TG2 Activity Research Articles

Loss of nitric oxide bioavailability promotes arterial stiffening in a tissue transglutaminase-dependent manner

Although arterial stiffening undeniably contributes to cardiovascular disease, the cellular and molecular mechanisms leading to increased arterial stiffness are not well understood. Increased arterial stiffness in disease states such as obesity and type 2 diabetes typically coexists with indices of endothelial dysfunction including diminished nitric oxide (NO) bioavailability. In fact, it is commonly assumed that endothelial dysfunction precedes, and causally contributes to, arterial stiffening; yet the molecular underpinnings for this causal relationship remain elusive. We and others previously showed that activation of tissue transglutaminase (TG2), a crosslinking enzyme that also works as a G-protein and cellular scaffold, promotes cytoskeletal actin polymerization and stiffening of vascular smooth muscle cells (VSMC), thus contributing to whole-artery stiffening. It is also reported that NO is an endogenous inhibitor of TG2. Accordingly, herein, we tested the hypothesis that increased exposure of VSMC to NO suppresses TG2 activity and actin polymerization. Conversely, we hypothesized that loss of NO bioavailability promotes arterial stiffening, and that this effect is mediated by TG2 activity and downstream activation of LIM kinase (LIMK), an enzyme that modulates fibrillar (F)-actin severing via cofilin phosphorylation. All differences reported herein are significant at P<0.05. In support of our hypotheses, we show that treatment of cultured human aortic VSMC with the NO donor S-nitrosoglutathione (200μM for 2 hours) reduces TG2 activity (N-epsilon gamma glutamyl Lysine presence) and F-actin formation (phalloidin staining) induced by TG2 activation (dithiothreitol, 200μM). Furthermore, we show that exposure of mouse isolated mesenteric arteries to the NO synthase inhibitor L-NAME (300μM for 18 hours) causes an increase in arterial stiffness ( i.e., increased incremental modulus of elasticity) as determined using pressure myography and videomicroscopy. Of note, L-NAME-induced arterial stiffening is prevented when arteries are co-treated with a TG2 inhibitor (cystamine, 1mM) or LIMK inhibitor (LIMKi, 1μM). Taken together, these findings support the notion that NO is a key downregulator of TG2 activity and demonstrate that reduced NO bioavailability promotes arterial stiffening in a VSMC TG2-LIMK-F-actin dependent manner. As such, this work provides insight into plausible molecular mechanisms by which endothelial dysfunction leads to arterial stiffening. R01HL153264 (to LAML and JP), and MU Research Excellence Program (REP) postdoctoral fellowship (to FIRP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of the role of TG2 on the expression of TGF-β, TIMP-1 and TIMP-2 in aged skin

Abstract Objectives Transglutaminase 2 (TG2) is a unique protein having enzymatic and nonenzymatic functions that have been implicated in various biological and pathological processes such as cell survival and apoptosis, cell signaling, differentiation, adhesion and migration, wound healing and inflammation. As reported in previous studies, TG2 expression and activity increase by age suggesting that TG2 possibly has roles in cellular aging process. In this study, we aimed to explore the role of TG2 in chronological skin aging through its impact on the expression of some important extracellular matrix (ECM) proteins including TGF-β, TIMP-1 and TIMP-2. Methods We have compared TG2 expression and activity in young and in vitro chronologically aged human dermal fibroblasts via Western blot and in situ TG2 activity assays. Afterwards, we inhibited TG2 expression via siRNA transfection and activity via active site inhibitor of TG2 separately in aged dermal fibroblasts and monitored the expression levels of TGF-β, TIMP-1 and TIMP-2 in these cells by Western blot and compared to that of untreated control cells. Results We obtained evidence that both TG2 expression and activity increase in aged cells. However, protein levels of TGF-β, TIMP-1 and TIMP-2 do not exhibit any significant difference in TG2 downregulated or TG2 activity inhibited aged cells compared to control cells. Conclusions Our results indicate that changes in the expression and activity of TG2 in (in vitro) chronologically aged human dermal fibroblasts do not impact the expression patterns of TGF-β, TIMP-1 and TIMP-2 proteins.

