COL5A1 promotes triple-negative breast cancer progression by activating tumor cell-macrophage crosstalk.

Triple-negative breast cancer (TNBC) is an exceptionally aggressive subtype of breast cancer. Despite the recognized interplay between tumors and tumor-associated macrophages in fostering drug resistance and disease progression, the precise mechanisms leading these interactions remain elusive. Our study revealed that the upregulation of collagen type V alpha 1 (COL5A1) in TNBC tissues, particularly in chemoresistant samples, was closely linked to an unfavorable prognosis. Functional assays unequivocally demonstrated that COL5A1 played a pivotal role in fueling cancer growth, metastasis, and resistance to doxorubicin, both in vitro and in vivo. Furthermore, we found that the cytokine IL-6, produced by COL5A1-overexpressing TNBC cells actively promoted M2 macrophage polarization. In turn, TGFß from M2 macrophages drived TNBC doxorubicin resistance through the TGFß/Smad3/COL5A1 signaling pathway, establishing a feedback loop between TNBC cells and macrophages. Mechanistically, COL5A1 interacted with TGM2, inhibiting its K48-linked ubiquitination-mediated degradation, thereby enhancing chemoresistance and increasing IL-6 secretion. In summary, our findings underscored the significant contribution of COL5A1 upregulation to TNBC progression and chemoresistance, highlighting its potential as a diagnostic and therapeutic biomarker for TNBC.

Transcriptomics Studies Reveal Functions of Transglutaminase 2 in Breast Cancer Cells Using Membrane Permeable and Impermeable Inhibitors.

Transglutaminase 2 (TG2) performs many functions both under physiological and pathological conditions. In cancer, its expression is associated with aggressiveness, propensity to epithelial-mesenchymal transition, and metastasis. Since TG2 performs key functions both outside and inside the cell, using inhibitors with different membrane permeability we analyzed the changes in the transcriptome induced in two triple-negative cell lines (MDA-MB-436 and MDA-MB-231) with aggressive features. By characterizing pathways and gene networks, we were able to define the effects of TG2 inhibitors (AA9, membrane-permeable, and NCEG2, impermeable) in relation to the roles of the enzyme in the intra- and extracellular space within the context of breast cancer. The deregulated genes revealed p53 and integrin signaling to be the common pathways with some genes showing opposite changes in expression. In MDA-MB-436, AA9 induced apoptosis, modulated cadherin, Wnt, gastrin and cholecystokinin receptors (CCKR) mediated signaling, with RHOB and GNG2 playing significant roles, and affected the Warburg effect by decreasing glycolytic enzymes. In MDA-MB-231 cells, AA9 strongly impacted HIF-mediated hypoxia, including AKT and mTOR pathway. These effects suggest an anti-tumor activity by blocking intracellular TG2 functions. Conversely, the use of NCEG2 stimulated the expression of ATP synthase and proteins involved in DNA replication, indicating a potential promotion of cell proliferation through inhibition of extracellular TG2. To effectively utilize these molecules as an anti-tumor strategy, an appropriate delivery system should be evaluated to target specific functions and avoid adverse effects. Additionally, considering combinations with other pathway modulators is crucial.

Mesenchymal Transglutaminase 2 Activates Epithelial ADAM17: Link to G-Protein-Coupled Receptor 56 (ADGRG1) Signalling.

