Transglutaminase 2 Expression Research Articles

MO748: The Effect of Warfarin-TG2-MVS Axis in Vascular Calcification

Abstract BACKGROUND AND AIMS Warfarin is a common anticoagulant drug. How to effectively reduce vascular calcification induced by warfarin while ensuring its anticoagulant effect is an urgent problem to be solved. Previous studies have found that warfarin enhances the expression and activity of transglutaminase 2 (TG2) in vascular smooth muscle cells (VSMCs), which mediates communication between cells and extracellular matrix (ECM). Matrix vesicles (MVs) are the center of hydroxyapatite crystal precipitation, which is released to ECM and interacts with ECM protein to initiate mineralization and form calcification core. This study observed the role of the warfarin-TG2-MVs axis in vascular calcification by culturing VSMCs in vitro. METHOD VSMCs were cultured in normal or osteogenic medium (OM) and stimulated with 10 μm warfarin for 3–14 days. The expressions of SM22α, Runx2, ALP, OPN and OPG were detected by RT-PCR and western blot. Alizarin red and von Kossa staining were performed to evaluate calcification, and calcium content was determined. Meanwhile, the expression and activity of TG2 were detected. Differential centrifugation was used to extract MVs and evaluate their release. Type I collagen was coated in the culture dish to determine the calcification of MV-collagen. RESULTS Warfarin stimulation promoted transdifferentiation of VSMCs, and the expression of osteogenic factors Runx2, ALPL and OPN were increased, while the expression of SM22α and calcification inhibitor OPG were decreased. When using OM, the above trend was more obvious. Alizarin red and von Kossa staining were performed when the cells were cultured for 14 days. The results of calcium staining in the warfarin intervention group were all positive. Warfarin promoted the expression and activity of TG2, and it gradually increased with the extension of the intervention time. The same number of cells were cultured for 7 days under different stimuli, and the medium was changed every other day. Then, the supernatant was collected for differential centrifugation. The amount of MVs produced by the warfarin and OM groups was significantly higher than that of other groups, and the ability to mineralize of MVs in type I collagen was increased. CONCLUSION Warfarin increased the expression and activity of TG2, promoted the release of MVs from VSMCs, and further cross-linked ECM to aggravate vascular calcification.

Multi-Omics Analysis of Glioblastoma and Glioblastoma Cell Line: Molecular Insights Into the Functional Role of GPR56 and TG2 in Mesenchymal Transition.

G protein-coupled receptor 56 (GPR56/ADGRG1) is an adhesion GPCR with an essential role in brain development and cancer. Elevated expression of GPR56 was observed in the clinical specimens of Glioblastoma (GBM), a highly invasive primary brain tumor. However, we found the expression to be variable across the specimens, presumably due to the intratumor heterogeneity of GBM. Therefore, we re-examined GPR56 expression in public domain spatial gene expression data and single-cell expression data for GBM, which revealed that GPR56 expression was high in cellular tumors, infiltrating tumor cells, and proliferating cells, low in microvascular proliferation and peri-necrotic areas of the tumor, especially in hypoxic mesenchymal-like cells. To gain a better understanding of the consequences of GPR56 downregulation in tumor cells and other molecular changes associated with it, we generated a sh-RNA-mediated GPR56 knockdown in the GBM cell line U373 and performed transcriptomics, proteomics, and phospho-proteomics analysis. Our analysis revealed enrichment of gene signatures, pathways, and phosphorylation of proteins potentially associated with mesenchymal (MES) transition in the tumor and concurrent increase in cell invasion and migration behavior of the GPR56 knockdown GBM cells. Interestingly, our analysis also showed elevated expression of Transglutaminase 2 (TG2) - a known interactor of GPR56, in the knockdown cells. The inverse expression of GPR56 and TG2 was also observed in intratumoral, spatial gene expression data for GBM and in GBM cell lines cultured in vitro under hypoxic conditions. Integrating all these observations, we propose a putative functional link between the inverse expression of the two proteins, the hypoxic niche and the mesenchymal status in the tumor. Hypoxia-induced downregulation of GPR56 and activation of TG2 may result in a network of molecular events that contribute to the mesenchymal transition of GBM cells, and we propose a putative model to explain this functional and regulatory relationship of the two proteins.

Role of TGM2 in T‑cell lymphoblastic lymphoma via regulation of IL‑6/JAK/STAT3 signalling.

