Abstract:

Fibroblasts produce and assemble fibronectin (FN) into an extensively branched extracellular matrix. In cancer, the ability to deposit FN into a matrix is often reduced or lost. Decreased FN assembly by the human fibrosarcoma cell line, HT1080, is recovered by treatments that inhibit intracellular signaling through MAP kinase or stimulate transcription using the glucocorticoid dexamethasone. One gene, identified by a microarray experiment, significantly upregulated by MAP kinase pathway inhibition or dexamethasone treatment is the diastrophic dysplasia sulfate transporter (DTDST). DTDST is a transmembrane sulfate/chloride antiporter. Mutations in DTDST in human patients reduce sulfate uptake by fibroblasts and result in an under-sulfation of cartilage proteoglycans, which manifests in several autosomal recessive chondrodysplasias. Work in this thesis found that blockade of DTDST expression with siRNAs significantly reduced detergent-insoluble FN matrix, whereas overexpression of DTDST enhanced matrix assembly and partially bypassed the dexamethasone requirement, functionally linking this sulfate transporter to FN matrix assembly.

Proteoglycans are extensively sulfated on their glucosaminoglycan (GAG) side chains and these chains interact electrostatically with the hepII domain of FN. Chlorate and xyloside treatments that inhibit sulfation or GAG chain synthesis, respectively, and competition experiments using soluble heparin or chondroitin sulfate chains caused a reduction in FN matrix assembly. By contrast, excess soluble N- or O-desulfated heparin failed to inhibit matrix assembly. Thus, sulfated GAG chains are necessary for formation of a detergent-insoluble FN matrix. Knock-down of the five different transmembrane proteoglycans individually showed that matrix assembly was diminished only with loss of syndecan-2, a known FN binding proteoglycan. We suggest a novel role for DTDST-mediated sulfation in providing the requisite charge that allows this interaction to occur with the functional outcome of FN matrix deposition. We speculate that a defect in FN deposition in the early stages of chondrogenesis may be an underlying cause of the diastrophic dysplasia disorders.