Expression Of Tissue Transglutaminase Research Articles

Effect of Ferulic Acid Loaded in Nanoparticle on Tissue Transglutaminase Expression Levels in Human Glioblastoma Cell Line.

Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.

Hypoxia and inflammation conditions differentially affect the expression of tissue transglutaminase spliced variants and functional properties of extravillous trophoblast cells.

Persistent hypoxia and inflammation beyond early pregnancy are involved in a bad outcome because of defective trophoblast invasiveness. Tissue transglutaminase (TG2) coregulates several cell functions. An aberrant expression and/or transamidation activity could contribute to placental dysfunction. The first-trimester trophoblast cell line (Swan-71) was used to study TG2 expression and cell functions in the absence or presence of inflammatory cytokines (TNF-α, IL-1β) or chemical hypoxia (CoCl2 ). We analyzed The concentration of cytokines in the supernatant by ELISA; Cell migration by scratch assay; NF-κB activation by detection of nuclear p65 by immunofluorescence or flow cytometry using a Swan-71 NF-κB-hrGFP reporter cell line. Tissue transglutaminase expression was analyzed by immunoblot and confocal microscopy. Expression of spliced mRNA variants of tissue transglutaminase was analyzed by RT-PCR. Transamidation activity was assessed by flow cytometry using 5-(biotinamido)-pentylamine substrate. Chemical hypoxia and TGase inhibition, but not inflammatory stimuli, decreased Swan-71 migration. IL-6 production was also decreased by chemical hypoxia, but increased by inflammation. Intracellular TGase activity was increased by all stimuli, but NF-κB activation was observed only in the presence of proinflammatory cytokines. TG2 expression was decreased by CoCl2 and TNF-α. Translocation of TG2 and p65 to nuclei was observed only with TNF-α, without colocalization. Differential relative expression of spliced variants of mRNA was observed between CoCl2 and inflammatory stimuli. The observed decrease in total TG2 expression and relative increase in short variants under hypoxia conditions could contribute to impaired trophoblast invasion and impact on pregnancy outcome.

Fast-Degrading Tissue-Engineered Vascular Grafts Lead to Increased Extracellular Matrix Cross-Linking Enzyme Expression.

Tissue-engineered vascular grafts (TEVGs) require adequate extracellular matrix (ECM) to withstand arterial pressure. Tissue transglutaminase (TG2) and lysyl oxidase (LOX) are enzymes that cross-link ECM proteins and play a pivotal role in the development of vascular stiffness associated with aging. The purpose of this study is to investigate the expression of ECM cross-linking enzymes and mechanisms of scaffold degeneration leading to vascular stiffness in TEVG remodeling. Fast- and slow-degrading electrospun TEVGs were fabricated using polydioxanone (PDO) and poly(L-lactide-co-caprolactone) (PLCL) copolymer, with a PDO/PLCL ratio of 9:1 for fast-degrading and 1:1 for slow-degrading graft. These grafts were implanted in rats (n = 5/group) as abdominal aortic interposition conduits. The grafts were harvested at 1 month to evaluate patency, mechanical properties, vascular neotissue formation, and the expression of ECM cross-linking enzymes. All TEVGs were patent without any aneurysmal formation at 1 month. ECM area, TG2-positive area, and LOX-positive area were significantly greater in fast-degrading TEVGs compared to slow-degrading TEVGs, with significantly less remaining scaffold. The mechanical properties of fast-degrading TEVGs were similar to that of native aorta, as demonstrated by strain-stress curve. In conclusion, at 1 month, fast-degrading TEVGs had rapid and well-organized ECM with greater TG2 and LOX expression and native-like mechanical properties, compared to slow-degrading TEVGs. Impact statement Around 1.4 million patients in the United States require arterial prostheses each year due to cardiovascular diseases. Current synthetic vascular grafts suffer from increased risk of infection, thrombosis, a lack of endothelialization, and compliance mismatch to the native vasculature. Tissue-engineered vascular graft (TEVGs) presented in this study exhibited tunable biodegradation profiles by controlling the polymer ratio of polydioxanone/poly(L-lactide-co-caprolactone). One month after implantation, the fast-degrading TEVGs exhibited mechanical properties similar to that of native aorta, formation of endothelium, and well-organized extracellular matrix (ECM) with increased expression of tissue transglutaminase and lysyl oxidases, which are critical to the ECM remodeling process.