P117 Transglutaminase 2 is elevated in Crohn’s disease associated strictures and exerts profibrotic activities in myofibroblasts and experimental intestinal fibrosis

Abstract Background Intestinal fibrosis is a significant clinical problem in Inflammatory bowel disease (IBD). This may lead to intestinal strictures induced by excessive accumulation of extracellular matrix (ECM) produced by human intestinal myofibroblasts (HIMF). Transglutaminase (TG)2 covalently cross-links ECM-associated proteins, increases ECM stiffness and has direct pro-fibrotic effects. We investigated the role of TG2 in intestinal fibrosis in human tissues, HIMF and transgenic mouse models. Methods TG2 was detected in human tissues from Crohn’s disease (CD) strictures (CDs; n=10), CD non-strictured (CDns; n=20), ulcerative colitis (UC; n=10) and non-IBD controls (NL; n=14) by immunofluorescence (IF), qPCR, in-situ hybridization (ISH), and biotinylated cadaverin incorporation (BCI). Relative contribution of TG2 was measured by BCI using a TG2-selective blocking antibody (TAb). The cellular source of TG2 was evaluated by IF. HIMF ECM production was measured using a novel ECM deposition assay and TG2-inhibitors. HIMF were exposed to anti-TG2 antibody or isotype control and bulk RNA-sequencing (RNAseq) analysis was performed. Tamoxifen-inducible global Cre/TG2 floxed conditional KO (cKO) mice were used in the DSS and TNBS fibrosis models. Results Total TG activity was increased in CDs and UC vs. NL in the submucosa (SM) and muscularis propria (MP; P≤ 0.04). Strikingly 90-99% of TG activity were attributable to TG2 in all groups. TG2 activity correlated with fibrosis scores (P≤0.02), but not inflammation scores. TG2 amount was elevated in CDs patients vs. NL in qPCR and ISH (P≤0.05). IF revealed HIMF as the major source of TG2 in the intestine as indicated by colocalization with alpha-SMA. HIMF, invitro, produced 82–97% of active TG2. Inhibition of TG2 by cell-permeable inhibitor or Tab reduced fibronectin (FN) and collagen (COL)1 and 3 deposition by HIMF. RNAseq showed TAb-treated cells expressed 432 downregulated and 402 upregulated genes. Functional enrichment analysis showed pathways associated with fibroblast activation/proliferation, ECM, and fibrosis-associated signaling (e.g. AKT) pathways to be downregulated in TAb-treated HIMF. Associated genes included nuclear protein (NUPR1) and sphingolipid signaling pathway proteins (SMPD1 and CTSD), which may be potential targets for TG2-mediated activity in HIMF. Deletion of TGM2 prior to inducing murine DSS or TNBS experimental fibrosis did not lead to any difference in inflammation, but decreased fibrosis (picrosirius red intensity, COL1/FN expression, wall thickness) in TG2 cKO mice vs. wild type (P≤0.05). Conclusion TG2 is involved in inflammation-independent progression of intestinal fibrosis and may represent a future anti-fibrotic target.

Tissue transglutaminase exacerbates renal fibrosis via alternative activation of monocyte-derived macrophages

