RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and α5β1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and ß1 integrin co-signaling pathway. By using a5 null cells, ß1 integrin functional blocking antibody, and a a5ß1 integrin targeting peptide A5-1, we demonstrate that the a5 and ß1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCa is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.

The role of tissue transglutaminase (TG2) in regulating the tumour progression of the mouse colon carcinoma CT26

The multifunctional enzyme tissue transglutaminase (TG2) is reported to both mediate and inhibit tumour progression. To elucidate these different roles of TG2, we established a series of stable-transfected mouse colon carcinoma CT26 cells expressing a catalytically active (wild type) and a transamidating-inactive TG2 (Cys277Ser) mutant. Comparison of the TG2-transfected cells with the empty vector control indicated no differences in cell proliferation, apoptosis and susceptibility to doxorubicin, which correlated with no detectable changes in the activation of the transcription factor NF-?B. TG2-transfected cells showed increased expression of integrin ß3, and were more adherent and less migratory on fibronectin than control cells. Direct interaction of TG2 with ß3 integrins was demonstrated by immunoprecipitation, suggesting that TG2 acts as a coreceptor for fibronectin with ß3 integrins. All cells expressed the same level of TGFß receptors I and II, but only cells transfected with active TG2 had increased levels of TGFß1 and matrix-deposited fibronectin, which could be inhibited by TG2 site-directed inhibitors. Moreover, only cells transfected with active TG2 were capable of inhibiting tumour growth when compared to the empty vector controls. We conclude that in this colon carcinoma model increased levels of active TG2 are unfavourable to tumour growth due to their role in activation of TGFß1 and increased matrix deposition, which in turn favours increased cell adhesion and a lowered migratory and invasive behaviour.

Fibronectin-tissue transglutaminase matrix rescues RGD-impaired celladhesion through syndecan-4 and β integrin co-signaling

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 Ca (PKCa) and its subsequent interaction with ß1 integrin since disruption of PKCa binding to ß1 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 PKCa leading to its association with ß1 integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

TG2, a novel extracellular protein with multiple functions

TG2 is multifunctional enzyme which can be secreted to the cell surface by an unknown mechanism where its Ca(2+)-dependent transamidase activity is implicated in a number of events important to cell behaviour. However, this activity may only be transient due to the oxidation of the enzyme in the extracellular environment including its reaction with NO probably accounting for its many other roles, which are transamidation independent. In this review, we discuss the novel roles of TG2 at the cell surface and in the ECM acting either as a transamidating enzyme or as an extracellular scaffold protein involved in cell adhesion. Such roles include its ability to act as an FN co-receptor for ß integrins or in a heterocomplex with FN interacting with the cell surface heparan sulphate proteoglycan syndecan-4 leading to activation of PKCa. These different properties of TG2 involve this protein in various physiological processes, which if not regulated appropriately can also lead to its involvement in a number of diseases. These include metastatic cancer, tissue fibrosis and coeliac disease, thus increasing its attractiveness as both a therapeutic target and diagnostic marker.

Analysis of Transglutaminase-2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Transglutaminase-2 (TG2) is a Ca2+-dependent protein crosslinking enzyme known to play an important role in apoptotic cell clearance by macrophages through an ill-defined mechanism. Several studies have implicated TG2 in the apoptosis programme e.g. raised TG2 levels in cells undergoing apoptosis; increased cell death with down-regulation of TG2; up-regulation of TG2 in monocytes upon differentiation into macrophages. Defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be elucidated. Here we aim to characterise the role of TG2 in macrophage function with a focus on apoptotic cell clearance. THP-1 monocytes were induced to differentiate to macrophage-like cells by three different stimulants and were analysed for the presence of TG2. Macrophage-apoptotic cell interaction studies in the presence and absence of irreversible TG2 inhibitors resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. TG2 was expressed at the macrophage cell surface and its association with ß3 integrin expression suggests the possible link between TG2 and ß3 integrins. Our current findings suggest that TG2 had got a crucial but yet to be defined role in apoptotic cell clearance.

The importance of the tissue transglutaminase-fibronectin heterocomplex in the RGD-independent cell adhesion and fibronectin matrix deposition

