It has been shown that osteopontin (OPN) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). However, the molecular mechanism of OPN action is yet to be elucidated. Splenic monocytes obtained from arthritic mice exhibited a significant capacity for cell migration toward thrombin-cleaved OPN but not toward full-length OPN. Migratory monocytes expressed α9 and α4 integrins. Since cleavage of OPN by thrombin exposes the cryptic epitope recognized by α9 and α4 integrins, we investigated the role of the cryptic epitope SLAYGLR in a murine RA model by using a specific antibody (M5) reacting to SLAYGLR sequence. The M5 antibody could abrogate monocyte migration toward the thrombin-cleaved form of OPN. Importantly, M5 antibody could inhibit the proliferation of synovium, bone erosion, and inflammatory cell infiltration in arthritic joints. Thus, we demonstrated that a cryptic epitope, the SLAYGLR sequence of murine OPN, is critically involved in the pathogenesis of a murine model of RA

Laminin, a major component of the basement membrane, has diverse biological activities. Recently, we identified various biologically active sequences on laminin-1 by using a large set of synthetic peptides. Chitosan, a polysaccharide, is biodegradable and has been used as a biomaterial. Here, we conjugated several biologically active laminin peptides onto chitosan membranes and measured the cell attachment activity of peptide-conjugated chitosan membranes with various cell types. The active laminin peptide-conjugated chitosan membranes promoted cell attachment with cell type specificity. A99 (AGTFALRGDNPQG)-chitosan membrane promoted cell attachment with well-organized actin stress fibers. This adhesion was inhibited by EDTA but not by heparin. AG73 (RKRLQVQLSIRT)-chitosan membrane promoted cell attachment with filopodia formation, and this adhesion was inhibited by heparin but not by EDTA. These data suggest that the A99-chitosan membrane interacted with an integrin cellular receptor and that the AG73-chitosan membrane promoted proteoglycan-mediated cell attachment, as previously reported. Furthermore, both AG73-chitosan and A99-chitosan membranes effectively promoted neurite outgrowth with PC12 rat pheochromocytoma cells. We conclude that conjugation on a chitosan membrane is applicable for testing quantitatively the biological activity of synthetic peptides and that these constructs have a potential ability to serve as bioadhesive materials for tissue regeneration and engineering.

No abstract

Laminins are a family of trimeric extracellular matrix proteins consisting of ␣, ␤, and ␥ chains. So far five different laminin ␣ chains have been identified. The laminin ␣4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin ␣4 chain. Here we have focused on the biological functions of the laminin ␣4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-␣4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-␣4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-␣4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVIS-LYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-␣4G. These results suggest that the four active sites are involved in the biological functions of the laminin ␣4 chain G domain. Furthermore, rec-␣4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.Laminins, a family of extracellular matrix proteins, consist of three different subunits, ␣, ␤, and ␥ chains. So far, five ␣, three ␤, and three ␥ chains have been identified, and at least 15 isoforms (laminin 1-15) are formed by various combinations of each subunit (1-4). Laminins have diverse biological activities including promotion of cell adhesion, migration, neurite outgrowth, angiogenesis, and tumor metastasis (5). More than 20 receptors have been reported for these laminin molecules (6). Several active sites on laminin-1 have been identified using proteolytic fragments, recombinant proteins, and synthetic peptides (7,8). Previously, we screened for cell adhesive sequences on laminin-1 using 673 overlapping synthetic peptides covering the entire protein (9 -12). Most of the active peptides were localized in the globular domains and found to play a critical role in binding to cell surface receptors in a peptide-and cell type-specific manner (13,14). Several peptides were found to interact with integrins and syndecans (15-18). Some of the peptides promoted neurite outgrowth, angiogenesis, and tumor metastasis (19 -22).The laminin ␣ chains are generally large (M r ϭ 400,000) and contain a C-terminal globular domain consisting of five globular modules LG1-LG5. The laminin ␣4 chain lacks the N-terminal short arm and is expressed in cells of mesenchymal origin, such as...