A wound healing model was developed to elucidate the role of mesenchymal-matrix-associated transglutaminase 2 (TG2) in keratinocyte re-epithelialisation. TG2 drives keratinocyte migratory responses by activation of disintegrin and metalloproteinase 17 (ADAM17). We demonstrate that epidermal growth factor (EGF) receptor ligand shedding leads to EGFR-transactivation and subsequent rapid keratinocyte migration on TG2-positive ECM. In contrast, keratinocyte migration was impaired in TG2 null conditions. We show that keratinocytes express the adhesion G-protein-coupled receptor, ADGRG1 (GPR56), which has been proposed as a TG2 receptor. Using ADAM17 activation as a readout and luciferase reporter assays, we demonstrate that TG2 activates GPR56. GPR56 activation by TG2 reached the same level as observed with an agonistic N-GPR56 antibody. The N-terminal GPR56 domain is required for TG2-regulated signalling response, as the constitutively active C-GPR56 receptor was not activated by TG2. Signalling required the C-terminal TG2 ß-barrel domains and involved RhoA-associated protein kinase (ROCK) and ADAM17 activation, which was blocked by specific inhibitors. Cell surface binding of TG2 to the N-terminal GPR56 domain is rapid and is associated with TG2 and GPR56 endocytosis. TG2 and GPR56 represent a ligand receptor pair causing RhoA and EGFR transactivation. Furthermore, we determined a binding constant for the interaction of human TG2 with N-GPR56 and show for the first time that only the calcium-enabled "open" TG2 conformation associates with N-GPR56.

Transglutaminase 2 Binds to the CD44v6 Cytoplasmic Domain to Stimulate CD44v6/ERK1/2 Signaling and Maintain an Aggressive Cancer Phenotype.

Transglutaminase 2 (TG2) is a key cancer cell survival protein in many cancer types. As such, efforts are underway to characterize the mechanism of TG2 action. In this study, we report that TG2 stimulates CD44v6 activity to enhance cancer cell survival via a mechanism that involves formation of a TG2/CD44v6/ERK1/2 complex that activates ERK1/2 signaling to drive an aggressive cancer phenotype. TG2 and ERK1/2 bind to the CD44v6 C-terminal intracellular cytoplasmic domain to activate ERK1/2 and stimulate cell proliferation and invasion. This is the same region that binds to ERM proteins and ankyrin to activate CD44v6-dependent cell proliferation, invasion, and migration. We further show that treatment with hyaluronan (HA), the physiologic CD44v6 ligand, stimulates CD44v6 activity, as measured by ERK1/2 activation, but that this response is severely attenuated in TG2 or CD44v6 knockdown or knockout cells. Moreover, treatment with TG2 inhibitor reduces tumor growth and that is associated with reduced CD44v6 level and ERK1/2 activity, and reduced stemness and epithelial-mesenchymal transition (EMT). These changes are replicated in CD44v6 knockout cells. These findings suggest that a unique TG2/CD44v6/ERK1/2 complex leads to increased ERK1/2 activity to stimulate an aggressive cancer phenotype and stimulate tumor growth. These findings have important implications for cancer stem cell maintenance and suggest that cotargeting of TG2 and CD44v6 with specific inhibitors may be an effective anticancer treatment strategy. IMPLICATIONS: TG2 and CD44v6 are important procancer proteins. TG2 and ERK1/2 bind to the CD44v6 C-terminal domain to form a TG2/CD44v6/ERK1/2 complex that activates ERK1/2 to stimulate the cancer phenotype.

TGM2 inhibits the proliferation, migration and tumorigenesis of MDCK cells.

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.

The Role and Mechanism of Transglutaminase 2 in Regulating Hippocampal Neurogenesis after Traumatic Brain Injury.

Traumatic brain injury usually results in neuronal loss and cognitive deficits. Promoting endogenous neurogenesis has been considered as a viable treatment option to improve functional recovery after TBI. However, neural stem/progenitor cells (NSPCs) in neurogenic regions are often unable to migrate and differentiate into mature neurons at the injury site. Transglutaminase 2 (TGM2) has been identified as a crucial component of neurogenic niche, and significantly dysregulated after TBI. Therefore, we speculate that TGM2 may play an important role in neurogenesis after TBI, and strategies targeting TGM2 to promote endogenous neural regeneration may be applied in TBI therapy. Using a tamoxifen-induced Tgm2 conditional knockout mouse line and a mouse model of stab wound injury, we investigated the role and mechanism of TGM2 in regulating hippocampal neurogenesis after TBI. We found that Tgm2 was highly expressed in adult NSPCs and up-regulated after TBI. Conditional deletion of Tgm2 resulted in the impaired proliferation and differentiation of NSPCs, while Tgm2 overexpression enhanced the abilities of self-renewal, proliferation, differentiation, and migration of NSPCs after TBI. Importantly, injection of lentivirus overexpressing TGM2 significantly promoted hippocampal neurogenesis after TBI. Therefore, TGM2 is a key regulator of hippocampal neurogenesis and a pivotal therapeutic target for intervention following TBI.

