Extracellular matrix modulates mesangial cell apoptosis and mRNA expression of cathepsin-B and tissue transglutaminase

Mesangial matrix is a dynamic structure which modulates mesangial cell function. Since accumulation of matrix precedes the development of focal glomerulosclerosis, we studied the effect of different matrices on mesangial cell (MC) apoptosis. Suspended mesangial cells became apoptotic in a time dependent manner. Collagen type III did not modulate MC apoptosis when compared to cells grown on plastic. MCs grown on Matrigel, collagen type I and IV showed an increased number of apoptotic cells when compared to MCs grown on plastic. DNA end-labeling further confirmed these observations. MCs grown on Matrigel showed enhanced (P < 0.05) mRNA expression for tissue transglutaminase (TTG) and cathepsin-B. Mesangial cells grown on Matrigel also showed enhanced expression of superoxide dismutase (SOD). We conclude that mesangial cells require attachment to the matrix for their survival and alteration of the quality of matrix modulates mesangial cell apoptosis. J. Cell. Biochem. 68:22–30, 1998. © 1998 Wiley-Liss, Inc.     

Tissue transglutaminase modulates alpha-synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated α-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant α-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective α-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/α-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all α-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal α-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal β-sheet-containing oligomers, the tTG/α-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant α-synuclein variants. We propose that tTG cross-linking imposes structural constraints on α-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.     

Tissue transglutaminase in mesenchymal tumour cells

During our search for novel transformation-sensitive proteins whose synthesis is abolished in tumour cells we found a cDNA clone coding for tissue transglutaminase. This enzyme was identified, at the protein as well as the mRNA level, in normal human fibroblasts, but was completely missing in their matched SV40 transformed counterparts. Since tissue transglutaminase has been implicated in cell cycle regulation and apoptosis, we investigated the possibility of whether this enzyme might represent a negative marker for tumour cells. We found that its synthesis varied largely among 10 cell lines derived from spontaneous mesenchymal tumours. While cells from a rhabdomyosarcoma and a chondrosarcoma did not produce it at all, an extremely high expression was observed in cells from an osteosarcoma and a liposarcoma. Thus, tissue transglutaminase is not a tumour-related marker. This study was supported by grants from the ETH Zurich (0-20-854-94) and from the Swiss National Science Foundation (31-40337.94).     

Tissue transglutaminase mRNA expression in apoptotic cell death

Introduction: Tissue transglutaminase (t.TG) is an enzyme that catalyzes the cross-linking of intracellular proteins, thus assembling a protein scaffold that prevents leakage of intracellular components. t.TG is activated during the apoptotic cell death cascade and plays a key role in the formation of apoptotic bodies. The aim of this study was to determine to what amount t.TG-mRNA becomes expressed during apoptosis and whether the t.TG-mRNA expression level could be used as trace marker of recent apoptosis and in individual cases for quantification of apoptosis. Methods: Expression of t.TG-mRNA was determined using TaqMan based, real-time RT-PCR, a semi-quantitative RT-PCR technique. The t.TG-mRNA expression was measured in cultured cells (MCF-7, human endothelial cells) and in peripheral blood mononuclear cells (PBMCs) before and after induction of apoptosis in vitro. Results: The TaqMan RT-PCR of t.TG proved to be reliable, reproducible (CV's inter and intraassay precisions of 0.8–2.8%, measured at two levels), and specific for apoptotic cell death. t.TG-mRNA expression increases in response to apoptosis induction and is not expressed during the process of necrotic cell death. The expression during apoptotic cell death changes in the dose dependent manner in cultured cells as well as in the PBMCs, treated in vitro. The increase t.TG-mRNA expression level was up to 20 times, depending on the intensity of the apoptosis induction treatment and incubation time afterwards. PBMCs of patients with myelodysplasia showed spontaneous expression of t.TG-mRNA in agreement with their increased apoptotic cell death in vivo. Conclusion: t.TG-mRNA expression increases significantly in response to apoptosis inducing treatment. The observed changes are dose and time dependent. This leads to the conclusion that t.TG expression can be used as a trace marker for detection and quantification of apoptosis.     

Type 2 transglutaminase differentially modulates striatal cell death in the presence of wild type or mutant huntingtin

Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt), is characterized by extensive loss of striatal neurons. The dysregulation of type 2 transglutaminase (TG2) has been proposed to contribute to the pathogenesis in HD as TG2 is up-regulated in HD brain and knocking out TG2 in mouse models of HD ameliorates the disease process. To understand the role of TG2 in the pathogenesis of HD, immortalized striatal cells established from mice in which mutant htt with a polyglutamine stretch of 111 Gln had been knocked-in and wild type (WT) littermates, were stably transfected with human TG2 in a tetracycline inducible vector. Overexpression of TG2 in the WT striatal cells resulted in significantly greater cell death under basal conditions as well as in response to thapsigargin treatment, which causes increased intracellular calcium concentrations. Furthermore, in WT striatal cells TG2 overexpression potentiated mitochondrial membrane depolarization, intracellular reactive oxygen species production, and apoptotic cell death in response to thapsigargin. In contrast, in mutant striatal cells, TG2 overexpression did not increase cell death, nor did it potentiate thapsigargin-induced mitochondrial membrane depolarization or intracellular reactive oxygen species production. Instead, TG2 overexpression in mutant striatal cells attenuated the thapsigargin-activated apoptosis. When in situ transglutaminase activity was quantitatively analyzed in these cell lines, we found that in response to thapsigargin treatment TG2 was activated in WT, but not mutant striatal cells. These data suggest that mutant htt alters the activation of TG2 in response to certain stimuli and therefore differentially modulates how TG2 contributes to cell death processes.     

