Syndecan-2 cytoplasmic domain upregulates Matrix Metalloproteinase-7 expression via Protein KinaseCγ mediated FAK/ERK signaling pathway in colon cancer

The syndecan family of heparan sulfate proteoglycans contribute to cell adhesion and communication by serving as co-receptors for cell signaling and extracellular matrix molecules. Syndecan-2 is located at the cell surface, and we previously reported that it induces matrix metalloproteinase-7 (MMP-7) expression in colon cancer cells. However, the underlying regulatory mechanisms are unknown. Here, we report that over-expression of syndecan-2 in HT-29 colon cancer cells increases the phosphorylation of focal adhesion kinase (FAK) and extracellular signalregulated kinase (ERK) in parallel with upregulated MMP-7 expression, but a syndecan-2 mutant lacking the cytoplasmic domain showed significant reductions in these effects. Consistent with this observation, FAK inhibition via FAKrelated nonkinase expression or inhibition of ERK with the ERK1/2 inhibitor SCH772984 diminished the syndecan-2–mediated upregulation of MMP-7. Activation of protein kinase C (PKC) enhanced syndecan-2-mediated MMP-7 expression, whereas inhibition of PKC had the opposite effect. Of note, the exogenous expression of syndecan-2 triggered localization of PKC to the membrane. Expression of syndecan-2 harboring a phospho-mimetic (S198E) mutation of the variable region of the cytoplasmic domain enhanced MMP-7 expression and FAK phosphorylation. Finally, experimental supression of shedding of the syndecan-2 extracellular domain did not significantly affect the syndecan-2–mediated upregulation of MMP-7 in the early period after syndecan-2 over-expression. Taken together, these findings suggest that syndecan-2’s cytoplasmic domain upregulates MMP-7 expression in colon cancer cells via PKCmediated activation of FAK/ERK signaling. The members of the syndecan family of heparan sulfate (HS) proteoglycans contribute to cell adhesion and cell communication by serving as co-receptors for cell signaling and extracellular matrix (ECM) molecules (1). Similar to many other receptors, the syndecans contain three domains (2) that have unique but related functions in signal transduction. The extracellular domain interacts http://www.jbc.org/cgi/doi/10.1074/jbc.M117.793752 The latest version is at JBC Papers in Press. Published on August 16, 2017 as Manuscript M117.793752 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from Role of the syndecan-2 cytoplasm on MMP-7 expression 2 with various extracellular ligands including growth factors, ECM molecules, cytokine, chemokines and enzymes (1) to accommodate the functional needs of the cells (3). These interactions initiate signal transduction to regulate cellular functions both inside and outside cells. In particular, extracellular ligand binding induces clustering of syndecan transmembrane domains to initiate intracellular signaling events. Notably, syndecans contain a GXXXG motif that promotes self-association with the core protein (4). In the context of intracellular signaling, however, the cytoplasmic domain sequence is the most critical for regulating syndecan-mediated adhesion and cytoskeletal organization. The cytoplasmic domain of syndecan is essentially composed of two regions of conserved amino acid sequences, C1 and C2. The C1 region participates in direct interactions with the ezrin-radixin-moesin (ERM) protein, tubulin, cortactin, and Src (5,6); in syndecan-1, this domain is involved in regulating endocytosis (7). The C2 region contains a PDZ-binding motif and is responsible for regulating syndecan recycling and exosome formation (8,9). The C1 and C2 regions are separated by a central variable sequence of amino acids that is distinct for each family member (V) and provides the unique function of each syndecan (10). The V region of syndecan-1 is known to be involved in the regulation of cell spreading and actin-fascin bundling (11). The syndecan-4 V region contains an NPXY sequence that binds to phosphatidylinositol 4, 5bisphosphate (PIP2) and regulates syndecan-4 cytoplasmic domain oligomerization and protein kinase C(PKCactivation(12-14). In syndecan-2, the V region has the sequence RKPSSAA, in which the two serine residues can be phosphorylated by PKC, - and -(15-17). Indeed, the associations of the syndecan-2 cytoplasmic domain with PKCs are important for syndecan-2-mediated functions. For example, PKC-II directly interacts with syndecan-2, enhances its membrane localization, and increases melanin synthesis in melanoma cells (18). Moreover, PKC has been shown to mediate the development of the left-right axis in the Xenopus heart (15). These previous results support the unique functional roles of the cytoplasmic domain of syndecan-2, and show that these functions may me mediated through serine phosphorylation. In colon cancers, various matrix metalloprotease (MMPs) are over expressed, and their increased expressions and activations have been correlated with cancer activity and poor prognosis (19,20). Human colon cancer cells reportedly overexpress MMP-1, -2, -3, -7, -9, -10, -11, -12, -13, and -14 (21). Of them, MMP-7 is consistently expressed in colon cancer cells (22), and its enzymatic activity has been closely associated with colon cancer progression and advanced clinical stages (23). MMP-7 is well known to directly cleave various ECM substrates, including elastin, type II collagen, fibronectin, vitronectin, aggrecan, and proteoglycan (24-26). When promoting cancer activity, MMP-7 also cleaves cell surface molecules; it has been shown to promote the shedding of pro-TNF-, Fas ligand, and HS proteoglycans in various cancers (27-29). We previously reported that syndecan-2 expression is increased in colon cancer cells (30), and that increased syndecan-2 expression enhances the tumorigenic potential of colon cancer cells (30-33). Therefore, the regulation of MMP-7 expression might be critical to colon cancer progression. We previously reported that syndecan-2 interacts with pro-MMP-7 at the cell surface, and that elevated syndecan-2 expression critically activates the processing of pro-MMP-7 into the active form (30). Accordingly, we proposed that syndecan-2 could function as a docking receptor for pro-MMP-7. However, it remained unknown how syndecan-2 might regulate the expression of MMP-7 as a cell surface receptor. Here, we analyzed the ability of the syndecan-2 cytoplasmic domain to act as a cell surface receptor in regulating MMP-7 expression. by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from Role of the syndecan-2 cytoplasm on MMP-7 expression