The transglutaminase 2 cancer cell survival factor maintains mTOR activity to drive an aggressive cancer phenotype.

Transglutaminase 2 (TG2) is an important cancer stem-like cell survival protein that is highly expressed in epidermal squamous cell carcinoma and drives an aggressive cancer phenotype. In the present study, we show that TG2 knockdown or inactivation results in a reduction in mammalian target of rapamycin (mTOR) level and activity in epidermal cancer stem-like cells which are associated with reduced spheroid formation, invasion, and migration, and reduced cancer stem cell and epithelial-mesenchymal transition (EMT) marker expression. Similar changes were observed in both cultured cells and tumors. mTOR knockdown or treatment with rapamycin phenocopies the reduction in spheroid formation, invasion, and migration, and cancer stem cell and EMT marker expression. Moreover, mTOR appears to be a necessary mediator of TG2 action, as a forced expression of constitutively active mTOR in TG2 knockdown cells partially restores the aggressive cancer phenotype and cancer stem cell and EMT marker expression. Tumor studies show that rapamycin reduces tumor growth and cancer stem cell marker expression and EMT. These studies suggest that TG2 stimulates mTOR activity to stimulate cancer cell stemness and EMT and drive aggressive tumor growth.

Cytosolic TGM2 promotes malignant progression in gastric cancer by suppressing the TRIM21-mediated ubiquitination/degradation of STAT1 in a GTP binding-dependent modality.

BACKGROUND: Previous studies have revealed the critical role of transglutaminase 2 (TGM2) as a potential therapeutic target in cancers, but the oncogenic roles and underlying mechanisms of TGM2 in gastric cancer (GC) are not fully understood. In this study, we examined the role and potential mechanism of TGM2 in GC. METHODS: Western blotting, immunohistochemistry, CCK8, colony formation and transwell assays were used to measure TGM2 expression in the GC cells and tissues and to examine the in vitro role of TGM2 in GC. Xenograft and in vivo metastasis experiments were performed to examine the in vivo role of TGM2 in GC. Gene set enrichment analysis, quantitative PCR and western blotting were conducted to screen for potential TGM2 targets involved in GC. Gain/loss-of-function and rescue experiments were conducted to detect the biological roles of STAT1 in GC cells in the context of TGM2. Co-immunoprecipitation, mass spectrometry, quantitative PCR and western blotting were conducted to identify STAT1-interacting proteins and elucidate their regulatory mechanisms. Mutations in TGM2 and two molecules (ZM39923 and A23187) were used to identify the enzymatic activity of TGM2 involved in the malignant progression of GC and elucidate the underlying mechanism. RESULTS: In this study, we demonstrated elevated TGM2 expression in the GC tissues, which closely related to pathological grade, and predicted poor survival in patients with GC. TGM2 overexpression or knockdown promoted (and inhibited) cell proliferation, migration, and invasion, which were reversed by STAT1 knockdown or overexpression. Further studies showed that TGM2 promoted GC progression by inhibiting STAT1 ubiquitination/degradation. Then, tripartite motif-containing protein 21 (TRIM21) was identified as a ubiquitin E3 ligase of STAT1 in GC. TGM2 maintained STAT1 stability by facilitating the dissociation of TRIM21 and STAT1 with GTP-binding enzymatic activity. A23187 abolished the role of TGM2 in STAT1 and reversed the pro-tumor role of TGM2 in vitro and in vivo. CONCLUSIONS: This study revealed a critical role and regulatory mechanism of TGM2 on STAT1 in GC and highlighted the potential of TGM2 as a therapeutic target, which elucidates the development of medicine or strategies by regulating the GTP-binding activity of TGM2 in GC.