Tissue Transglutaminase Expression Associates With Progression of Multiple Sclerosis.

ObjectiveThe clinical course of multiple sclerosis (MS) is variable and largely unpredictable pointing to an urgent need for markers to monitor disease activity and progression. Recent evidence revealed that tissue transglutaminase (TG2) is altered in patient-derived monocytes. We hypothesize that blood cell–derived TG2 messenger RNA (mRNA) can potentially be used as biomarker in patients with MS.MethodsIn peripheral blood mononuclear cells (PBMCs) from 151 healthy controls and 161 patients with MS, TG2 mRNA was measured and correlated with clinical and MRI parameters of disease activity (annualized relapse rate, gadolinium-enhanced lesions, and T2 lesion volume) and disease progression (Expanded Disability Status Scale [EDSS], normalized brain volume, and hypointense T1 lesion volume).ResultsPBMC-derived TG2 mRNA levels were significantly associated with disease progression, i.e., worsening of the EDSS over 2 years of follow-up, normalized brain volume, and normalized gray and white matter volume in the total MS patient group at baseline. Of these, in patients with relapsing-remitting MS, TG2 expression was significantly associated with worsening of the EDSS scores over 2 years of follow-up. In the patients with primary progressive (PP) MS, TG2 mRNA levels were significantly associated with EDSS, normalized brain volume, and normalized gray and white matter volume at baseline. In addition, TG2 mRNA associated with T1 hypointense lesion volume in the patients with PP MS at baseline.ConclusionPBMC-derived TG2 mRNA levels hold promise as biomarker for disease progression in patients with MS.Classification of EvidenceThis study provides Class II evidence that in patients with MS, PBMC-derived TG2 mRNA levels are associated with disease progression.

Osteopontin contributes to late-onset asthma phenotypes in adult asthma patients

Patients with late-onset asthma (LOA) have poor clinical outcomes. Osteopontin (OPN) is associated with airway inflammation and remodeling. To investigate the role of OPN in LOA compared to early-onset asthma (EOA), serum OPN levels were compared between 131 adult asthma patients (48 LOA and 83 EOA patients) and 226 healthy controls (HCs). BALB/c mice were sensitized with ovalbumin with/without polyinosinic-polycytidylic acid (poly(I:C)) from week 6 (A6 mice) or week 12 (A12 mice) after birth. Airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF), cell counts, histology, and Spp1 expression were assessed. The levels of OPN, transforming growth factor β1 (TGF-β1), chitinase 3-like 1 (CH3L1), and interleukin (IL) 5 were measured by ELISA. The expression of Smad3 phosphorylation and tissue transglutaminase 2 (TGM2) was evaluated by Western blot. The serum OPN levels were significantly higher in asthma patients than in HCs and in LOA patients than in those with EOA (P < 0.05) and were positively correlated with serum TGF-β1 and CH3L1 (r = 0.174, r = 0.264; P < 0.05). A12 mice showed elevated AHR with increased levels of OPN/TGF-β1/IL-5 in BALF and Spp1 compared to A6 mice. Poly(I:C) induced remarkable TGF-β1, CH3L1, Th2 cytokine, and OPN levels in BALF and the expression of phosphorylated Smad3, TGM2, and Spp1 in the lungs. OPN triggered TGF-β1/Smad3 signaling in the lungs, which was suppressed by dexamethasone and anti-IL5 antibody. In conclusion, aging and exposure to viral infections may induce OPN release and consequently modulate inflammation and TGF-β1/Smad3-related remodeling, contributing to the development of LOA.