Transglutaminase 2 (TGM2) is a Ca2+-dependent enzyme that is closely associated with cancer progression; however, the function of TGM2 in T-cell lymphoma remains unclear. In the present study, TGM2 was identified as an upregulated gene by bioinformatics analysis of the microarray datasets GSE132550 and GSE143382 from the Gene Expression Omnibus database. The effects and mechanisms of TGM2 on T-cell lymphoma cells were evaluated using the Cell Counting Kit-8, colony formation assay, 5-ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction, western blotting and gene set enrichment analysis (GSEA). TGM2 expression was shown to be elevated in formalin-fixed paraffin-embedded skin biopsies from patients with T-cell lymphoma relative to skin tissue from healthy cases. TGM2 expression was also increased in T-cell lymphoma cell lines compared with that in CD4+ T cells. Transfection with TGM2 small interfering RNAs (siRNAs) decreased the number of EdU-positive cells, and the viability and colony formation of T-cell lymphoma cells. Furthermore, TGM2 siRNAs enhanced the apoptosis of T-cell lymphoma cells potentially via cleavage of caspase-3 and poly ADP-ribose polymerase. GSEA identified the IL-6/JAK/STAT3 pathway as a potential downstream signalling pathway of TGM2. Notably, the effects of TGM2 siRNAs on T-cell lymphoma cells were attenuated by IL-6 and accelerated by IL-6/JAK/STAT3 inhibitor AG490. These findings indicated that TGM2 siRNAs inhibited the proliferation of T-cell lymphoma cells by regulating the IL-6/JAK/STAT3 signalling pathway; therefore, TGM2 may function as a potential therapeutic target for T-cell lymphoma.

Transglutaminase 2 mediates UVB-induced matrix metalloproteinase-1 expression by inhibiting nuclear p65 degradation in dermal fibroblasts.

Matrix metalloproteinases (MMPs) play a key role in tissue remodelling by cleaving extracellular matrix (ECM) components. In the skin, UV irradiation increases expression of MMPs that causes dysregulation of ECM homeostasis in dermis, leading to acceleration of skin aging. However, the mediator(s) that links UV irradiation to the upregulation of MMPs have not been fully defined. Previously, we showed that UVB irradiation activated transglutaminase 2 (TG2) in keratinocytes, eliciting an inflammatory response by activating NF-κB signalling. In this study, we reported the role of TG2 in mediating the UVB-induced expression of MMP-1. In human dermal fibroblasts, UVB irradiation enhanced the expression and activity of TG2, which in turn promotes the expression of MMP-1. Analyses of MMP-1 promoter showed that activation of the NF-κB signalling pathway, rather than AP-1, was responsible for the TG2-mediated upregulation of MMP-1. Moreover, Western blot analysis revealed that TG2 increased the activity of NF-κB by inhibiting degradation of p65 in the nucleus. Furthermore, ex vivo skin from TG2-knockout mice exhibited significantly reduced levels of MMP-1 compared to that from wild-type mice. These results indicate that TG2 functions as a mediator for the UVB-induced expression of MMP-1 in dermal fibroblasts, providing a new target for preventing skin photodamage.

Topical retinoic acid induces corneal strengthening by upregulating transglutaminase 2 in murine cornea

Transglutaminase 2 (TG2) is the most abundant crosslinking enzyme in murine and human cornea, while retinoids are well-known inducers of TG2 expression. This study aims to determine if the retinoic acid supplementation can increase corneal stiffness by crosslinking through upregulating the corneal TG2 expression. The right eyes of C57BL/6 mice were treated with 2 × 10−2M retinol palmitate (VApal) eyedrops or control eyedrops and hold for 30 min, once a day for 28 consecutive days. The WB and qPCR results showed increased expression of TG2 in murine cornea with the prolongation of VApal eyedrop application. After 28 days of VApal eyedrop treatment, the increased TG2 were found catalytically active and distributed in corneal epithelium and stroma as detected by 5-(biotinamido) pentylamine (5-BP) incorporation method and immunofluorescence staining. The transmission electron microscope image revealed that VApal treated cornea manifested with increased collagen density in anterior and middle layer of stroma. The higher elastic module was found among VApal treated cornea by nano-indentation test. In cultured corneal epithelial cells and keratocytes, all-trans retinoid acid (ATRA) treatment increased the content of TG2 in cell lysis and in culture medium. These results indicate that retinoic acid induce the reinforcement of the cornea by TG2 mediated crosslinking via increasing the TG2 expression in corneal epithelium and keratocyte. As TG2 was found to be less in the cornea of keratoconus patients in several RNA-sequencing studies, retinoic acid could serve as a non-invasive prevention method for keratoconus progression.