TG2 as a novel breast cancer prognostic marker promotes cell proliferation and glycolysis by activating the MEK/ERK/LDH pathway.

BACKGROUND: Breast cancer (BC) is the most common malignant tumor among women worldwide. Tissue transglutaminase 2 (TG2) has been reported as a major player across several types of cancer. However, the effects of TG2 in breast cancer are less known. METHODS: The expression of TG2 in patients with BC was detected by immunochemistry staining and RT-qPCR. The correlation of TG2 expression and clinicopathological factors or overall survival (OS) was analyzed by Chi-square test, Kaplan-Meier, and Cox-regression analysis. The effects of TG2 on cell proliferation and glycolysis were investigated in vivo and in vitro by gain- and loss-of-function experiments. RESULT: Both mRNA and protein levels of TG2 were overexpressed in BC tissues and cultured cells. Clinical stage (p = 0.011), molecular subtype (p<0.001) and survival status (p<0.001) were significantly correlated with TG2 expression. Specifically, TG2 expression was positively associated with the clinical stage (r = 0.193, p = 0.005) and OS (r = 0.230, p = 0.001), while negatively associated with molecular subtype (r = - 0.161, p = 0.020). Overexpressed TG2 was a prognostic factor of poor OS by Cox-regression analysis. Gain- and loss-of-function experiments indicated that cell proliferation and glycolysis were regulated by TG2 via the MEK/ERK/LDH pathway. TG2-induced activation of the MEK/ERK/LDH pathway and glycolysis were attenuated by MEK inhibitor U0126. CONCLUSION: TG2 is overexpressed in BC, which can serve as an independent prognostic factor for OS. TG2 promotes tumor cell proliferation and increases glycolysis associated with the activation of the MEK/ERK/LHD pathway.

Aberrant expression of transglutaminase 2 in pancreas and thymus of NOD mice underscores the importance of deamidation in neoantigen generation.

Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2, the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1ß and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD ß-cells underscores the role of deamidation in amplifying T-cell reactivity.

Type 2 Transglutaminase in Coeliac Disease: A Key Player in Pathogenesis, Diagnosis and Therapy.

Type 2 transglutaminase (TG2) is the main autoantigen in coeliac disease (CD), a widespread inflammatory enteropathy caused by the ingestion of gluten-containing cereals in genetically predisposed individuals. As a consequence, serum antibodies to TG2 represent a very useful marker in CD diagnosis. However, TG2 is also an important player in CD pathogenesis, for its ability to deamidate some Gln residues of gluten peptides, which become more immunogenic in CD intestinal mucosa. Given the importance of TG2 enzymatic activities in CD, several studies have sought to discover specific and potent inhibitors that could be employed in new therapeutical approaches for CD, as alternatives to a lifelong gluten-free diet. In this review, we summarise all the aspects regarding TG2 involvement in CD, including its enzymatic reactions in pathogenesis, the role of anti-TG2 antibodies in disease management, and the exploration of recent strategies to reduce deamidation or to use transamidation to detoxify gluten.

Tissue transglutaminase activates integrin-linked kinase and ß-catenin in ovarian cancer.