Innate immunity is a late event in the onset of gliadin-specific enteropathy in the HLA-DQ8 mice

Celiac disease (CD) is a food enteropathy that occurs in genetically susceptible individuals following the ingestion of gluten. Both gluten cytotoxicity and immunity activation play a role in CD pathogenesis; however, the chronological assessment of the different pathogenic mechanisms remains elusive. The models developed so far have only partially addressed this issue. Herein, Ab°DQ8 transgenic mice were administered wheat gliadin and indomethacin for 10 days to induce enteropathy. Gliadin-induced alteration of the small intestinal architecture was associated with increased expression of tissue transglutaminase in the lamina propria and a marked hypoxic environment. Enteropathic mice showed activation of innate immunity, featuring an increase of pro-inflammatory IFN-γ and IL-15 mRNAs, as well as CD11c+CD103+, CD11b+CD11c+, and CD11b+CD103+ dendritic cell subsets. However, the temporal assessment of examined parameters indicated that the induction of innate immunity during the generation of the mucosal lesion, occurred belatedly, highlighting a major role of gliadin intrinsic cytotoxicity in the pathogenic mechanism of this model. These results have important implications for the use of this model to test the impact of biotechnological interventions to reduce the cytotoxicity of gliadin.

Gut antibody deficiency in CD19−/− mice results in a spontaneous Celiac-Like sprue

Abstract Primary immunodeficiencies are heritable disorders of the immune response that most commonly manifest as an inability to generate immunoglobulin A (IgA). Despite its prevalence, IgA deficiency is considered a benign form of antibody deficiency due to a lack of easily observed clinical symptoms. CD19 is an important co-receptor that facilitates B cell receptor signaling and B cell development into antibody-secreting plasma cells. CD19 deficiency is a known genetic risk factor for the development of common variable immunodeficiency (CVID). Here, we utilize the CD19−/− mouse model to quantify the role of IgA deficiency on gut homeostasis. Our results indicate that CD19−/− mice develop a severe gut IgA deficiency associated with aberrant anti-commensal IgA responses that result in the outgrowth of anaerobic bacteria in the gut. More importantly, anaerobic expansion results in intestinal malabsorption under steady-state conditions. Intestinal malabsorption is associated with defects in normal hepatobiliary circulation, vitamin deficiency, and dyslipidemia. Additionally, results from histological analysis and RNAseq experiments indicate that CD19−/− mice develop intestinal epithelial cell (IEL) lymphocytosis mediated by CD8aa+TCRgd lymphocytes. Finally, CD19−/− mice, while deficient in anti-gliadin antibody (AGA) production compared to WT mice, demonstrate a significantly stronger relationship between serum AGA titers and total serum IgG and IgA production compared to WT animals and have significantly elevated ileal expression of tissue transglutaminase 2. Collectively, we believe that we have identified a novel mouse model of sero-negative celiac disease that will be of great value to the celiac research community.

Biological Activity of Olive Leaf Extract and Regulation of Tissue Transglutaminase Expression in Diabetic Wound Healing

Biological Activity of Olive Leaf Extract and Regulation of Tissue Transglutaminase Expression in Diabetic Wound Healing

Alternative RNA splicing of leucocyte tissue transglutaminase in coeliac disease

Tissue transglutaminase is a ubiquitous and multifunctional protein that contributes to several processes such as apoptosis/survival, efferocytosis, inflammation and tissue repairing under physiological and pathological conditions. Several activities can be associated with well-established functional domains; in addition, four RNA alternative splice variants have been described, characterized by sequence divergences and residues deletion at the C-terminal domains. Tissue transglutaminase is recognized as the central player in the physiopathology of coeliac disease (CD) mainly through calcium-dependent enzymatic activities. It can be hypothesized that differential regulation of tissue transglutaminase splice variants expression in persons with CD contributes to pathology by altering the protein functionality. We characterized the expression pattern of RNA alternative splice variants by RT-PCR in peripheral cells from patients with CD under free gluten diet adhesion; we considered inflammatory parameters and specific antibodies as markers of the stage of disease. We found significant higher expression of both the full length and the shortest C-truncated splice variants in leucocytes from patients with CD in comparison with healthy individuals. As tissue transglutaminase expression and canonical enzymatic activity are linked to inflammation, we studied the RNA expression of inflammatory cytokines in peripheral leucocytes of persons with CD in relation with splice variants expression; interestingly, we found that recently diagnosed patients showed significant correlation between both the full length and the shortest alternative spliced variants with IL-1 expression. Our results points that regulation of alternative splicing of tissue transglutaminase could account for the complex physiopathology of CD.