Propofol Suppresses Microglia Inflammation by Targeting TGM2/NF-κB Signaling.

Background Propofol is a known intravenous hypnotic drug used for induction and maintenance of sedation and general anesthesia. Emerging studies also reveal a neuroprotective effect of propofol in diverse diseases of neuronal injuries via modulating microglia activation. In this study, we aimed to uncover the downstream targets of propofol in this process. Methods RNA sequencing analysis to identify genes implicated in the propofol-mediated neuroprotective effect. Quantitative real-time PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze inflammatory gene expression, cytokine levels, and TGM2. BV2 cells and primary microglia were used for functional verification and mechanism studies. Results The multifunctional enzyme transglutaminase 2 (TGM2) was identified as a putative functional mediator of propofol. TGM2 was significantly upregulated in lipopolysaccharide- (LPS-) primed BV2 cells. Genetic silencing of TGM2 abolished LPS-induced microglial activation. Notably, gain-of-function experiments showed that the proinflammatory effects of TGM2 were dependent on its GTP binding activity instead of transamidase activity. Then, TGM2 was revealed to activate the NF-κB signaling pathway to facilitate microglial activation. Propofol can inhibit TGM2 expression and NF-κB signaling in BV2 cells and primary microglia. Ectopic expression of TGM2 or constitutively active IKKβ (CA-IKKβ) can compromise propofol-induced anti-inflammatory effects. Conclusions Our findings suggest that TGM2-mediated activation of NF-κB signaling is an important mechanism in the propofol-induced neuroprotective effect that prevents microglial activation.

Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein–protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

TGFβ1-Induced Transglutaminase-2 Triggers Catabolic Response in Osteoarthritic Chondrocytes by Modulating MMP-13.

Transforming growth factor beta 1 (TGFβ1) plays an essential role in maintaining cartilage homeostasis. TGFβ1 is known to upregulate anabolic processes in articular cartilage, but the role of TGFβ1 in chondrocyte catabolism remains unclear. Thus, we examined whether TGFβ1 increases catabolic processes in the osteoarthritic joint via transglutaminase 2 (TG2). In this study, we investigated whether interplay between TGFβ1 and TG2 mediates chondrocyte catabolism and cartilage degeneration in osteoarthritis. To investigate the role of TGFβ1 and TG2 in osteoarthritis, we performed immunostaining to measure the levels of TGFβ1 and TG2 in 6 human non-osteoarthritic and 16 osteoarthritic joints. We conducted quantitative reverse transcription polymerase chain reaction and western blot analysis to investigate the relationship between TGFβ1 and TG2 in chondrocytes and determined whether TG2 regulates the expressions of matrix metalloproteinase (MMP)-13, type II, and type X collagen. We also examined the extent of cartilage degradation after performing anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgery in TG2 knock-out mice. We confirmed the overexpression of TGFβ1 and TG2 in human osteoarthritic cartilage compared with non-osteoarthritic cartilage. TGFβ1 treatment significantly increased the expression of TG2 via p38 and ERK activation. TGFβ1-induced TG2 also elevated the level of MMP-13 and type X collagen via NF-κB activation in chondrocytes. Cartilage damage after ACLT and DMM surgery was less severe in TG2 knock-out mice compared with wild-type mice. TGFβ1 modulated catabolic processes in chondrocytes in a TG2-dependent manner. TGFβ1-induced TG2 might be the therapeutic target for treating cartilage degeneration and osteoarthritis.

Tranglutaminase 2 contributes to the asthmatic inflammation by modulating activation of alveolar macrophages.