Ovarian cancer (OC) is the most lethal gynecological cancer. OC cells have high proliferative capacity, are invasive, resist apoptosis, and tumors often display rearrangement of extracellular matrix (ECM) components, contributing to accelerated tumor progression. The multifunctional protein tissue transglutaminase (TG2) is known to be secreted in the tumor microenvironment, where it interacts with fibronectin (FN) and the cell surface receptor integrin ß1. However, the mechanistic role of TG2 in cancer cell proliferation is unknown. Here, we demonstrate that TG2 directly interacts with and facilitates the phosphorylation and activation of the integrin effector protein integrin-linked kinase (ILK) at Ser246. We show that TG2 and p-Ser246-ILK form a complex that is detectable in patient-derived OC primary cells grown on FN-coated slides. In addition, we show that coexpression of TGM2 and ILK correlates with poor clinical outcome. Mechanistically, we demonstrate that TG2-mediated ILK activation causes phosphorylation of glycogen synthase kinase-3α/ß, allowing ß-catenin nuclear translocation and transcriptional activity. Furthermore, inhibition of TG2 and ILK using small molecules, neutralizing antibodies, or shRNA-mediated knockdown blocks cell adhesion to the FN matrix, as well as the Wnt receptor response to the Wnt-3A ligand, and ultimately, cell adhesion, growth, and migration. In conclusion, we demonstrate that TG2 directly interacts with and activates ILK in OC cells and tumors and define a new mechanism that links ECM cues with ß-catenin signaling in OC. These results suggest a central role of TG2-FN-integrin clusters in ECM rearrangement and indicate that downstream effector ILK may represent a potential new therapeutic target in OC.

Transglutaminase 2 enhances hepatocyte growth factor signaling to drive the mesothelioma cancer cell phenotype.

Transglutaminase 2 (TG2) is an important mesothelioma cancer cell survival protein. However, the mechanism whereby TG2 maintains mesothelioma cell survival is not well understood. We present studies showing that TG2 drives hepatocyte growth factor (HGF)-dependent MET receptor signaling to maintain the aggressive mesothelioma cancer phenotype. TG2 increases HGF and MET messenger RNA and protein levels to enhance MET signaling. TG2 inactivation reduces MET tyrosine kinase activity to reduce cancer cell spheroid formation, invasion and migration. We also confirm that HGF/MET signaling is a biologically important mediator of TG2 action. Reducing MET level using genetic methods or treatment with MET inhibitors reduces spheroid formation, invasion and migration and this is associated with reduced MEK1/2 and ERK1/2. In addition, MEK1/2 and ERK1/2 inhibitors suppress the cancer phenotype. Moreover, MET knockout mesothelioma cells form 10-fold smaller tumors compared to wild-type cells and these tumors display reduced MET, MEK1/2, and ERK1/2 activity. These findings suggest that TG2 maintains HGF and MET levels in cultured mesothelioma cells and tumors to drive HGF/MET, MEK1/2, and ERK1/2 signaling to maintain the aggressive mesothelioma cancer phenotype.

Histological changes and transglutaminase 2 expression in the oviduct of advanced pregnant cows.

The oviduct is a dynamic organ that has not been assigned specific functions during advanced pregnancy. However, since changes in the oviductal epithelium during the estrous cycle are attributed mainly to variations in estradiol (E2) levels, and E2 levels increase along pregnancy, we hypothesized that advanced pregnant cows should present changes in the oviductal epithelium. In advanced pregnant cows, the oviducts showed higher leaf-like folds and lower mucosa width and epithelium height than those of cycling animals. Also, PAS-positive apical protrusions and TUNEL-positive extruded cytoplasmic material were observed in advanced pregnant cows. Oviductal fluid from advanced pregnant cows showed lower protein concentration than that from cycling cows. Transglutaminase 2 (TG2) was detected exclusively in oviductal fluid of pregnant cows but not in cells from any stage, whereas its mRNA was detected in different amounts in cells from all stages. This protein was identified by LC/MS-MS and its identity was corroborated by Western blot. The observations in histology of the epithelium and the presence of TG2 in oviductal fluid correlate with high levels of E2 in serum. In conclusion, important histological changes in the oviductal epithelium and secretion of TG2 to the oviductal fluid appear to be triggered by the high E2 levels exclusive of advanced pregnancy.

Transglutaminase 2 crosslinks zona pellucida glycoprotein 3 to prevent polyspermy.