Herba Artemisiae Capillaris Extract Prevents the Development of Streptozotocin-Induced Diabetic Nephropathy of Rat.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease throughout the world; until now there is no specific drug available. In this work, we use herba artemisiae capillaris extract (HACE) to alleviate renal fibrosis characterized by the excessive accumulation of extracellular matrix (ECM) in rats, aiming to investigate the protective effect of the HACE on DN. We found that the intragastric treatment of high-dose HACE could reverse the effect of streptozotocin not only to decrease the level of blood glucose and blood lipid in different degree but also further to improve renal functions. It is worth mentioning that the effect of HACE treatment was comparable to the positive drug benazepril. Moreover, we found that HACE treatment could on one hand inhibit oxidative stress in DN rats through regulating enzymatic activity for scavenging reactive oxygen species and on the other hand increase the ECM degradation through regulating the activity of metalloproteinase-2 (MMP-2) and the expression of tissue transglutaminase (tTG), which explained why HACE treatment inhibited ECM accumulation. On the basis of above experimental results, we conclude that HACE prevents DN development in a streptozotocin-induced DN rat model, and HACE is a promising candidate to cure DN in clinic.

TMIC-07. HIGH ALDEHYDE DEHYDROGENASE ACTIVITY IDENTIFIES BRAIN METASTASIS-INITIATING CELLS IN BREAST CANCER

Brain metastases (BM) from breast cancer (BC) are associated with significant morbidity and mortality. However, the mechanism of how BC cells initiate and propagate BM tumors remains elusive. In this study, we established a set of patient-derived models of circulating tumor cells (CTCs) from either hormone receptor positive (HR+) or negative (HR-) cells, and subsequent BM cells in vivo. Our brain-primed BC derivative lines exhibited a trend to form metastatic tumors in the brain more frequently than in the bones or lungs. In contrast, the naive BC cells rarely formed BM tumors. Micro-MRI of mouse brains after cardiac injection of brain-primed BC derivatives detected the persistent attachment and subsequent colonization of these circuiting cells, whereas naive BC cells, when injected into arterial circulation, were quickly eradicated from the brain. Transcriptomic expression analysis showed that ALDH isoforms including ALDH1A3 are highly upregulated in BM cells in contrast to BC cells. Silencing of ALDH1A3 by lentiviral shRNA infection specifically attenuated in vivo BM formation from the BC derivatives through arterial circulation, while it did not affect formation of the metastases in the lungs and bones. RNA sequencing detected that the expression of tissue transglutaminase (TG2), constitutive activator of transcription factor-kB (NF-kB), is higher in BM but not in lung and bone metastasis and its expression was attenuated by reducing ALDH1A3. Clinically, matched pairs of human primary BC and BM tissue samples showed the elevation of ALDH1A3 expression in BM tumors. In addition, ALDH1A3 expression was significantly correlated with poor prognosis. To develop therapeutics for ALDH1A3, we designed and synthesized a small molecule ALDH1A3 inhibitor, termed GA11. Systemic treatment of the CTC-BM mouse models with GA11 yielded a prominent inhibitory effect on BM formation. Taken together, our results suggest that ALDH1A3 might be a promising therapeutic target for BM tumors.

Tissue transglutaminase expression is necessary for adhesion, metastatic potential and cancer stemness of renal cell carcinoma

ABSTRACTTissue transglutaminase (TG2) is the ubiquitously expressed member of transglutaminase family and shown to play a critical role in the development and progression of drug resistance malignancies. We have previously showed the association of TG2 upregulation with progression and metastasis of renal cell carcinoma (RCC) and low disease-free survival. In the present study we further investigate the role of TG2 in cell adhesion, migration and invasion of RCC by silencing TG2 expression in Caki-2 and A-498 primary site and Caki-1 and ACHN metastatic site RCC cell lines. Downregulation of TG2 expression led up to a 60% decrease in actin stress fiber formation and adhesion to β 1 integrin (ITGB1) substrates fibronectin, collagen type I and laminin in both primary and metastatic site RCC cell lines. In addition, treatment with siRNAs against TG2 impaired the migration capacity and cellular invasiveness of ITGB1 substrates in all 4 RCC cell lines. Lastly, the knockdown of TG2 in metastatic Caki-1 cells diminished the expression of CD44, CD73-and CD105 cancer stem cell-like markers. We conclude, for the first time, that TG2 expression is critical for cancer cell adhesion, migration, invasiveness and cancer cell-stemness during RCC progression and dissemination. Therefore, combined targeting of TG2 with drugs widely used in the treatment of RCC may be a promising therapeutic strategy for RCC.

Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells.

The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK) 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA). However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs), cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs) to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG) but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE) or myeloperoxidase (MPO) attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC), nonsevere asthma (NSA), and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1gfp/gfp mice

Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1gfp/gfp mice during EAE, visualizing CX3CR1-GFP+ monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP+ monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP+ monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology.

Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System.

Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system.

Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System.

Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system.

May the assessment of baseline mucosal molecular pattern predict the development of gluten related disorders among microscopic enteritis?

AIMTo evaluate mucosal baseline mRNA expression of tissue transglutaminase 2 (tTG2), interferon gamma (IFNγ), toll-like receptor 2 (TLR2) and Myeloid Differentiation factor 88 (MyD88) in patients with microscopic enteritis (ME).METHODSWe retrospectively enrolled 89 patients with ME of different etiology, which was defined within a 2-year mean period of follow-up. Baseline histological examination was performed on Hematoxylin-Eosin stained sections and CD3 lymphocyte immunohistochemistry was used for intraepithelial lymphocyte count (IELs). ME was defined according to the criteria of Bucharest Consensus Conference. For each patient, formalin embedded biopsy samples of the duodenum referred to the period of ME diagnosis were retrieved. Real-time polymerase chain reaction (RT-PCR) was used to detect the amount of mRNA coding for tTG2, IFNγ, TLR2 and MyD88, and the quantity was expressed as fold change compared to controls. Control group was represented by duodenal normal specimens from 15 healthy subjects undergoing endoscopy for functional symptoms. Comparisons among continuous variables were performed by One way analysis of variance (ANOVA) and Bonferroni’s test. The χ2 test was used for categorical variables. Pearson’s test was used to evaluate correlations. Receiver operating curves were drawn for all four markers to estimate sensitivity and specificity in discriminating the development of CD and GS.RESULTSAfter a period of follow up of 21.7 ± 11.7 mo, the following diagnoses were achieved: gluten related disorders in 48 subjects (31 CD; 17 GS) and non-gluten related ones in 41 (29 Irritable Bowel Syndrome - IBS; 12 Others). CD patients had the highest tTG2 levels (8.3 ± 4.5). The ANOVA plus Bonferroni analysis showed that CD > Other ME > GS = IBS > negative controls. A cut off value of 2.258 was able to discriminate between CD and GS with a sensitivity of 52.94% and a specificity of 87.1%. Additionally, CD patients had the highest IFNγ levels (8.5 ± 4.1). ANOVA plus Bonferroni demonstrated CD > Other ME > GS = IBS > negative controls. A cut off of 1.853 was able to differentiate CD and GS with a sensitivity of 47.06% and a specificity of 96.77%. Patients with non gluten-related causes of ME exhibited the highest TLR2 levels (6.1 ± 1.9) as follows: Other ME > CD = GS = IBS > negative controls. TLR2 was unable to discriminate CD from GS. Patients with CD overexpressed MyD88 levels similarly to non gluten-related causes of DL (7.8 ± 4.9 and 6.7 ± 2.9), thus CD = Other ME > GS = IBS > negative controls. A cut off of 3.722 was able to differentiate CD from GS with a sensitivity of 52.94% and a specificity of 74.19%. IELs count (15-25 and more than 25/100 enterocytes) strongly correlated with mRNA levels of all tested molecules (P < 0.0001).CONCLUSIONOur results confirm that a single marker is unable to predict a discrimination among ME underlying conditions as well as between CD and GS. Mucosal high levels of tTG and IFNγ mRNA may predict the development of CD more than GS with high specificity, despite an expected low sensitivity. TLR2 does not discriminate the development of CD from GS. MyD88 levels indicate that intestinal permeability is more increased when a severe intestinal damage underlies ME in both gluten related and unrelated conditions. Therefore, the results of the present paper do not seem to show a clear translational value.

Tissue transglutaminase expression during neural differentiation of human mesenchymal stem cells.