BackgroundTransglutaminase 2 (TG2), a multifunctional calcium‐dependent acyltransferase, is upregulated in asthmatic airways and reported to play a role in the pathogenesis of allergic asthma. However, the underlying mechanism is not fully understood.ObjectiveTo investigate the role of TG2 in alternative activation of alveolar macrophages by using murine asthma model.MethodsTG2 expression was assessed in induced sputum of 21 asthma patients and 19 healthy controls, and lung tissue of ovalbumin (OVA)‐induced murine asthma model. To evaluate the role of TG2 in asthma, we developed an OVA asthma model in both TG2 null and wild‐type mice. The expression of M2 macrophage markers was measured by fluorescence‐activated cell sorting (FACS) after OVA sensitization and challenge. To evaluate the effect of TG2 inhibition in vitro, interleukin 4 (IL‐4) or IL‐13‐stimulated expression of M2 macrophage markers was measured in CRL‐2456 cells in the presence and absence of a TG2 inhibitor.ResultsThe expression of both TG2 and M2 markers was increased in the sputum of asthmatics compared with that of healthy controls. The expression of TG2 was increased in macrophages of OVA mice. Airway hyperresponsiveness, and the number of inflammatory cells, including eosinophils, was significantly reduced in TG2 null mice compared with wild‐type mice. Enhanced expression of M2 markers in OVA mice was normalized by TG2 knockout. IL‐4 or IL‐13‐stimulated expression of M2 markers in alveolar macrophages was also attenuated by TG2 inhibitor treatment in vitro.ConclusionOur results suggest that TG2‐mediated modulation of alveolar macrophage polarization plays important roles in the pathogenesis of asthma.

Cystamine Treatment Fails to Prevent the Development of Pulmonary Hypertension in Chronic Hypoxic Rats

Introduction: Pulmonary hypertension is characterized by vasoconstriction and remodeling of pulmonary arteries, leading to right ventricular hypertrophy and failure. We have previously found upregulation of transglutaminase 2 (TG2) in the right ventricle of chronic hypoxic rats. The hypothesis of the present study was that treatment with the transglutaminase inhibitor, cystamine, would inhibit the development of pulmonary arterial remodeling, pulmonary hypertension, and right ventricular hypertrophy. Methods: Effect of cystamine on transamidase activity was investigated in tissue homogenates. Wistar rats were exposed to chronic hypoxia and treated with vehicle, cystamine (40 mg/kg/day in mini-osmotic pumps), sildenafil (25 mg/kg/day), or the combination for 2 weeks. Results: Cystamine concentration-dependently inhibited TG2 transamidase activity in liver and lung homogenates. In contrast to cystamine, sildenafil reduced right ventricular systolic pressure and hypertrophy and decreased pulmonary vascular resistance and muscularization in chronic hypoxic rats. Fibrosis in the lung tissue decreased in chronic hypoxic rats treated with cystamine. TG2 expression was similar in the right ventricle and lung tissue of drug and vehicle-treated hypoxic rats. Discussion/Conclusions: Cystamine inhibited TG2 transamidase activity, but cystamine failed to prevent pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial muscularization in the chronic hypoxic rat.

The NEAT1_2/miR-491 Axis Modulates Papillary Thyroid Cancer Invasion and Metastasis Through TGM2/NFκb/FN1 Signaling.

NEAT1 (nuclear paraspeckle assembly transcript 1) is an oncogenic long non-coding RNA (lncRNA) that facilitates tumorigenesis in multiple cancers. In papillary thyroid cancer (PTC), the molecular mechanism by which NEAT1 affects invasion and metastasis remains elusive. RNA sequencing was used to discover differentially expressed NEAT1_2 downstream genes. Protein and RNA expression analyses and immunohistochemistry detected the expression of NEAT1_2, Transglutaminase 2 (TGM2), and microRNA-491 (miR-491) among PTC and non-cancerous tissues. Transwell and wound healing assays, and a mouse model of lung metastasis were used for further functional analyses. Bioinformatics was performed to predict miRNAs binding to both NEAT1_2 and TGM2. Rescue experiments and dual-luciferase reporter assays were performed. In PTC tissues, NEAT1_2 expression was markedly increased and regulated TGM2 expression. TGM2 was overexpressed in PTC, correlating positively with exthyroidal extension and lymph node metastasis. TGM2 knockdown significantly inhibited invasion and metastasis. NEAT1_2 sponged miR-491, acting as a competing endogenous RNA to regulate TGM2 expression. Fibronectin 1 (FN1) was predicted as a TGM2 target. TGM2 could transcriptionally activate FN1 by promoting nuclear factor kappa B (NFκb) p65 nuclear translocation, ultimately promoting PTC invasion/metastasis. These findings identify that NEAT1_2 sponges miR-491 to regulate TGM2 expression. TGM2 activates FN1 via NFκb to promote PTC invasion and metastasis.

Transglutaminase 2 as a Marker for Inflammation and Therapeutic Target in Sepsis.