Soon after fertilization, the block mechanisms are developed in the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, penetration, and fusion. However, the molecular basis and underlying mechanism for the post-fertilization block to sperm penetration through ZP has not yet been determined. Here, we find that transglutaminase 2 (Tgm2), an enzyme that catalyzes proteins by the formation of an isopeptide bond within or between polypeptide chains, crosslinks zona pellucida glycoprotein 3 (ZP3) to result in the ZP hardening after fertilization and thus prevents polyspermy. Tgm2 abundantly accumulates in the subcortical region of the oocytes and vanishes upon fertilization. Both inhibition of Tgm2 activity in oocytes by the specific inhibitor in vitro and genetic ablation of Tgm2 in vivo cause the presence of additional sperm in the perivitelline space of fertilized eggs, consequently leading to the polyploid embryos. Biochemically, recombinant Tgm2 binds to and crosslinks ZP3 proteins in vitro, and incubation of oocytes with recombinant Tgm2 protein inhibits the polyspermy. Altogether, our data identify Tgm2 as a participant of zona block to the post-fertilization sperm penetration via hardening ZP surrounding fertilized eggs, extending our current understanding about the molecular basis of block to polyspermy.

Role of TGM2 in T­cell lymphoblastic lymphoma via regulation of IL­6/JAK/STAT3 signalling.

Transglutaminase 2 (TGM2) is a Ca2+­dependent enzyme that is closely associated with cancer progression; however, the function of TGM2 in T­cell 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 T­cell lymphoma cells were evaluated using the Cell Counting Kit­8, colony formation assay, 5­ethynyl­2'­deoxyuridine (EdU) assay, flow cytometry, reverse transcription­quantitative polymerase chain reaction, western blotting and gene set enrichment analysis (GSEA). TGM2 expression was shown to be elevated in formalin­fixed paraffin­embedded skin biopsies from patients with T­cell lymphoma relative to skin tissue from healthy cases. TGM2 expression was also increased in T­cell lymphoma cell lines compared with that in CD4+ T cells. Transfection with TGM2 small interfering RNAs (siRNAs) decreased the number of EdU­positive cells, and the viability and colony formation of T­cell lymphoma cells. Furthermore, TGM2 siRNAs enhanced the apoptosis of T­cell lymphoma cells potentially via cleavage of caspase­3 and poly ADP­ribose polymerase. GSEA identified the IL­6/JAK/STAT3 pathway as a potential downstream signalling pathway of TGM2. Notably, the effects of TGM2 siRNAs on T­cell lymphoma cells were attenuated by IL­6 and accelerated by IL­6/JAK/STAT3 inhibitor AG490. These findings indicated that TGM2 siRNAs inhibited the proliferation of T­cell lymphoma cells by regulating the IL­6/JAK/STAT3 signalling pathway; therefore, TGM2 may function as a potential therapeutic target for T­cell lymphoma.

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity.

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking. GTP-binding/GTPase activity is required for TG2 maintenance of the aggressive cancer phenotype. Thus, identifying agents that convert TG2 from the closed to the open GTP-binding/GTPase inactive conformation is an important cancer prevention/treatment strategy. Sulforaphane (SFN) is an important diet-derived cancer prevention agent that is known to possess a reactive isothiocyanate group and has potent anticancer activity. Using a biotin-tagged SFN analog (Biotin-ITC) and kinetic analysis we show that SFN covalently and irreversibly binds to recombinant TG2 to inhibit transamidase activity and shift TG2 to an open/extended conformation, leading to a partial inhibition of GTP binding. We also show that incubation of cancer cells or cancer cell extract with Biotin-ITC results in formation of a TG2/Biotin-ITC complex and that SFN treatment of cancer cells inhibits TG2 transamidase activity and shifts TG2 to an open/extended conformation. These findings identify TG2 as a direct SFN anticancer target in epidermal squamous cell carcinoma.

Respiratory syncytial virus infection induces the release of transglutaminase 2 from human airway epithelial cells.