Herein, we assessed the expression of transglutaminase-2 (TG2) during in vitro neural differentiation of human Mesenchymal Stem Cells (hMSCs) induced by some Growth Factors (GFs). The amount and distribution of mRNAs for some transglutaminase (TGs) isoforms (TG1, TG2, TG3, TG5) were also evaluated. In addition, we tested some neural lineage markers (Glial Acid Fibrillary Protein, GFAP; Neurofilament Protein, NF; Nestin; β-Tubulin III). A progressive increase of all neural markers during GFs neural differentiation of hMSCs was found. A significant overexpression of TG1 and TG2 mRNA was observed in undifferentiated hMSCs. GFs-treatment induced a progressive decrease of TG1 mRNA during hMSCs differentiation. A significant down-regulation of TG2 mRNA in 2 days in vitro (DIV) GFs-treated cells was observed. TG2 mRNA progressively increased during GFs hMSCs differentiation, reaching after 6 DIV of GFs-treatment of similar values to those observed in undifferentiated cells. TG3 mRNA levels were express at very low only in undifferentiated hMSCs, whereas TG5 mRNA was undetectable in undifferentiated and differentiated hMSCs. Immunocytochemical and Western blot analysis showed an overexpression of TG2 in undifferentiated hMSCs. A dramatic reduction of the protein levels in 2 DIV GFs-treated cells was observed. Its expression levels progressively increased during the differentiation of hMSCs with GFs, reaching after 6 DIV of the treatment similar values to those observed in undifferentiated cells. Our findings demonstrate the presence of multiple TGs in hMSCs during neural differentiation in vitro induced by GFs, and suggest that TG2 may be part of the downstream events associated to neural differentiation.

Expression of Tissue Transglutaminase in Human Thyroid Cancer Cell Lines: Effect of Novel Enantiopure Triazole Derivatives

A synthesis of (-)5b and (-)6d according to enzymatic resolution of racemic triazole derivatives 5b and 6d was performed with Lipozyme in 1,4-dioxane, using vinyl benzoate as the acyl donor. The effect of the compounds on TG2 overexpression in human thyroid cancer cell lines cultures at two degrees of malignancyand invasiveness was assessed. The involvement of TG2 in the activation of the apoptotic pathway is also shown. We found that the obtained compounds are able to reduce TG2 overexpression in follicular (FTC-133) and anaplastic (8305C) human thyroid cancer cell lines either in the cytosol, or in the nuclear compartment, activating also the apoptotic pathway. Our data suggest that they may represent new anticancer agents for human thyroid cancer.

Elongation factor-2 kinase regulates TG2/β1 integrin/Src/uPAR pathway and epithelial-mesenchymal transition mediating pancreatic cancer cells invasion.

Pancreatic ductal adenocarcinoma is one of the lethal cancers with extensive local tumour invasion, metastasis, early systemic dissemination and poorest prognosis. Thus, understanding the mechanisms regulating invasion/metastasis and epithelial–mesenchymal transition (EMT), is the key for developing effective therapeutic strategies for pancreatic cancer (PaCa). Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase that we found to be highly up-regulated in PaCa cells. However, its role in PaCa invasion/progression remains unknown. Here, we investigated the role of eEF-2K in cellular invasion, and we found that down-regulation of eEF-2K, by siRNA or rottlerin, displays impairment of PaCa cells invasion/migration, with significant decreases in the expression of tissue transglutaminase (TG2), the multifunctional enzyme implicated in regulation of cell attachment, motility and survival. These events were associated with reductions in β1 integrin/uPAR/MMP-2 expressions as well as decrease in Src activity. Furthermore, inhibition of eEF-2K/TG2 axis suppresses the EMT, as demonstrated by the modulation of the zinc finger transcription factors, ZEB1/Snail, and the tight junction proteins, claudins. Importantly, while eEF-2K silencing recapitulates the rottlerin-induced inhibition of invasion and correlated events, eEF-2K overexpression, by lentivirus-based expression system, suppresses such rottlerin effects and potentiates PaCa cells invasion/migration capability. Collectively, our results show, for the first time, that eEF-2K is involved in regulation of the invasive phenotype of PaCa cells through promoting a new signalling pathway, which is mediated by TG2/β1 integrin/Src/uPAR/MMP-2, and the induction of EMT biomarkers which enhance cancer cell motility and metastatic potential. Thus, eEF-2K could represent a novel potential therapeutic target in pancreatic cancer.