 may be important in combating “stress situations” (
). Impaired cell migration may be because of the inability of cells to either generate enough force to propel themselves over an underlying substrate or to disengage established adhesion contacts to promote new adhesions (
). FAK contains multiple tyrosine residues that, upon their phosphorylation, are capable of binding proteins containing SH2 domains. Tyrosine 397 (Tyr397) is a critical phosphorylation site that results from autophosphorylation upon antibody-induced clustering of cell-surface integrins or cell adhesion to extracellular matrix proteins (such as collagen, fibronectin, and vitronectin) (Refs.
) demonstrated that FAK-null cells exhibit constitutive activation of Rho and this activity level is inversely correlated with focal adhesion turnover. They reintroduced FAK to the deficient cells and showed that Rho activity was restored to normal levels. This suggests that FAK is responsible for the transient inhibition of Rho during early cell spreading (
), we were interested in determining what effect syndecan-4 signaling might have on the autophosphorylation site of FAK. We now demonstrate that increased phosphorylation of FAK Tyr397 is dependent on syndecan-4 ligation, and that the syndecan-4 signal may be superseded by direct activation of Rho.
The heparan sulfate proteoglycan syndecan-4 has two main cellular functions. It acts as a co-receptor for heparin-binding growth factors (such as the family of fibroblast growth factors and heparin-binding vascular endothelial growth factor isoforms) regulating the ligand-dependent activation of the primary receptor (
). Interestingly, transfection of cells with syndecan-4 constructs that lack cytoplasmic domains also exhibit decreased cell migration although they do not form stress fibers or extensive focal adhesions when seeded on a FN or vitronectin substrate (
). The impaired migration in syndecan-4-null cells may result from the inability of the adhesions to generate enough tension required for migration or from the inability to disengage established contacts so that new adhesions may form (
). Alternatively, although not mutually exclusive from the former, syndecan-4 may regulate components of a signaling pathway involved in cell migration and the absence of syndecan-4 disrupts the efficiency with which the pathway acts.
It has previously been shown that integrin ligation to the CBD of FN does not induce tyrosine phosphorylation of FAK to the same level as cells that are ligated to full-length FN (
). We analyzed the autophosphorylated form of FAK and now demonstrate that FAK Tyr397 phosphorylation is lower in the absence of the heparin-binding domain of FN. Incubation of CBD-adherent FN-null cells with the tyrosine phosphatase inhibitor sodium vanadate augmented the phosphorylation levels of FAK Tyr397 to that of vanadate-treated cells seeded on full-length FN suggesting that syndecan-4 may influence FAK Tyr397 phosphorylation by regulating the activity of a cellular tyrosine phosphatase. Candidates for such tyrosine phosphatases are: PTEN (
), which have been shown to dephosphorylate FAK in vitro. Incubation of our cells with the Shp-2 inhibitor calpeptin did not enhance phosphorylation of FAK Tyr397 (data not shown) suggesting that under our conditions Shp-2 is not involved in modulating the phosphorylation state of FAK Tyr397.
Syndecan-4-regulated focal adhesion formation is primarily associated with two signaling molecules: the serine/threonine kinase PKC and the small GTPase Rho. Fibroblasts seeded on CBD can be stimulated to generate actin stress fibers and focal adhesions by incubating the cells with either PMA (to directly activate PKC) (
). It is unclear whether these signaling molecules collaborate in one signaling pathway or if they work in parallel but separate pathways. Our study shows that increased phosphorylation of FAK Tyr397 is associated with the activation of the Rho pathway and not the PKC pathway, as only LPA and not PMA increased Tyr397 phosphorylation in our conditions. Similarly, LPA, but not PMA, treatment also induced the localization of FAK phosphorylated on Tyr397 to vinculin-containing focal adhesions in syndecan-4-null cells plated on FN. Therefore, our data indicate that syndecan-4-mediated FAK phosphorylation occurs only through a Rho-mediated process and not through a collaboration of both Rho and PKC stimulation.
PKC activation augmented general tyrosine phosphorylation in CBD-adherent cells but this stimulation did not translate into increased phosphorylation of FAK Tyr397. The lack of effect with PMA treatment does not imply that a syndecan-4-PKC pathway does not exist. Indeed, syndecan-4 and PKC have a close functional association. PKC recruits syndecan-4 to focal adhesion sites (
It is possible that PKC promotes focal adhesion formation through an alternative cell-surface heparan sulfate proteoglycan. Syndecan-4-null cells will generate actin stress fibers and focal adhesions when seeded on a combination substrate of CBD and heparin-binding domain FN fragments, and this effect can be inhibited with the addition of heparin to the cell medium (
). These data indicate that another cell-surface heparan sulfate proteoglycan can compensate for the lack of syndecan-4 to promote focal adhesion and stress fiber formation.