Macrophages are important components in modulating homeostatic and inflammatory responses and are generally categorized into two broad but distinct subsets: classical activated (M1) and alternatively activated (M2) depending on the microenvironment. Fibrosis is a chronic inflammatory disease exacerbated by M2 macrophages, although the detailed mechanism by which M2 macrophage polarization is regulated remains unclear. These polarization mechanisms have little in common between mice and humans, making it difficult to adapt research results obtained in mice to human diseases. Tissue transglutaminase (TG2) is a known marker common to mouse and human M2 macrophages and is a multifunctional enzyme responsible for crosslinking reactions. Here we sought to identify the role of TG2 in macrophage polarization and fibrosis. In IL-4-treated macrophages derived from mouse bone marrow and human monocyte cells, the expression of TG2 was increased with enhancement of M2 macrophage markers, whereas knockout or inhibitor treatment of TG2 markedly suppressed M2 macrophage polarization. In the renal fibrosis model, accumulation of M2 macrophages in fibrotic kidney was significantly reduced in TG2 knockout or inhibitor-administrated mice, along with the resolution of fibrosis. Bone marrow transplantation using TG2-knockout mice revealed that TG2 is involved in M2 polarization of infiltrating macrophages derived from circulating monocytes and exacerbates renal fibrosis. Furthermore, the suppression of renal fibrosis in TG2-knockout mice was abolished by transplantation of wild-type bone marrow or by renal subcapsular injection of IL4-treated macrophages derived from bone marrow of wild-type, but not TG2 knockout. Transcriptome analysis of downstream targets involved in M2 macrophages polarization revealed that ALOX15 expression was enhanced by TG2 activation and promoted M2 macrophage polarization. Furthermore, the increase in the abundance of ALOX15-expressing macrophages in fibrotic kidney was dramatically suppressed in TG2-knockout mice. These findings demonstrated that TG2 activity exacerbates renal fibrosis by polarization of M2 macrophages from monocytes via ALOX15.

Peculiar Ca2+ Homeostasis, ER Stress, Autophagy, and TG2 Modulation in Celiac Disease Patient-Derived Cells.

Celiac disease (CD) is an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals by genetically predisposed individuals. Constitutive differences between cells from CD patients and control subjects, including levels of protein phosphorylation, alterations of vesicular trafficking, and regulation of type 2 transglutaminase (TG2), have been reported. In the present work, we investigated how skin-derived fibroblasts from CD and control subjects responded to thapsigargin, an endoplasmic reticulum ER stress inducer, in an attempt to contribute to the comprehension of molecular features of the CD cellular phenotype. We analyzed Ca2+ levels by single-cell video-imaging and TG2 activity by a microplate assay. Western blots and PCR analyses were employed to monitor TG2 levels and markers of ER stress and autophagy. We found that the cytosolic and ER Ca2+ level of CD cells was lower than in control cells. Treatments with thapsigargin differently activated TG2 in control and CD cells, as well as caused slightly different responses regarding the activation of ER stress and the expression of autophagic markers. On the whole, our findings identified further molecular features of the celiac cellular phenotype and highlighted that CD cells appeared less capable of adapting to a stress condition and responding in a physiological way.

Organophosphates modulate tissue transglutaminase activity in differentiated C6 neural cells.

The organophosphate compounds chlorpyrifos (O, O-diethyl O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate, CPF) and phenyl saligenin phosphate (PSP) have been widely implicated in developmental neurotoxicity and neurodegeneration. However, the underlying mechanism remains unclear. Transglutaminase (TG)2 is a calcium ion (Ca2+)-dependent enzyme with an important role in neuronal cell outgrowth and differentiation and in neurotoxin activity and is modulated by organophosphates. We studied TG2 activity modulation by CPO and PSP during differentiation in C6 glioma cells. We studied the effects of CPO or PSP treatment with or without the TG2 inhibitor Z-DON and identified potential TG2 protein substrates via mass spectrometry. PSP and CPO did not affect cell viability but affected TG2 activity in differentiating cells. Our results indicate that the organophosphate-induced amine incorporation activity of TG2 may have a direct effect on neuronal outgrowth, differentiation, and cell survival by modifying several essential microtubule proteins, including tubulin. Inhibiting TG2 reduced neurite length but not cell survival. TG2 inhibitors can protect against organophosphate-induced neuropathy and could be used for developing novel therapeutic strategies for treating brain cancer and neurodegenerative disorders.

