Paul F. Goetinck

Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4

The syndecans make up a family of transmembrane heparan sulfate proteoglycans that act as coreceptors with integrins and growth factor tyrosine kinase receptors. Syndecan-4 is upregulated in skin dermis after wounding, and, in cultured fibroblasts adherent to the ECM protein fibronectin, this proteoglycan signals cooperatively with beta1 integrins. In this study, we generated mice in which the syndecan-4 gene was disrupted by homologous recombination in embryonic stem cells to test the hypothesis that syndecan-4 contributes to wound repair. Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from wild-type littermates. Compared with wild-type littermates, mice heterozygous or homozygous for the disrupted gene have statistically significant delayed healing of skin wounds and impaired angiogenesis in the granulation tissue. These results indicate that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis and that syndecan-4 is haplo-insufficient in these processes.

Phosphoinositide 3-Kinase Signaling to Akt Promotes Keratinocyte Differentiation Versus Death

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of “late” differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

Syndecan-4 modulates focal adhesion kinase phosphorylation.

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.

Matrix Contraction by Dermal Fibroblasts Requires Transforming Growth Factor-β/Activin-Linked Kinase 5, Heparan Sulfate-Containing Proteoglycans, and MEK/ERK: Insights into Pathological Scarring in Chronic Fibrotic Disease

Scarring is characterized by excessive synthesis and contraction of extracellular matrix. Here, we show that fibroblasts from scarred (lesional) areas of patients with the chronic fibrotic disorder diffuse scleroderma [diffuse systemic sclerosis (dSSc)] show an enhanced ability to adhere to and contract extracellular matrix, relative to fibroblasts from unscarred (nonlesional) areas of dSSc patients and dermal fibroblasts from normal, healthy individuals. The contractile abilities of normal and dSSc dermal fibroblasts were suppressed by blocking heparin sulfate-containing proteoglycan biosynthesis or antagonizing transforming growth factor-beta receptor type I [activin-linked kinase (ALK5)] or ras/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Compared with both normal and nonlesional fibroblasts, lesional dSSc fibroblasts overexpressed the heparin sulfate-containing proteoglycan syndecan 4. We also found that the procontractile signals from transforming growth factor (TGF)-beta were integrated through syndecan 4 and MEK/ERK because the ability of TGFbeta to induce contraction of dermal fibroblasts was prevented by MEK antagonism. TGFbeta could not induce a contractile phenotype or phosphorylate ERK in syndecan 4(-/-) dermal fibroblasts. These results suggest that integrating TGFbeta and ERK signals via syndecan 4 is essential for the contractile ability of dermal fibroblasts. We conclude that antagonizing MEK/ERK, TGFbeta1/ALK5, or syndecan 4 may alleviate scarring in chronic fibrotic disease.

Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4

Pulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4-null (Sdc4-/-) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4-/- mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis.

Proteochondroitin sulfate synthesis and chondrogenic expression

Abstract Embryonic limb bud cells were cultured under conditions in which chondrogenic expression takes place. Quantitative and qualitative estimates were made of the proteochondroitin sulfate synthesized under these conditions. A tenfold increase in incorporation into proteochondroitin sulfate is observed over the culture period during which chondrogenic expression takes place. This increase is reflected in a preferential increase of a particular fraction identified on 1% agarose chomatography. Low levels of this chromatographic fraction can be identified in limb buds before any evidence of cartilage expression is evident. We postulate that this preferential augmentation is a cartilage specific event.

Biochemical and ultrastructural studies of collagen and proteochondroitin sulfate in normal and nanomelic cartilage