Sepsis results in lethal organ malfunction due to dysregulated host response to infection, which is a condition with increasing prevalence worldwide. Transglutaminase 2 (TG2) is a crosslinking enzyme that forms a covalent bond between lysine and glutamine. TG2 plays important roles in diverse cellular processes, including extracellular matrix stabilization, cytoskeletal function, cell motility, adhesion, signal transduction, apoptosis, and cell survival. We have shown that the co-culture of Candida albicans and hepatocytes activates and induces the translocation of TG2 into the nucleus. In addition, the expression and activation of TG2 in liver macrophages was dramatically induced in the lipopolysaccharide-injected and cecal ligation puncture-operated mouse models of sepsis. Based on these findings and recently published research, we have reviewed the current understanding of the relationship between TG2 and sepsis. Following the genetic and pharmacological inhibition of TG2, we also assessed the evidence regarding the use of TG2 as a potential marker and therapeutic target in inflammation and sepsis.

Lipotoxic hepatocytes promote nonalcoholic fatty liver disease progression by delivering microRNA-9-5p and activating macrophages.

M1-polarized macrophages are involved in chronic inflammatory diseases, including nonalcoholic fatty liver disease (NAFLD). However, the mechanisms responsible for the activation of macrophages in NAFLD have not been fully elucidated. This study aimed at investigating the physiological mechanisms by which extracellular vesicles (EVs)-encapsulated microRNA-9-5p (miR-9-5p) derived from lipotoxic hepatocytes might activate macrophages in NALFD. After blood sample and cell collection, EVs were isolated and identified followed by co-culture with macrophages. Next, the palmitic acid-induced cell and high fat diet-induced mouse NALFD models were established to explore the in vitro and in vivo effects of EVs-loaded miR-9-5p on NAFLD as evidenced by inflammatory cell infiltration and inflammatory reactions in macrophages. Additionally, the targeting relationship between miR-9-5p and transglutaminase 2 (TGM2) was identified using dual-luciferase reporter gene assay. miR-9-5p was upregulated in the NAFLD-EVs, which promoted M1 polarization of THP-1 macrophages. Furthermore, miR-9-5p could target TGM2 to inhibit its expression. Downregulated miR-9-5p in NAFLD-EVs alleviated macrophage inflammation and M1 polarization as evidenced by reduced levels of macrophage inflammatory factors, positive rates of CD86+ CD11b+, and levels of macrophage surface markers in vitro. Moreover, the effect of silencing of miR-9-5p was replicated in vivo, supported by reductions in TG, TC, AST and ALT levels and attenuated pathological changes. Collectively, lipotoxic hepatocytes-derived EVs-loaded miR-9-5p downregulated the expression of TGM2 and facilitated M1 polarization of macrophages, thereby promoting the progression of NAFLD. This highlights a potential therapeutic target for treating NAFLD.

Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein\u2013protein interactions of the fused protein

Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein–protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST4QN) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST4QN as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein–protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST4QN tag could improve the reproducibility and reliability of GST pulldown experiments.

Study of tumor transglutaminase 2 expression in gallbladder cancer - Is it a novel predictor of survival?

Backgrounds/AimsTransglutaminase 2 (TG2) is known to be an important mediator of inflammation induced carcinogenesis pathway. Chronic inflammation is the most important causative factor in Gallbladder cancer (GBC) carcinogenesis. We analyzed the expression of TG2 in GBC and its role as potential prognostic marker, first of its kind study.MethodsWe analyzed TG2 expression in 100 cases of GBC and 28 cases of non-cancer gallbladder specimen (calculus cholecystitis). We studied TG2 expression in GBC in comparison to control group and evaluated its role as a potential prognostic marker.ResultsTG2 score (1-9) was calculated by multiplying percentage cytoplasmic expression (P) with intensity of expression (I) in tumor cells. Positive TG-2 expression was observed in 62% of GBC patients compared to only 21% (n=6) in control group (p=0.001). In curative resection subgroup (n=54), TG2 positive patients showed shorter disease free survival rate (p=0.04) and higher rate of recurrence (p=0.03) compared to TG2 negative patients. TG2 positive expression was observed in 15/16 of patients with recurrent disease. In palliative treatment subgroup, patients with strong TG2 positive expression had poorer disease specific survival (p=0.01) as compared to weakly positive group. On multivariate analysis, lymph node status (p=0.03) and TG2 expression (p=0.037), were found to be significant predictor of recurrence and eventual survival.ConclusionsPositive TG2 expression was related to higher recurrence rates post curative surgery, shorter disease free and overall survival and ultimately portended poor prognosis. It may be helpful in better prognostication and tailoring therapeutic approach for better management of GBC.