Transglutaminase Modification of Tendon Collagen

Category:AnkleIntroduction/Purpose:Achilles tendinopathy is one of the most common overuse conditions encountered in foot and ankle surgery. Various etiological risk factors for this disease have been explored: age, body-mass index, and patient specific biomechanics. However, the pathogenesis remains poorly understood. Healthy tendon is composed primarily of type III and I collagen, which undergo post-translationally modification by lysyl oxidase and transglutaminase (TG) enzymes. The expression and activity in the latter of these two is poorly characterized in Achilles tendon. This study aims to describe the function of a specific transglutaminase, TG2, within solubilized collagen and Achilles tenocyte cultures, and to demonstrate evidence of TG2 activity within pathologic human Achilles tendon tissue.Methods:Acellular Collagen: Type I collagen gels were aliquoted into a silicone well plate. Gels were then seeded with either 2.4 ng of TG2 or a control. Two sets of gels were prepared; one undergoing a 10% stretch at 2Hz for 24 hours and the other a static control. TG2 substrates were labelled, and the relative quantity of higher molecular weight collagens produced by TG2 activity was compared. Tenocytes: Human tenocytes were grown in 3D cultures as well as traditional cultures in a conditioned fibroblast growth media. Proteins expressed by tenocytes in both cultures were extracted and examined for TG modification. Pathologic Tendon: After receiving Institutional Review Board approval, tissue was collected from patients undergoing debridement of Achilles tendinopathy. Collagen was extracted in 0.1M acetic acid and Western blot analysis was used to identify TG2 crosslink epitopes.Results:In each of three experimental paradigms: acellular, cellular, and human tendon extracts, evidence for transglutaminase modification of collagen I was noted. Dynamic conditions increased TG2 modification of collagen in acellular cultures, as evidenced by higher molecular weight collagen forms. Western blot analysis of protein expression demonstrated that tenocytes cultured in 3-D produced higher levels of TG-modified proteins than those in 2-D. Similarly, western blot analysis demonstrated presence of TG2 modification epitopes in the harvested human Achilles tendon tissue. However, the enzyme itself did not appear to be strongly expressed and could not be detected from the samples.Conclusion:Transglutaminase 2 expression by tenocytes in 3D culture and evidence for transglutaminase-dependent modification of collagen I by TG2 under dynamic loading supports a key role for transglutaminase in tendon biology and in repair. Though evidence of TG2 modification was found in diseased human samples, the absence of detectable TG2 expression suggests that it may play a significant role in the healthy physiologic turnover of Achilles tendon collagen.

Nitrosylation of tissue transglutaminase enhances fibroblast migration and regulates MMP activation

In wound healing, the TG2 enzyme plays a dual functional role. TG2 has been shown to regulate extracellular matrix (ECM) stabilization by its transamidase activity while increasing cell migration by acting as a cell adhesion molecule. In this process, nitric oxide (NO) plays a particularly important role by nitrosylation of free cysteine ​​residues on TG2, leading to the irreversible inactivation of the catalytic activity.In this study, transfected fibroblasts expressing TG2 under the control of the tetracycline-off promoter were treated with NO donor S-nitroso-N-acetyl penicillamine (SNAP) to analyze the interplay between NO and TG2 in the regulation of cell migration/invasion as well as TGF-β1-dependent MMP activation. Our results demonstrated that inhibition of TG2 cross-linking activity by SNAP promoted the migration and invasion capacity of fibroblasts by hindering TG2-mediated TGF-β1 activation. While the inhibition of TG2 activity by NO downregulated the biosynthesis and activity of MMP-2 and MMP-9, that of MMP-1a and MMP-13 was shown to be upregulated in a TGF-β1-dependent manner under the same conditions. In the presence of SNAP, interaction of TG2 with its cell surface binding partners Integrin-β1 and Syndecan-4 was reduced, which was paralleled by an increase in TG2 and PDGF association. These findings suggests that migratory phenotype of fibroblasts can be regulated by the interplay between nitric oxide and TG2 activity.

