1987; Shin et al. al. 1992, Bernfield et al. 1999). All adhesive cells express at least one syndecan, and most express multiple syndecans (Kim et al. 1994). The core proteins of each of these four distinct gene products place the HS chains distal from the plasma membrane. Each syndecan contains at its COOH terminus a short and highly homologous cytoplasmic domain with serine and tyrosine residues at conserved positions. By way of their HS chains, syndecans bind a wide variety of soluble and insoluble ligands, such as follows: extracellular matrix components, cell adhesion molecules, growth factors, cytokines, proteinases and proteinase inhibitors, lipid metabolism proteins, and microbial pathogens (Bernfield et al. 1992; Carey 1997; Bernfield et al. 1999). Syndecans facilitate the formation of signaling complexes by acting as coreceptors, concentrating and presenting ligands to the cell surface receptors, or internalizing them via endocytosis, thus, modulating ligand activities (Bernfield et Pexmetinib (ARRY-614) al. 1999). Because the HS chains of the cell surface and shed syndecans can bind the same ligands, syndecan ectodomain shedding is a mechanism for producing soluble HSPG effectors that can compete for the same ligands as their cell surface counterparts. Shedding of syndecan-1 and -4 can be accelerated via receptor activation (e.g., thrombin and EGF family members) and by direct action of proteases (e.g., plasmin and thrombin; Subramanian et al. 1997). These ectodomains are in fluids accumulating following injury and inflammation (Subramanian et al. 1997; Kato et al. 1998), but not in normal human plasma (Subramanian et al. 1997). The soluble syndecan-1 ectodomain potently inhibits heparin-mediated FGF-2 mitogenicity (Kato et al. 1998), which is consistent with studies indicating that the shed ectodomains can inhibit cell proliferation (Mali et al. 1994; Forsten et al. 1997; Dhodapkar and Sanderson 1999), and binds neutrophil-derived elastase and Pexmetinib (ARRY-614) cathepsin G, reducing the action of their physiological inhibitors (Kainulainen et al. 1998). These activities are consistent with a role for the soluble syndecan ectodomains in the response to tissue injury. While syndecan ectodomain shedding is known to be activated by physiological stimulants (Subramanian et al. 1997) and the ectodomains are being ascribed pathophysiological roles, little is known about how their release from the cell surface is regulated. Therefore, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that syndecan shedding is regulated at multiple levels, based on the following findings: (1) that in addition to proteases and receptor ligands, agents that mediate cellular Pexmetinib (ARRY-614) responses to stress accelerate shedding; (2) shedding accelerated by various physiological agents involves activation of distinct intracellular signaling pathways; (3) the proteolytic activity responsible for cleavage of syndecan core proteins is associated with the cell surface, and is a TIMP-3Csensitive MP that can act on unstimulated adjacent cells; (4) the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and (5) the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding. These results MMP19 demonstrate the existence of highly regulated mechanisms that convert syndecans from cell surface receptors or coreceptors to soluble HSPG effectors. Regulation of shedding by physiological mediators suggests that syndecan ectodomains are shed in response to specific developmental and pathophysiological cues. Now soluble, the shed syndecan ectodomains likely have roles in morphogenesis, tissue repair, and host defense. Preliminary reports of this study have been presented in abstract form (Fitzgerald, M.L., J.-S. Chun, and M. Bernfield, American Society of Cell Biology. 1994. 1813 (Abstr.); Fitzgerald, M.L., and M. Bernfield, American Society of Cell Biology. 1997. 2286 (Abstr.); Fitzgerald, M.L., Z. Wang, and M. Bernfield, American Society of Cell Biology. 1998. 326 (Abstr.)). Materials and Methods Materials and Chemicals Ceramide (d-erythro-Sphingosine, at 4C to remove unbound mAb, and incubated (106 cells/tube) for 30 min at 37C with or without 0.5 M PMA. All washes and incubations were done in serum-free RPMI 1640 media. After treatment, cells were fixed in 4% paraformaldehyde in Pexmetinib (ARRY-614) PBS for 15 min at 4C, washed in PBS, and incubated with FITC-conjugated streptavidin for 30 min at room temperature. Cells labeled with FITC-streptavidin only were included as controls for nonspecific staining..
