Fibronectin-Tissue Transglutaminase Matrix Rescues RGD-impaired Cell Adhesion through Syndecan-4 and β1 Integrin Co-signaling

Abstract

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase C␣ (PKC␣) and its subsequent interaction with ␤ 1 integrin since disruption of PKC␣ binding to ␤ 1 integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKC␣ leading to its association with ␤ 1 integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation. Tissue transglutaminase (TG2)2 belongs to a family of enzymes that in the presence of Ca 2ϩ catalyze the post-translational modification of proteins either by covalent cross-linking or through the incorporation of primary amines. TG2 has a GTP binding/hydrolysis site that negatively modulates the Ca 2ϩ -dependent transamidating activity of the enzyme by obstructing access to the active site (1). As a consequence, the enzyme is likely to be inactive under normal Ca 2ϩ homeostasis. TG2 localizes mainly in the cytoplasm, yet recent reports also suggest its presence in the nucleus, mitochondria, at the cell surface, and in the extracellular matrix (ECM) (1-3). TG2 is translocated to the plasma membrane and was subsequently deposited into the ECM via a non-classical secretory mechanism reportedly dependent on active site conformation and on an intact N-terminal ␤-sandwich domain (4, 5) as well as on its possible association with integrins (6). Deposition of the enzyme into the ECM after cell damage and stress is important in the remodeling and/or stabilization of the several ECM proteins, such as FN (7,8). FN is particularly interesting since TG2 binds to this ECM protein with high affinity promoting wideranging effects on cell-matrix interactions, including the regulation of cell adhesion and migration, matrix assembly, and adhesion-dependent signaling (6,7,9).Cell adhesion to FN involves a series of coordinated signaling events orchestrated by numerous transmembrane receptors including the integrins and the superfamily of cell-surface proteoglycans (10, 11). The identity of the receptor-ligand pairing defines the composition of f...

RGD-independent Cell Adhesion via a Tissue Transglutaminase-Fibronectin Matrix Promotes Fibronectin Fibril Deposition and Requires Syndecan-4/2 and α5β1 Integrin Co-signaling

“…Following serum starvation for 16 h, cells were detached as described above and then washed three times with serum-free medium to remove any traces of serum or trypsin inhibitor and pretreated with GRGDTP peptide or its control peptide GRADSP in serum-free medium for 20 min. Based on previous toxicity findings (7,22), adhesion experiments were performed using 100 g/ml RGD synthetic peptide, leading to partial inhibition in cell adhesion. Cells were then seeded onto 96-well plates (2.5 ϫ 10 4 /well) coated with human plasma FN (FN matrix) with or without immobilized guinea pig liver TG (gplTG) (TG-FN matrix) as described previously (7,22).…”

“…Wild type T98G glioblastoma cells and transfected T98G cells with syndecan-2 or syndecan-4 vectors were grown as reported previously (29). Wild type, syndecan-4 null, and ␤1 integrin null mouse embryo fibroblast (MEF) cells were grown as described previously (22). Parental Chinese hamster ovary (CHO) and CHO-K1 cells were purchased from ATCC and grown in Ham's F-12 medium according to the supplier's instructions.…”

“…Cell Adhesion Assay-Cell adhesion assays were performed as described previously (7,22). Unless otherwise stated, cells were detached with 0.25% (w/v) trypsin, and the trypsin was neutralized either with trypsin inhibitor or by the addition of serum-containing medium.…”

“…We demonstrated that the TG-FN heterocomplex can compensate for the RGD peptideinduced loss of cell adhesion through a syndecan-4 and ␤1 integrin co-signaling pathway supporting both cell adhesion and cell survival (22). We illustrated how this mechanism could be physiologically important by showing that a TG2-rich conditioned matrix produced by TG2-transfected Swiss 3T3 cells could also compensate for RGD peptide-induced loss of cell adhesion (22). We also illustrated how this compensation mechanism is particularly important for cell survival during situations involving matrix remodeling (e.g.…”

“…In our previous work, we demonstrated that formation of the TG-FN heterocomplex can modulate cell behavior, including cell adhesion and spreading and their relevant signal transduction pathways (7,18,21). We demonstrated that the TG-FN heterocomplex can compensate for the RGD peptideinduced loss of cell adhesion through a syndecan-4 and ␤1 integrin co-signaling pathway supporting both cell adhesion and cell survival (22). We illustrated how this mechanism could be physiologically important by showing that a TG2-rich conditioned matrix produced by TG2-transfected Swiss 3T3 cells could also compensate for RGD peptide-induced loss of cell adhesion (22).…”