Palmitate induces insulin resistance in monocytes and increases expression of the integrin CD11b

Obesity and insulin resistance are important risk factors for atherosclerosis, and elevated level of plasma NEFA is a common feature in individuals with obesity and insulin resistance. Palmitate, one of the most abundant non-esterified SFA in plasma, has been reported to induce insulin resistance in adipose tissues and skeletal muscles and to cause an increased inflammatory response in monocytes. The present study investigated whether palmitate can induce insulin resistance in monocytes and its effect on monocyte adhesion molecular expression (CD11b). Insulin resistance was measured by in vitro uptake of insulin-stimulated 3H-labelled 2-deoxy-D-glucose into THP-1 cells, cell surface CD11b expression was measured by flow cytometry. The data showed that palmitate-induced insulin resistance in THP-1 monocytes was concentration and time dependent (Figure 1). The insulin-stimulated glucose uptake was significantly decreased in cells treated with 300 mM-palmitate compared with control cells (P<0.001) and was observed within 6 h, but was not a result of palmitate toxicity. There was no significant increase in caspase 3 activation (P>0.05). Treatment with 300 mM-palmitate for 24 h also caused a significant increase in surface CD11b expression in both U937 and THP-1 monocytic cell lines and human primary monocytes compared with the control (P<0.001). Both these effects were inhibited by co-incubation with Fumonisin B1, an inhibitor of de novo ceramide synthesis. In conclusion, these data show that palmitate, at physiological concentrations, can cause insulin resistance in monocytes and increase monocyte surface integrin CD11b expression, which is in part the result of the synthesis of ceramide.

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.

Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFa production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the I?Ba. Low basal levels of I?Ba explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-?B. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.

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.

Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding. Function-blocking antibodies against both alpha7 and beta1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal beta1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the alpha7beta1 integrin with tenascin-C peptides containing the VFDNFVLK sequence but not with peptides with alterations in FD and/or FV. These findings are the first to provide evidence that the alpha7beta1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the alpha7beta1 integrin receptor in CNS neurons.

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.

C-reactive protein mediates CD11b expression in monocytes through the non-receptor tyrosine kinase, Syk, and calcium mobilization but not through cytosolic peroxides

Objective: C-Reactive protein (CRP) can modulate integrin surface expression on monocytes following Fcγ receptor engagement. We have investigated the signal transduction events causing this phenotypic alteration. Methods: CRP-induced signalling events were examined in THP-1 and primary monocytes, measuring Syk phosphorylation by Western blotting, intracellular Ca2+ ([Ca2+]i) by Indo-1 fluorescence and surface expression of CD11b by flow cytometry. Cytosolic peroxides were determined by DCF fluorescence. Results: CRP induced phosphorylation of Syk and an increase in [Ca2+]i both of which were inhibitable by the Syk specific antagonist, piceatannol. Piceatannol also inhibited the CRP-induced increase in surface CD11b. In addition, pre-treatment of primary monoytes with the Ca2+ mobiliser, thapsigargin, increased CD11b expression; this effect was accentuated in the presence of CRP but was abolished in the presence of the [Ca2+]i chelator, BAPTA. CRP also increased cytosolic peroxide levels; this effect was attenuated by antioxidants (ascorbate, α-tocopherol), expression of surface CD11b not being inhibited by antioxidants alone. Conclusion: CRP induces CD11b expression in monocytes through a peroxide independent pathway involving both Syk phosphorylation and [Ca2+]i release. © Birkhäuser Verlag, 2005.

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.

A novel RGD-independent cel adhesion pathway mediated by fibronectin-bound tissue transglutaminase rescues cells from anoikis

Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies alpha5beta1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Calpha, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress.

Characterization of tissue 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. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in a number of cell functions. Up-regulation of TG2 is thought to be involved in monocyte to macrophage differentiation and defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be fully elucidated. Cell surface-associated TG2 is now recognized as being important in regulating cell adhesion and migration, via its association with cell surface receptors such as syndecan-4, ß1 and ß3 integrin, but its extracellular role in the clearance of apoptotic cells is still not fully explored. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function within the framework of apoptotic cell clearance. Both THP-1 cell-derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors (both cell permeable and impermeable, irreversible and active site directed) resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. Macrophage cell surface TG2 and, in particular, its cell surface crosslinking activity was found to be crucial in dictating apoptotic cell clearance. Our further studies demonstrate syndecan-4 association with TG2 and imply possible cooperation of these proteins in apoptotic cell clearance. Knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance which seems to involve protein cross linking and interaction with other cell surface receptors.

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.

Cells, surfaces and adhesion

This thesis concerns cell adhesion to polymer surfaces with an experimental emphasis on hydrogels. The thesis begins with a review of the literature and a synthesis of recent evidence to describe the process of cell adhesion in a given situation. The importance of understanding integrin-adhesion protein interactions and adhesion protein-surface interactions is emphasised. The experimental chapters describe three areas of investigation. Firstly, in vitro cell culture techniques are used to explore a variety of surfaces including polyethylene glycol methacrylate (PEGMA) substituted hydrogels, sequence distribution modified hydrogels and worn contact lenses. Cell adhesion to PEGMA substituted gels is found to decrease with increases in polyethylene oxide chain length and correlations are made between sequence distribution and adhesion. Worn contact lenses are investigated for their cell adhesion properties in the presence of antibodies to specific adhesion proteins, demonstrating the presence of vitronectin and fibronectin on the lenses. The second experimental chapter addresses divalent cation regulation of integrin mediated cell adhesion. Several cell types and various cations are used. Zinc, previously not regarded as an important cation in the process, is found to inhibit 3T3 cell adhesion to vitronectin that is promoted by other divalent cations. The final experimental chapter concerns cell adhesion and growth on macroporous hydrogels. A variety of freeze-thaw formed porous gels are investiated and found generally to promote cell growth rate.Interpenetrating networkbased gels (IPN) are made porous by elution of dextrin particles of varying size and loading density. These materials provide the basis for synthetic cartilage. Cartilage cells (chondrocytes) plated onto the surface of the porous IPN materials maintain a rounded shape and hence phenotypic function when a critical pore size and density is achieved. In this way, a prospective implant, made porous at the perpendicular edges contacting natural cartilage can be both mechanically stabilised and encourage the maintenance of normal matrix production at the tissue interface.

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.

A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis

The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr1214 and its downstream effectors Akt and ERK1/2, and importantly its association with b1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity. © 2013 Macmillan Publishers Limited. All rights reserved.

Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300µM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300µM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.