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1–10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

Constitutive Differential Features of Type 2 Transglutaminase in Cells Derived from Celiac Patients and from Healthy Subjects.

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31–43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31–43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31–43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31–43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.

Upregulation of Tissue Transglutaminase in Transforming Growth Factor-b Induced Epithelial Mesenchymal TG2Transdifferentiation In Rat Tubular Epithelial Cells

AbstractBackground: Epithelial-Mesenchymal Transdifferentiation (EMT) is believed to be a key step in the course of glomerular and tubulointerstitial fibrosis. Many of the regulators of this process have been identified and Transforming Growth Factor-b (TGF-b) plays a central role. Tissue transglutaminase (TG2) has a crucial role in extracellular matrix formation and stabi-lization.Aim of Study: This study was designed to explore whether TG2 is essential in EMT induced by TGF-b in renal tubular epithelial cells.Material and Methods: EMT was achieved by treating NRK52-E renal tubular epithelial cells with high concentration of rhTGF-b (10ng/ml) for 24, 48 and 72h. Development of mesenchymal cell markers Fibroblast Specific Protein (FSP-1) and a Smooth Muscle Actin (a-SMA) and decrease in tubular epithelial cell surface marker E-Cadherin were used to confirm EMT. The NTU283; TG2 inhibitor was used for the inhibition assays. TG2 activity was quantified using a 14C putrescine incorporation assay. Protein expression and localisation of different markers were assessed by western blot and immunofluorescent dual immunofluorescent staining. Phase analysis was used to quantify the amount of staining for each of the markers used.Results: A significant increase in TG2 activity and protein expression were both observed with TGF-b induced transdif-ferentiation at 48h. This observation was associated with loss of epithelial cell marker E-Cadherin and appearance of intra-cellular a-SMA and FSP-1, characteristic of myofibroblasts. Dual staining showed high TG2 levels in cells (0.254±0.07), compared to the control (0.02±0.008). This also showed loss of epithelial phenotype, decrease of E-cadherin and acquisition of myofibroblast characteristics a-SMA (0.671±0.13) com-pared to the control (0.076±0.016) and FSP-1 (0.492±0.017) compared to control (0.03±0.017). Cells also demonstrated high degree of co-localization of Collagen I (ColI) and a-SMA with TG2. The NTU283; TG2 inhibitor ameliorated TGF-b induced EMT. This was also associated with changed pattern of collagen I and a-SMA co-localization.Conclusion: This data demonstrated upregulation of TG2 during the process of TGF-b induced EMT. TG2 inhibitor showed non-significant reduction of both TG2 and mesenchy-mal markers but demonstrated changes in the molecular configuration of mesenchymal markers a-SMA and Col I, raising the possibility of a role of TG2 in EMT.

Procyanidin B2 rich cocoa extracts inhibit inflammation in Caco-2 cell model of in vitro celiac disease by down-regulating interferon-gamma- or gliadin peptide 31-43-induced transglutaminase-2 and interleukin-15

Abstract The objective of this research was to investigate the efficacy of procyanidin B2 rich cocoa extracts on interferon-gamma (IFN-γ) or gliadin peptide p31-43-induced transglutaminase-2 and interleukin-15 (IL-15) in Caco-2 cell model of in vitro celiac disease. Cysteamine was used as a positive control inhibitor of TG2. Procyanidin B2-rich extract reduced IFN-γ- and gliadin p31-43-induced TG2 activity by 77% and 45%, respectively. Cocoa extract containing 4.9–289 µg/mL of procyanidin B2 dose-dependently reduced IFN-γ-induced IL-15 secretion to the level of IL-15 in control cells. Similarly, cocoa extract containing 4.9–289 µg/mL of procyanidin B2 dose-dependently reduced gliadin p31-43-induced IL-15 to the level in control cells. Procyanidin B2-rich cocoa extracts reduced the activities of other inflammatory biomarkers including COX-2, IL-1β, IL-6, and IL-8 in both IFN-γ and p31-43-treated Caco-2 cells. Caffeine or theobromine, at the concentration found in the cocoa extracts, did not contribute to the activity of procyanidin B2 against TG2 or IL-15.

Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

Abstract P275: Transglutaminase-2 Contributes to Angiotensin II-Induced Reactive Oxygen Species Production in Mice

Transglutaminase type II (TG2) is a pleiotropic enzyme that exhibits various activities and it is involved in diverse biological functions, including cell signaling, cytoskeleton rearrangements, displaying enzymatic activities. We previously demonstrated that TG2 may contribute to angiotensin II-induced reduction of NO bioavailability as well as to the impaired vascular functional and structural alterations induced by angiotensin II. Here we hypothesized that TG2 may contribute to increased production of reactive oxygen species (ROS) in the vasculature of angiotensin-II-treated mice. TG2-knockout mice (TG2-K/O, 8 weeks old, n=6) and age-matched wild type (WT) control mice were treated or not with angiotensin-II (400ng/kg/min) for 14 days. TG2 activity in aorta was measured by ELISA. ROS production in aorta was evaluated by dihydroethidium staining. The expression of angiotensin type1 receptor (AT1R), TG2, NOX-1, and ERp72 (the positive modulator of NOX-1) was evaluated in aorta by immunoblotting, coimmunoprecipitation analysis was also performed. As expected, TG2-K/O lacked TG2 expression and activity. Angiotensin-II significantly increased (2-fold) TG2 expression and activity only in WT. AT1R expression in aorta was not influenced by Angiotensin II treatment in both WT and TG2K/O mice. ROS production was similar in WT and TG2-K/O and increased only in angiotensin-II-treated WT (+9%, p<0.01). NOX-1 and ERp72 expression was similar in WT and TG2-K/O. Angiotensin-II significantly increased NOX-1 (+23%, p<0.01) and ERp72 (+29%, p<0.01) only in WT. Only in aorta from WT and not from TG2-K/O, TG2 was successfully immunoprecipitated by AT1 and ERP72, indicating that TG2 is able to interact with both proteins, and suggesting that it may be involved in angiotensin II- induced NOX modulation and ROS production. In conclusion, Angiotensin-II increased ROS production and NOX-1 expression and activation only in presence of TG2 in WT. TG2 interacts with both AT1R and ERp72. Thus, TG2 may contribute to NOX-induced ROS production in mice treated with angiotensin-II.

Analysis of IL17A and IL21 Expression in the Small Intestine of Celiac Disease Patients and Correlation with Circulating Thioredoxin Level

Th17 cells and their related cytokines play an important role in the pathogenesis of celiac disease (CD), and thioredoxin (Trx) is an extracellular TG2 activity regulator. This study evaluated Trx serum levels and the expression levels of IL17A, IL21, and Trx genes in biopsies of treated (gluten-free diet) and naïve (untreated) CD patients compared with healthy individuals. Duodenal biopsies were collected from treated CD patients (n = 60), healthy controls (n = 60), and eight newly diagnosed celiac patients. IL17A, IL21, and Trx gene expression was assessed by quantitative polymerase chain reaction (qPCR) and compared with serum Trx levels assessed by enzyme-linked immunosorbent assay (ELISA). Expression levels of the IL21 and Trx genes were not significantly modulated in the CD group compared to the control group, whereas the IL17A gene in CD patients was transcribed at significantly higher levels among the CD group. Serum concentrations of Trx were significantly increased in treated CD patients compared to the control group. We observed that IL17A gene is more highly expressed in duodenal biopsies of CD patients than controls, and that the serum levels of Trx are significantly higher in treated CD patients than controls. Therefore, the expression levels of these genes and gene products, respectively, could potentially be used as diagnostic biomarkers for CD patients, although more studies are needed to unravel the underlying molecular mechanisms.