Abstract Biochemical and ultrastructural analysis of the sternal cartilage of chick embryos homozygous for the autosomal recessive gene nanomelia suggest that the mutant cells are functional chondrocytes in all respects except in proteochondroitin sulfate synthesis. Proteochondroitin sulfate synthesized by normal and mutant sterna in vitro was chromatographed on 1% agarose. Two distinct fractions of proteochondroitin sulfate were resolved from both normal and mutant cartilage. In normal cartilage, the major fraction represents approximately 90% of the total material, and in the mutant, this fraction is reduced to 10%, while the second fraction remains unchanged. It is suggested that at the onset of chondrogenesis in the mutant, an augmentation in the syntheis of the major fraction does not occur. Collagen synthesis in the mutant cartilage was analyzed by hydroxyproline determination, carboxymethylcellulose chromatography, and amino acid analysis to determine the percentage hydroxylation of lysine residues. By these procedures, collagen synthesis in the mutant was found to be both quantitatively and qualitatively similar to normal. Ultrastructural studies on the mutant sterna revealed that while the mutant chondrocytes were normal in appearance, the amount of extracellular matrix was decreased. In conjunction with this decrease, there is a severe reduction in the number of proteochondroitin sulfate matrix granules. No differences were observed in the collagen fibrils.

Studies on limb morphogenesis II. Experiments with the polydactylous mutant eudiplopodia

Abstract The development of the rear limb buds in embryos homozygous for eudiplopodia was studied. Eudiplopod embryos are unique among polydactylous forms in having their supernumerary digits located dorsally to the normal toe complement, which is always present. Histological examination of eudiplopod limbs from stages 17 to 26 indicated that a secondary apical ectodermal ridge (AER) forms in the dorsal ectoderm at stages 22 or 23. Prior to these stages (17–21) the histological picture was normal in all respects. Reciprocal recombinations between eudiplopod and normal limb bud components indicated that the genetic defect in this mutant is localized in the ectoderm. Limb buds composed of eudiplopod ectoderm and normal mesoderm developed a two-plane duplication typical of eudiplopod limbs while those constituted from eudiplopod mesoderm and normal ectoderm developed into normal limbs. These results are discussed in context of the role of the AER in normal limb development and its interaction with the mesoderm in the formation of terminal limb parts.

Mice Lacking Matrilin-1 (Cartilage Matrix Protein) Have Alterations in Type II Collagen Fibrillogenesis and Fibril Organization

Matrilin‐1 (cartilage matrix protein) is a homotrimeric protein that forms collagen‐dependent and collagen‐independent fibrils in the extracelluar matrix of cartilage. In the growth plate of developing long bones, the gene for matrilin‐1 is transcribed exclusively by the chondrocytes of the zone of maturation which is situated between the zones of proliferation and hypertrophy. When associated with the cartilage collagen fibril, which consists of collagens type II, IX, and XI, matrilin‐1 displays a periodicity of 59.3 nm. Matrilin‐1 also interacts with the proteoglycan, aggrecan. Because of its association with the collagen fibril, we tested the hypothesis that matrilin‐1 may play a role in collagen fibril formation and cartilage matrix assembly by generating mice with targeted mutations in the matrilin‐1 gene. Ultrastructural studies of the cartilage of growth plates of matrilin‐1 null mice reveal an abnormal type II collagen fibrillogenesis and fibril organization in the matrix of the zone of maturation. These results represent the first report on the regulation of the heterotypic type II collagen fibril by a non‐collagenous protein. The abnormal fibrillogenesis had no obvious effects on skeletal development, on the organization of chondrocytes in the growth plate and on the deposition of aggrecan and the hypertrophic‐specific type X collagen in the cartilaginous matrix. Dev Dyn 1999;216:434–441. ©1999 Wiley‐Liss, Inc.

Syndesmos, a Syndecan-4 Cytoplasmic Domain Interactor, Binds to the Focal Adhesion Adaptor Proteins Paxillin and Hic-5

Syndecan-4 and integrins are the primary transmembrane receptors of focal adhesions in cells adherent to extracellular matrix molecules. Syndesmos is a cytoplasmic protein that interacts specifically with the cytoplasmic domain of syndecan-4, and it co-localizes with syndecan-4 in focal contacts. In the present study we sought possible interactors with syndesmos. We find that syndesmos interacts with the focal adhesion adaptor protein paxillin. The binding of syndesmos to paxillin is direct, and these interactions are triggered by the activation of protein kinase C. Syndesmos also binds the paxillin homolog, Hic-5. The connection of syndecan-4 with paxillin through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperative signaling of these two receptors in the assembly of focal adhesions and actin stress fibers.