Respiratory syncytial virus (RSV) is an important human pathogen that causes severe lower respiratory tract infections in young children, the elderly, and the immunocompromised, yet no effective treatments or vaccines are available. The precise mechanism underlying RSV-induced acute airway disease and associated sequelae are not fully understood; however, early lung inflammatory and immune events are thought to play a major role in the outcome of the disease. Moreover, oxidative stress responses in the airways play a key role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium (Ca2+) levels, which in turn activate Ca2+-dependent enzymes, including transglutaminase 2 (TG2). Transglutaminase 2 is a multifunctional cross-linking enzyme implicated in various physiological and pathological conditions; however, its involvement in respiratory virus-induced airway inflammation is largely unknown. In this study, we demonstrated that RSV-induced oxidative stress promotes enhanced activation and release of TG2 from human lung epithelial cells as a result of its translocation from the cytoplasm and subsequent release into the extracellular space, which was mediated by Toll-like receptor (TLR)-4 and NF-κB pathways. Antioxidant treatment significantly inhibited RSV-induced TG2 extracellular release and activation via blocking viral replication. Also, treatment of RSV-infected lung epithelial cells with TG2 inhibitor significantly reduced RSV-induced matrix metalloprotease activities. These results suggested that RSV-induced oxidative stress activates innate immune receptors in the airways, such as TLRs, that can activate TG2 via the NF-κB pathway to promote cross-linking of extracellular matrix proteins, resulting in enhanced inflammation.

TGM2 positively regulates myoblast differentiation via enhancing the mTOR signaling.

Myoblast differentiation is an essential process for the control of muscle regeneration. However, the intrinsic mechanisms underlying this dynamic process are still not well clarified. Herein, we identified transglutaminase type 2 (TGM2) as a novel regulator of muscle differentiation and regeneration in vitro and in vivo. Specifically, knockdown of TGM2 suppresses whereas overexpression of TGM2 promotes myoblast differentiation in differentiating C2C12 cells. Mechanistic studies revealed that TGM2 promotes C2C12 myoblast differentiation via enhancing GPR56 mediated activation of the mTOR signaling. Additionally, lentivirus mediated knockdown of TGM2 hinders the regeneration of muscles in a BaCl2 induced skeletal muscle injury model of mice. Finally, we found that both TGM2 and activation of the mTOR signaling are up-regulated in muscles of patients with immune-mediated necrotizing myopathy (IMNM), especially in the regenerating myofibers. Collectively, our research demonstrates that TGM2 positively regulates muscle differentiation and regeneration through facilitating the myogenic mTOR signaling, which might be a potential target of therapy for skeletal muscle injury.

Deletion or Inhibition of Astrocytic Transglutaminase 2 Promotes Functional Recovery after Spinal Cord Injury.

Following CNS injury, astrocytes become "reactive" and exhibit pro-regenerative or harmful properties. However, the molecular mechanisms that cause astrocytes to adopt either phenotype are not well understood. Transglutaminase 2 (TG2) plays a key role in regulating the response of astrocytes to insults. Here, we used mice in which TG2 was specifically deleted in astrocytes (Gfap-Cre+/- TG2fl/fl, referred to here as TG2-A-cKO) in a spinal cord contusion injury (SCI) model. Deletion of TG2 from astrocytes resulted in a significant improvement in motor function following SCI. GFAP and NG2 immunoreactivity, as well as number of SOX9 positive cells, were significantly reduced in TG2-A-cKO mice. RNA-seq analysis of spinal cords from TG2-A-cKO and control mice 3 days post-injury identified thirty-seven differentially expressed genes, all of which were increased in TG2-A-cKO mice. Pathway analysis revealed a prevalence for fatty acid metabolism, lipid storage and energy pathways, which play essential roles in neuron-astrocyte metabolic coupling. Excitingly, treatment of wild type mice with the selective TG2 inhibitor VA4 significantly improved functional recovery after SCI, similar to what was observed using the genetic model. These findings indicate the use of TG2 inhibitors as a novel strategy for the treatment of SCI and other CNS injuries.