Abstract 2843: Calcium blockers modulate the steroid resistance in acute lymphoblastic leukemia

Abstract Background: Acute lymphoblastic leukemia (ALL) is one of common cancers in children and adults, and a fair number of ALL patients still die although the overall survival rate is approaching 90% with risk-directed therapy in many developed countries. Response to steroid is the most important prognostic factor of patients with ALL, and the development of steroid resistance is lethal to the ALL patients. To improve survival for steroid-resistant ALL patients, it is necessary to develop new concepts to overcome the steroid resistance of ALL. Methods: We generated the steroid-adapted subclones through prolonged cultures of ALL cell lines with gradually increased steroid concentration.Also, we used biochemical and molecular methodologies to demonstrate the signaling axis of tissue transglutaminase (TG2)-nuclear factor-kappa B (NF-kappa B) and cell cycle to evaluate the effect of calcium blockers on the steroid-resistant ALL cells. Results: Steroid-adapted ALL cells that were generated by culture with gradually increased steroid concentration were much more resistant to steroid than their parent ALL cells. We found that calcium blockers altered TG2 activity and nuclear factor-kappa B (NF-kappa B) expression in these steroid-resistant ALL cells. Also, calcium blockers induced the change of expression of NF-kappa B target genes in steroid-adapted ALL cells. In addition, calcium blockers induced the change of ABC transporter expression and the quiescence of steroid-resistant ALL cells. We also found that calcium blockers induced apoptosis of steroid-resistant ALL cells and reduced the steroid resistance of steroid-resistant ALL cells. Conclusion: This study shows that calcium blockers improved the cytotoxicity of steroid on steroid-resistant ALL cells via manipulation of TG2 and NF-kappa B activities and cell cycle. The combination of calcium blockers with chemotherapeutic agents may be a new alternative strategy to overcome the steroid resistance of ALL. Citation Format: Hyun Joo Jung. Calcium blockers modulate the steroid resistance in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2843.

Isozyme-specific comprehensive characterization of transglutaminase-crosslinked substrates in kidney fibrosis

Chronic kidney disease is characterized by prolonged decline in renal function, excessive accumulation of ECM, and progressive tissue fibrosis. Transglutaminase (TG) is a crosslinking enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues, and is involved in the induction of renal fibrosis via the stabilization of ECM and the activation of TGF-β1. Despite the accumulating evidences indicating that TG2 is a key enzyme in fibrosis, genetic knockout of TG2 reduced by only 50% the elevated protein crosslinking and fibrous protein in renal fibrosis model, whereas treatment with TG inhibitor almost completely reduced these levels. Here, we also clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific crosslinked substrates for both TG1 and TG2 in fibrotic kidney. We found that TG1 activity was markedly enhanced in renal tubular epithelium and interstitial areas, whereas TG2 activity increased only in the extracellular space. In total, 47 and 67 possible candidates were identified as TG1 and TG2 substrates, respectively, only in fibrotic kidney. Among them, several possible substrates related to renal disease and fibrosis were identified. These findings provide novel insights into the mechanisms of renal fibrosis through the targeting of isozyme-specific TG substrates.

Characterization of Transglutaminase 2 activity inhibitors in monocytes in vitro and their effect in a mouse model for multiple sclerosis.