Tissue transglutaminase (TG2) has been reported as a wound response protein. Once over-expressed by cells under stress such as during wound healing or following tissue damage, TG2 can be secreted and deposited into extracellular matrix, where it forms a heterocomplex (TG-FN) with the abundant matrix protein fibronectin (FN). A further cellular response elicited after tissue damage is that of matrix remodelling leading to the release of the Arg-Gly-Asp (RGD) containing matrix fragments by matrix matelloproteinases (MMPs). These peptides are able to block the interaction between integrin cell surface receptors and ECM proteins, leading to the loss of cell adhesion and ultimately Anoikis. This study provides a mechanism for TG2, as a stress-induced matrix protein, in protecting the cells from the RGD-dependent loss of cell adhesion and rescuing the cells from Anoikis. Mouse fibroblasts were used as a major model for this study, including different types of cell surface receptor knockout mouse embryonic fibroblasts (MEFs) (such as syndecan-4, a5, ß1 or ß3 integrins). In addition specific syndecan-2 targetting siRNAs, ß1 integrin and a4ß1 integrin functional blocking antibodies, and a specific targeting peptide against a5ß1 integrin A5-1 were used to investigate the involvement of these receptors in the RGD-independent cell adhesion on TG-FN. Crucial for TG-FN to compensate the RGD-independent cell adhesion and actin cytoskeleton formation is the direct interaction between the heparan sulfate chains of syndecan-4 and TG2, which elicits the inside-out signalling of a5ß1 integrin and the intracellular activation of syndecan-2 by protein kinase C a (PKCa). By using specific inhibitors, a cell-permeable inhibiting peptide and the detection of the phosphorylation sites for protein kinases and/or the translocation of PKCa via Western blotting, the activation of PKCa, focal adhesion kinase (FAK), ERK1/2 and Rho kinase (ROCK) were confirmed as downstream signalling molecules. Importantly, this study also investigated the influence of TG-FN on matrix turnover and demonstrated that TG-FN can restore the RGD-independent FN deposition process via an a5ß1 integrin and syndecan-4/2 co-signalling pathway linked by PKCa in a transamidating-independent manner. These data provide a novel function for TG2 in wound healing and matrix turnover which is a key event in a number of both physiological and pathological processes.

Importance of syndecan-4 and syndecan -2 in osteoblast cell adhesion and survival mediated by a tissue transglutaminase-fibronectin complex

Tissue transglutaminase (TG2) has been identified as an important extracellular crosslinking enzyme involved in matrix turnover and in bone differentiation. Here we report a novel cell adhesion/survival mechanism in human osteoblasts (HOB) which requires association of FN bound TG2 with the cell surface heparan sulphates in a transamidase independent manner. This novel pathway not only enhances cell adhesion on FN but also mediates cell adhesion and survival in the presence of integrin competing RGD peptides. We investigate the involvement of cell surface receptors and their intracellular signalling molecules to further explore the pathway mediated by this novel TG-FN heterocomplex. We demonstrate by siRNA silencing the crucial importance of the cell surface heparan sulphate proteoglycans syndecan-2 and syndecan-4 in regulating the compensatory effect of TG-FN on osteoblast cell adhesion and actin cytoskeletal formation in the presence of RGD peptides. By use of immunoprecipitation and inhibitory peptides we show that syndecan-4 interacts with TG2 and demonstrate that syndecan-2 and the a5ß1 integrins, but not a4ß1 function as downstream modulators in this pathway. Using function blocking antibodies, we show activation of a5ß1 occurs by an inside out signalling mechanism involving activation and binding of protein kinase PKCa and phosphorylation of focal adhesion kinase (FAK) at Tyr861 and activation of ERK1/2.

Joining S100 proteins and migration:for better or for worse, in sickness and in health

The vast diversity of S100 proteins has demonstrated a multitude of biological correlations with cell growth, cell differentiation and cell survival in numerous physiological and pathological conditions in all cells of the body. This review summarises some of the reported regulatory functions of S100 proteins (namely S100A1, S100A2, S100A4, S100A6, S100A7, S100A8/S100A9, S100A10, S100A11, S100A12, S100B and S100P) on cellular migration and invasion, established in both culture and animal model systems and the possible mechanisms that have been proposed to be responsible. These mechanisms involve intracellular events and components of the cytoskeletal organisation (actin/myosin filaments, intermediate filaments and microtubules) as well as extracellular signalling at different cell surface receptors (RAGE and integrins). Finally, we shall attempt to demonstrate how aberrant expression of the S100 proteins may lead to pathological events and human disorders and furthermore provide a rationale to possibly explain why the expression of some of the S100 proteins (mainly S100A4 and S100P) has led to conflicting results on motility, depending on the cells used. © 2013 Springer Basel.

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing. © 2014 Springer-Verlag.

Angiopoietin-1 induces migration of monocytes in a tie-2 and integrin-independent manner

Angiopoietin-1 (Ang-1) is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wall remodeling. The recent finding of the potential proatherogenic effects of Ang-1 prompted us to investigate whether Ang-1 promotes monocyte chemotaxis, endothelial binding, and transendothelial migration, key events in the progression of atherosclerosis. Here, we show that Ang-1 induces chemotaxis of monocytes in a manner that is independent of Tie-2 and integrin binding but dependent on phosphoinositide 3-kinase and heparin. In addition, Ang-1 promoted phosphoinositide 3-kinase-dependent binding of monocytes to endothelial monolayers and stimulated transendothelial migration. Fluorescence-activated cell sorting analysis showed that exogenous Ang-1 adheres directly to monocytes as well as to human umbilical endothelial cells, but neither Tie-2 mRNA nor protein were expressed by primary monocytes. Although Ang-1 binding to human umbilical endothelial cells was partially Tie-2 and integrin dependent, Ang-1 binding to monocytes was independent of these factors. Finally, preincubation of monocytes with soluble heparin abrogated Ang-1 binding to monocytes and migration, and partially prevented Ang-1 binding to human umbilical endothelial cells. In summary, Ang-1 induces chemotaxis of monocytes by a mechanism that is dependent on phosphoinositide 3-kinase and heparin but independent of Tie-2 and integrins. The ability of Ang-1 to recruit monocytes suggests it may play a role in inflammatory angiogenesis and may promote atherosclerosis.

Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.