Constitutive- and accelerated shedding of murine syndecan-1 is mediated by cleavage of its core protein at a specific juxtamembrane site

Syndecan-1 is one of four members of the syndecan family of heparan sulfate proteoglycans (1, 2) Most cells express at least one type of syndecan, and their expression pattern is highly regulated (1, 3). Syndecans are abundant on the surface of all adherent mammalian cells. About 1% of membrane-anchored proteins undergo regulated proteolytic cleavage near the plasma membrane, resulting in release of their ectodomains in a process known as shedding (4, 5). The syndecan ectodomain is shed constitutively by cultured cells, though the release can be accelerated by growth factor receptor activation, e.g. by thrombin and epidermal growth factor (6, 7). In vivo, stress or skin injury/wounding can lead to increased levels of soluble syndecan-1 ectodomain in biological fluids (7, 8). The process of ectodomain shedding not only reduces the number of surface receptors, an effective way to down-regulate signal transduction through these receptors, it also converts the membrane-bound cell surface receptors into soluble effectors that can effectively compete for the same ligand as dominant negative modulators as well as act as paracrine effectors at a remote location. The significance of this has been shown in vivo in syndecan-1-deficient and syndecan-1 overexpressing mice, which lack or contain excessive amounts of soluble ectodomains in their tissues, respectively. Syndecan-1 deficient mice are resistant to Pseudomonas aeruginosa lung infections, due to the absence of soluble syndecan-1 (9). Soluble syndecan-1, complexed to chemokines, is apparently also required for the formation of chemotactic gradients in a model of lung inflammation (10), which may also be the underlying cause for increased leukocyte-endothelial interactions and angiogenesis in these mice (11-13). In contrast, syndecan-1 overexpressing mice accumulate excessive amounts of shed syndecan-1 in skin wound fluids, which leads to a delay in wound repair concomitant with enhanced elastolytic activity, reduced cell proliferation rates and abnormal blood vessel morphology (8). Finally, shedding of syndecan ectodomains appears to modulate feeding behaviour and weight in rodents (14). The precise mechanism of syndecan-1 ectodomain shedding has not been elucidated, yet there is accumulating evidence which suggests that diverse signal transduction pathways, such as the protein kinase C (PKC), protein tyrosine kinase (PTK) and MAP kinase pathways, are involved (6, 15, 16). Inhibitors of PKC, PTK or MAP kinase can selectively inhibit the syndecan-1 ectodomains shedding (6). Previous work suggested that syndecan-1 ectodomain shedding appears to involve several metalloproteinases, since both a peptide hydroxamate metalloproteinases inhibitor and tissue inhibitor of metalloprotease-3 (TIMP-3) can specifically inhibit syndecan-1 ectodomain shedding (6). Peptide hydroxamates inhibit both constitutive and PMA-accelerated syndecan-1 ectodomain shedding, though TIMP-3 appears to inhibit only the PMA-accelerated shedding. This result implies that constitutive and PMA-stimulated shedding of syndecan-1 is mediated by different metalloproteinases. This finding appears to be corroborated in an in vivo model demonstrating matrilysin (MMP-7)-mediated murine syndecan-1 ectodomain shedding (10). Furthermore, MT1-MMP and MT3-MMP appear to be involved in human syndecan-1 ectodomain shedding as demonstrated in an in vitro study (17). The syndecan-1 ectodomain shedding cleavage site is thought to be the dibasic peptide near the plasma membrane (16, 18), though current evidence does not support this hypothesis. In the case of human syndecan-1, a point mutant, G245L, was shed at 50% reduced extend in MT-1-MMP overexpressing 293T cells compared to mock-transfected cells (17). Thus, we sought to identify the cleavage site of murine syndecan-1, both in vitro and in vivo. Our study found that the syndecan-1 ectodomain generated by constitutive and PMA-accelerated shedding are cleaved at the same site, about 9 amino acids from the plasma membrane. This same cleavage site used in vivo for constitutively shed syndecan-1. Our results demonstrate that under basal or phorbol ester (PMA) stimulated conditions, syndecan-1 is cleaved at a site 9 amino acids from the membrane. These results are based on a molecular and biophysical analysis of the syndecan-1 obtained from both in vitro and in vivo sources. These results lay the foundation for the identification of the metalloprotease responsible for murine syndecan-1 shedding.