Alternatively, the PKC pathway may be activated following the association of syndecan-4 with a heparin-binding growth factor receptor. Growth factor ligation (such as fibroblast growth factor-2) may promote the association of syndecan-4 with PKC, leading to its activation and the subsequent formation of focal adhesions and actin stress fibers. Syndecan-4 acts as a co-receptor for several heparin-binding growth factors, regulating the ligand-dependent activation of the primary receptors (
To demonstrate conclusively that the signaling mechanism affecting the phosphorylation of FAK Tyr397 acts specifically through syndecan-4, syndecan-4-null fibroblasts were used. The cells were seeded on a FN substrate as they have been shown to develop focal adhesions under these conditions. The lack of syndecan-4 expression in these cells dictates that adhesion to a FN substrate will not stimulate a syndecan-4 signaling pathway but will activate the unknown complementary pathway, if it is involved (
). Our experiments reveal that there is less FAK phosphorylated on Tyr397 in syndecan-4-null fibroblasts than wild type fibroblasts under basal conditions and this can be rescued through re-expression of syndecan-4, demonstrating that syndecan-4 is directly involved in influencing FAK Tyr397 phosphorylation. The lack of FAK phosphorylated on Tyr397 in the syndecan-4-null cells was not because of an inability of Tyr397 to be phosphorylated, as treatment with LPA or sodium vanadate (data not shown) increased Tyr397phosphorylation to syndecan-4-WT control levels, but was attributable to a decrease in the levels of active Rho in the cells. Correspondingly, direct inactivation of Rho using C3 exotransferase resulted in the loss of FAK phosphorylated at Tyr397 in the focal adhesions of syndecan-4-WT cells. The syndecan-4-null fibroblasts still generate vinculin-containing focal complexes so the limited level of GTP-bound Rho present in the null cells is sufficient to generate stress fibers and focal adhesions. Incubation of syndecan-4-null fibroblasts with LPA augmented the level of active Rho, indicating that the molecule is capable of functioning appropriately but is either not activated to the same degree as in wild type cells or is unable to maintain the active state for the “normal” length of time. Rho activation has been shown to increase FAK phosphorylation (
). This is the first description, to our knowledge, that the heparan sulfate proteoglycan syndecan-4 influences the level of active Rho in cells.
Rho cycles between an active GTP-bound state and an inactive GDP-bound state. While active it can bind to its effector molecules: Dia, ROCKs (Rho kinases), and phosphatidylinositol-4-phosphate 5-kinase to induce actin polymerization, cell body contraction, and stress fiber and focal adhesion formation (
) it seems likely that syndecan-4 signaling is associated with cell migration. Decreased cell migration is associated with either lack of syndecan-4 expression or by overexpressing full-length syndecan-4 or syndecan-4 containing cytoplasmic deletion mutants (
). That both ends of the spectrum (overexpression of syndecan-4 and lack of syndecan-4) inhibit cell migration suggests that a homeostasis is generated through syndecan-4 signaling and a balance may be required for optimal cell migration. We hypothesize that under conditions in which syndecan-4 is not ligated (FN-null cells seeded on the CBD or syndecan-4-null cells seeded on full-length FN) integrin ligation induces the phosphorylation of FAK Tyr397. Tyrosine phosphatase activity leads to the subsequent dephosphorylation of this tyrosine residue. The levels of GTP-bound Rho are also low. This situation is most likely not a static event but probably encourages the cycling of FAK between a phosphorylated and nonphosphorylated state on Tyr397. Ligation of syndecan-4 (whether in FN-null or syndecan-4-WT cells plated on full-length FN) results in the increased activation of Rho. This attenuates the tyrosine phosphatase activity causing FAK Tyr397 to remain phosphorylated longer. The tyrosine phosphatase activity is not completely inhibited, therefore, cycling between phosphorylation and dephosphorylation of Tyr397probably occurs but under a different kinetic. This is pictured diagrammatically in Fig. 6.
We thank Hui Su for confocal assistance and Stefania Saoncella, Tokuro Iwabuchi, and Enzo Calautti for helpful discussions.