Neuronal and Endothelial Transglutaminase-2 Expression during Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

Transglutiminase-2 (TG2) is a multifunctional enzyme that has been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) using global knockout mice and TG2 selective inhibitors. Previous studies have identified the expression of TG2 in subsets of macrophages–microglia and astrocytes after EAE. The aims of the current investigation were to examine neuronal expression of TG2 in rodent models of chronic-relapsing and non-relapsing EAE and through co-staining with intracellular and cell death markers, provide insight into the putative role of TG2 in neuronal pathology during disease progression. Here we report that under normal physiological conditions there is a low basal expression of TG2 in the nucleus of neurons, however following EAE or MS, robust induction of cytoplasmic TG2 occurs in most neurons surrounding perivascular lesion sites. Importantly, TG2-positive neurons also labeled for phosphorylated Extracellular signal-regulated kinase 1/2 (ERK1/2) and the apoptotic marker cleaved caspase-3. In white and gray matter lesions, high levels of TG2 were also found within the vasculature and endothelial cells as well as in tissue migrating pericytes or fibroblasts, though rarely did TG2 colocalize with cells identified with glial cell markers (astrocytes, oligodendrocytes and microglia). TG2 induction occurred concurrently with the upregulation of the blood vessel permeability factor and angiogenic molecule Vascular Endothelial Growth Factor (VEGF). Extracellular TG2 was found to juxtapose with fibronectin, within and surrounding blood vessels. Though molecular and pharmacological studies have implicated TG2 in the induction and severity of EAE, the cell autonomous functions of this multifunctional enzyme during disease progression remains to be elucidated.

TGM2 Novel Biomarker Of Radiation Response After Neo-Adjuvant TMZ In Glioblastoma

It is becoming increasingly evident that treatment drives rapid changes in the molecular state of tumor cells to provide a pro-survival phenotype to cells. Transglutaminase-2 (TGM2) is a membrane protein which has previously been implicated in driving radiation resistance in multiple cancers. We hypothesized that Transglutaminase-2 (TGM2) level is a predictor of response to radiation in Glioblastoma (GBM). We have investigated how exposure to neo-adjuvant Temozolomide (TMZ) in glioma stem cells (GSCs) with different levels of TGM2 would affect the response to subsequent radiation in these GSCs Primary Glioma Stem cells lines (TGM2-high: 1123, 83; TGM2-low: 528, OPK49) were used to explore the role of TGM2 expression in radiation response and modulation of expression by TMZ. We utilized pre-clinical in vivo models to assess whether a novel regime which included neo-adjuvant TMZ prior to chemoradiation was superior to upfront concurrent chemoradiation regimes. We provide evidence that TGM2 drives radioresistance in GBM through restriction of p53 mediated repression of expression RAD51, a DNA damage repair gene. We demonstrate that exposure of GBM to the standard TMZ drives rapid downregulation of TGM2 in vitro and that this phenomenon is recapitulated in vivo. Interestingly, we confirm previous reports that radiation (RT) is able to drive reciprocal changes in TGM2 and promotes reactivation of TGM2 in TGM2-high tumors but not TGM2-low tumors. Given these observations, we hypothesized that exposure to neo-adjuvant TMZ in TGM2-low tumors would increase the efficacy of subsequent RT in these tumors. Comparison of the effect of standard treatment consisting of 3 weeks of concurrent TMZ and RT (Stupp) to a novel regime (neo-Stupp) consisting of 1 week of neo-adjuvant TMZ followed by two weeks of TMZ and hypofractionated RT revealed a superior survival benefit of this novel regime in in TGM2-low tumors but not in TGM2-high tumors. Utilization of the TGM2 inhibitor GK921 in combination with neo-Stupp prevented rapid relapse previously observed in TGM2-high tumors. We propose that neo-Stupp treatment regimen is superior to standard chemoradiation regimes in a subset of tumors identified by low TGM2 expression

Increased Transglutaminase 2 Expression and Activity in Rodent Models of Obesity/Metabolic Syndrome and Aging.

