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Syndecans, a family of transmembrane heparan sulphate proteoglycans, contribute to various biological processes, including adhesion, motility, proliferation, differentiation and morphogenesis. We document here the involvement of syndecan-2 acting alone or co-operatively with integrin α5β1, for regulation of actin-cytoskeletal organization on cell adhesion to fibronectin, using fibronectin-recombinant polypeptides containing the ligands for either or both of these receptors as substrata. Lewis lung carcinoma-derived low-metastatic P29 cells binding to the substrata by both receptors formed actin stress fibres, whereas those binding by syndecan-2 or integrin α5β1 alone formed filopodia or cortex actin. In contrast, higher metastatic LM66-H11 cells formed cortex actin even on substrata containing both ligands. Northern-blot and flow-cytometric analyses revealed that syndecan-2 expression in LM66-H11 cells was significantly lower (1/4.5 in mRNA and 1/8 in cell-surface expression) than in P29 cells, whereas expression levels of integrin α5β1 and other syndecans were similar in both cell types. These results suggest that the failure of LM66-H11 to form stress fibres is due to a lower expression of syndecan-2 than that due to a threshold for its function. This was confirmed by the finding that overexpression of syndecan-2 by transfection of its cDNA into LM66-H11 cells caused the formation of stress fibres on the fibronectin substratum. These in vitro cellular responses of the two clones might reflect their in vivo situation in primary tumours in which P29 cells with a stroma-inducing capacity were immediately surrounded by fibronectin-rich matrix formed by the induced stromal cells, whereas LM66-H11 cells without such capacity were not surrounded by a similar matrix.

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of λ phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11 kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5ʹ-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5ʹ flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

We previously reported that a mouse Lewis lung carcinoma-derived stroma-inducing clone, P29, highly expresses a syndecan-like proteoglycan exhibiting specific binding to fibronectin, a major constituent of the interstitial matrix formed by the induced stromal cells, via its heparan sulphate chains [Itano, Oguri, Nakanishi and Okayama (1993) J. Biochem. (Tokyo) 114, 862–873]. On metabolic labelling of the proteoglycan with [32P]Pi, followed by identification of the radiolabelled material using glycanases, almost all the isotope was found to have been incorporated into a core portion of molecular mass 48 kDa, which was generated by digestion with heparan sulphate lyase I plus chondroitin ABC lyase. Immunoblotting of the core protein with a monoclonal antibody, F58-6G12, demonstrated that the proteoglycan was mouse syndecan-2. CsCl-density-gradient centrifugation after mild treatment of liposome-intercalated 32P-labelled syndecan-2 with trypsin resulted in clear separation of the radioactivity into a bottom fraction containing all the glycosaminoglycans (accounting for 40% of the total radioactivity) and a top fraction containing liposome-associated peptides (60%). The former isotope was shown to be linked covalently to both heparan sulphate and chondroitin sulphate chains, probably at their bridge regions. The latter was mostly attributed to phosphoserine, the one and only phosphorylated amino acid released on acid hydrolysis of this proteoglycan, strongly suggesting that the phosphorylation occurs at a specific serine residue(s) in the cytoplasmic domain of the core protein.

This study addresses the characterization of heparan sulphates of the basement-membrane proteoglycans in tumour formed after the subcutaneous implantation of Lewis-lung-carcinoma-derived different metastatic clones (P29, LM12-3 and LM60-D6 clones with low, medium and high metastatic potentials respectively). Heparan sulphate proteoglycans (125-158 micrograms of hexuronate/g dry weight of tissue) were isolated from chondroitin ABC lyase digests of a proteoglycan fraction obtained after DEAE-Sephacel chromatography of tissue extracts. The proteoglycans were separated into three molecular species by Sepharose CL-4B chromatography followed by CsCl-density-gradient centrifugation: large proteoglycans with an estimated M(r) of 820,000-130,000, which consisted of two components with low (< 1.34 g/ml; PGII-M) and high (> 1.37 g/ml; PGII-B) density, and a small proteoglycan with an M(r) of less than 80,000 (PGIII). Of these, only the PGII-M proteoglycan (34-37 micrograms of hexuronate/g dry weight) reacted with the antiserum against proteoglycan of Engelbreth-Holm-Swarm-tumour basement membrane, and represented, therefore, a basement-membrane proteoglycan. Digestion with heparan sulphate lyases I and II of the heparan sulphates (M(r) 36,000) from the PGII-M proteoglycan of the three tumours resulted in almost complete depolymerization to give six unsaturated disaccharides identified as 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-D-glucose and 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-6-O-sulpho-D-glucose. Comparison of the relative amounts of these disaccharides produced from the three tumour-derived heparan sulphates demonstrated that the degree of sulphation of the heparan sulphates correlated with the degree of morphological organization of the tumour basement membranes; the heparan sulphate from the more highly metastatic tumour with more highly organized basement membrane exhibited a higher degree of overall sulphation along the glycosaminoglycan chains, which was due to an increased content of the three repeating disaccharides having 6-O-sulphated glucosamine residues.