The neurodegenerative disease multiple sclerosis (MS) is pathologically characterized by the massive influx of immune cells into the central nervous system. This contributes to demyelination and axonal damage which causes symptoms such as motor and cognitive dysfunctions. The migration of leukocytes from the blood vessel is orchestrated by a multitude of factors whose determination is essential in reducing cellular influx in MS patients and the experimental autoimmune encephalomyelitis (EAE) animal model. The here studied enzyme tissue Transglutaminase (TG2) is present intracellularly, on the cell surface and extracellularly. There it contributes to cellular adhesion and migration via its transamidation activity and possibly by facilitating cellular interaction with the extracellular matrix. Previous data from our group showed reduced motor symptoms and cellular infiltration after using a pharmacological TG2 transamidation activity inhibitor in a rat EAE model. However, it remained elusive if the cross-linking activity of the enzyme resulted in the observed effects. To follow-up, we now characterized two new small molecule TG2 activity inhibitors, BJJF078 and ERW1041E. Both compounds are potent inhibitor of recombinant human and mouse Transglutaminase enzyme activity, mainly TG2 and the close related enzyme TG1. In addition they did not affect the binding of TG2 to the extracellular matrix substrate fibronectin, a process via which TG2 promotes cellular adhesion and migration. We found, that ERW1041E but not BJJF078 resulted in reduced EAE disease motor-symptoms while neither caused apparent changes in pathology (cellular influx), Transglutaminase activity or expression of inflammation related markers in the spinal cord, compared to vehicle treated controls. Although we cannot exclude issues on bioavailability and in vivo efficacy of the used compounds, we hypothesize that extracellular TG1/TG2 activity is of greater importance than (intra-)cellular activity in mouse EAE pathology.

Activation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: A role in cell survival and neurite outgrowth

NGF (nerve growth factor) and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 transamidase activity by NGF in retinoic acid-induced differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. The role of TG2 in NGF-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with NGF in N2a and SH-SY5Y cells. NGF mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON (Z-ZON-Val-Pro-Leu-OMe; Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-l-valinyl-l-prolinyl-l-leucinmethylester) and R283 (1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride) and by pharmacological inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB) and protein kinase C (PKC), and removal of extracellular Ca2+. Fluorescence microscopy demonstrated NGF induced in situ TG2 activity. TG2 inhibition blocked NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth in both cell lines. Together, these results demonstrate that NGF stimulates TG2 transamidase activity via a ERK1/2, PKB and PKC-dependent pathway in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. Furthermore, NGF-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results suggest a novel and important role of TG2 in the cellular functions of NGF.

Tissue transglutaminase in astrocytes is enhanced by inflammatory mediators and is involved in the formation of fibronectin fibril-like structures

BackgroundDuring multiple sclerosis (MS) lesion formation, inflammatory mediators are produced by microglial cells and invading leukocytes. Subsequently, hypertrophic astrocytes fill the lesion and produce extracellular matrix (ECM) proteins that together form the astroglial scar. This is beneficial because it seals off the site of central nervous system (CNS) damage. However, astroglial scarring also forms an obstacle that inhibits remyelination of brain lesions. This is possibly an important cause for incomplete remyelination of the CNS in early stage MS patients and for failure of remyelination when the disease progresses. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme that can cross-link proteins, appears in astrocytes in inflammatory MS lesions and may contribute to the rearrangement of ECM protein deposition and aggregation.MethodsThe effect of different inflammatory mediators on TG2 and fibronectin, an ECM protein, protein levels was examined in primary rat microglia and astrocytes by western blotting. Also, TG2 activity was analyzed in primary rat astrocytes by a TG activity assay. To determine the role of TG2 in the deposition and cross-linking of fibronectin, a TG2 inhibitor and TG2 knockdown astrocytes were used.ResultsOur data show that under inflammatory conditions in vitro, TG2 production is enhanced in astrocytes and microglia. We observed that in particular, astrocytes produce fibronectin that can be cross-linked and aggregated by exogenous TG2. Moreover, inflammatory stimulus-induced endogenously produced TG2 is involved in the appearance of morphological fibril-like fibronectin deposits but does not lead to cross-linked fibronectin aggregates.ConclusionsOur in vitro observations suggest that during MS lesion formation, when inflammatory mediators are produced, astrocyte-derived TG2 may contribute to ECM rearrangement, and subsequent astroglial scarring.