Diastolic dysfunction of the heart and decreased compliance of the vasculature and lungs (i.e., increased organ tissue stiffness) are known features of obesity and the metabolic syndrome. Similarly, cardiac diastolic dysfunction is associated with aging. Elevation of the enzyme transglutaminase 2 (TG2) leads to protein cross-linking and enhanced collagen synthesis and participates as a candidate pathway for development of tissue stiffness. With these observations in mind we hypothesized that TG2 may be elevated in tissues of a rat model of obesity/metabolic syndrome (the ZSF 1 rat) and a mouse model of aging, i.e., the senescent SAMP8 mouse. In the experiments reported here, TG2 expression and activity were found for the first time to be spontaneously elevated in organs from both the ZSF1 rat and the SAMP8 mouse. These observations are consistent with a hypothesis that a TG2-related pathway may participate in the known tissue stiffness associated with cardiac diastolic dysfunction in these two rodent models. The potential TG2 pathway needs better correlation with physiologic dysfunction and may eventually provide novel therapeutic insights to improve tissue compliance.

Abstract 395: The Role of Transglutaminase 2 (tg2) in Regulation of Macrophage Phenotype

Transglutaminase 2 (TG2) expression is increased in infarcted and remodeling hearts and modulates cellular responses through enzymatic effects and non-enzymatic actions. In monocytic cells, TG2 is predominantly expressed by M2 macrophages in both mice and humans. Although TG2 is considered a preserved and reliable marker of M2 macrophages, its role in regulation of macrophage phenotype remains unknown. In order to study the role of TG2 in macrophage function in homeostasis and disease, we generated mice with myeloid cell-specific loss of TG2 (MyTG2KO), and examined their response to experimental myocardial infarction (MI). Using myeloid cell reporter CSF1R EGFP mice, we found that TG2 is highly expressed in the majority of spleen, liver and lung macrophages, and is markedly upregulated in M2-like Arg1+ macrophages infiltrating the infarcted heart in contrast to infarct neutrophils. MyTG2KO mice had no baseline defects. Following MI, MyTG2KO mice and TG2 fl/fl controls had comparable chamber dilation, systolic dysfunction and infarct size, but exhibited attenuated diastolic dysfunction and reduced atrial size, reflecting lower filling pressures. Macrophages harvested from infarcted MyTG2KO mice had comparable pro-inflammatory and anti-inflammatory cytokine synthesis, but markedly increased expression of caveolin-1, the integrins a1, b1 and b3, and of the anti-fibrotic proteoglycan decorin. In vitro, we examined the effects of TG2 loss on the transcriptomic profile of bone marrow macrophages using RNAseq. Reactome and KEGG analysis showed that TG2 loss did not affect the baseline macrophage transcriptome differences. In contrast, following TGF-b stimulation, TG2 KO and WT cells had differential expression of genes regulating focal adhesion formation and the actin cytoskeleton. Consistent with the in vivo findings, TGF-b stimulation resulted in higher caveolin-1 and decorin in TG2 KO macrophages. In summary, TG2 expression is induced in M2-like macrophages infiltrating the infarcted heart and mediates diastolic dysfunction. The effects of TG2 in macrophages are not due to actions on their inflammatory profile, but may involve modulation of focal adhesion formation and cell migration, and subsequent expression of anti-fibrotic genes.

Essential Role for CD30-Transglutaminase 2 Axis in Memory Th1 and Th17 Cell Generation.

Memory helper T (Th) cells are crucial for secondary immune responses against infectious microorganisms but also drive the pathogenesis of chronic inflammatory diseases. Therefore, it is of fundamental importance to understand how memory T cells are generated. However, the molecular mechanisms governing memory Th cell generation remain incompletely understood. Here, we identified CD30 as a molecule heterogeneously expressed on effector Th1 and Th17 cells, and CD30hi effector Th1 and Th17 cells preferentially generated memory Th1 and Th17 cells. We found that CD30 mediated signal induced Transglutaminase-2 (TG2) expression, and that the TG2 expression in effector Th cells is essential for memory Th cell generation. In fact, Cd30-deficiency resulted in the impaired generation of memory Th1 and Th17 cells, which can be rescued by overexpression of TG2. Furthermore, transglutaminase-2 (Tgm2)-deficient CD4 T cells failed to become memory Th cells. As a result, T cells from Tgm2-deficient mice displayed impaired antigen-specific antibody production and attenuated Th17-mediated allergic responses. Our data indicate that CD30-induced TG2 expression in effector Th cells is essential for the generation of memory Th1 and Th17 cells, and that CD30 can be a marker for precursors of memory Th1 and Th17 cells.