Tissue transglutaminase expression in quail epiphyseal chondrocytes.

Tissue transglutaminase (tTGase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification via epsilon(gamma-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme.tTGase activity is present in primary cultures of epiphyseal chondrocytes and increases transiently upon retinoic acid (RA) treatment. Increase in enzyme activity occurs upon RA addition and is accompanied by a parallel increase in protein and mRNA levels. Stimulation of tTGase expression by RA correlates with suppression of cell growth and occurs independently of cell adhesion and cell differentiation.tTGase expression is not observed in MC2, a permanent chondrocyte cell line derived from retrovirus infected chondrocytes. RA treatment fails to activate tTGase expression in MC2 cells and to completely suppress cell proliferation.Our findings lend support to the idea that tTGase might play a role in non-dividing cultured chondrocytes.     

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.     

Tissue transglutaminase overexpression in the brain potentiates calcium-induced hippocampal damage

Tissue transglutaminase (tTG) post-translationally modifies proteins in a calcium-dependent manner by incorporation of polyamines, deamination or crosslinking. Moreover, tTG can also bind and hydrolyze GTP. tTG is the major transglutaminase in the mammalian nervous system, localizing predominantly in neurons. Although tTG has been clearly demonstrated to be elevated in neurodegenerative diseases and in response to acute CNS injury, its role in these pathogenic processes remains unclear. Transgenic mice that overexpress human tTG (htTG) primarily in CNS neurons were generated to explore the role of tTG in the nervous system and its contribution to neuropathological processes. tTG transgenic mice were phenotypically normal and were born with the expected Mendelian frequency. However, when challenged systemically with kainic acid, tTG transgenic mice, in comparison to wild-type (WT) mice, developed more extensive hippocampal neuronal damage. This was evidenced by a decreased number of healthy neurons, and increased terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) labeling as an indicator of neuronal cell death in the kainic acid-treated transgenic mice. Moreover, the duration and severity of seizures developed by htTG transgenics in response to kainic acid administration were significantly more pronounced than those observed in WT mice. These data indicate for the first time that tTG may play an active role in excitatory amino acid-induced neuronal cell death, which has been postulated to be an important component of acute CNS injury and chronic CNS neurodegenerative conditions.     

组织型转谷氨酰胺酶与肾脏纤维化

组织型转谷氨酰胺酶(tTG)是一个Ca2 依赖的具有转酰胺基作用的酶,它分布广泛,在许多生理和病理条件下发挥重要作用。近年来它参与组织纤维化的作用逐渐引起重视。tTG分泌到细胞外能够使很多细胞外基质蛋白成分之间发生交联,形成牢固结构,抵抗降解,从而促使细胞外基质沉积,促进组织纤维化发展。本文简要叙述tTG的分子特征和生理及病理学意义,并着重介绍tTG和肾脏纤维化的联系。     

Increase in extracellular cross-linking by tissue transglutaminase and reduction in expression of MMP-9 contribute differentially to focal segmental glomerulosclerosis in rats

Tissue transglutaminase (tTG) is a Ca²⁺-dependent enzyme which stabilizes the extracellular matrix (ECM) through post-translational modification, and may play an important role in the pathogenesis of focal and segmental glomerulosclerosis (FSGS). Here, we have investigated whether tTG contributes to the glomerular ECM expansion in the puromycin aminonucleoside (PAN)-injection-induced experimental rat model of FSGS. The localization and expression of tTG, MMP-9 gelatinase, and the ECM component fibronectin (FN) in kidneys was determined by immunohistochemistry and measured by semi-quantitative analysis. Protein levels of tTG and MMP-9 were also analyzed by Western blotting.In situtransglutaminase activity was assayed by measurement of incorporated substrate and the immunofluorescence staining for the cross-linking product, ε-(γ-glutamyl) lysine. Prominent proteinuria, a typical pathological feature of FSGS, was observed in PAN injection group rats. tTG immunoreactivity was located markedly in glomeruli and the levels of this protein in whole-kidney homogenates of PAN injection group rats were significantly increased (361± 106% control, P< 0.05). Similarly, transglutaminase activity and ε-(γ-glutamyl) lysine were also predominately located within glomeruli and were much more intense in the PAN-injected group than that in control animals. MMP-9 was also located primarily within glomeruli. In PAN-injected kidneys, protein levels of active MMP-9 were significantly reduced (59± 27% control, P< 0.01), while pro-MMP-9 levels increased (148± 42% control, P< 0.05). Remarkable expression of glomerular fibronectin (FN) was found in PAN injection group rats. Semi-quantitative analysis demonstrated this increased intensity of FN staining in the PAN-injected rats was 149± 23% of the control values (P< 0.05). Enhanced cross-linking of ECM by tissue transglutaminase and decreased degradation due to reduced active MMP-9 expression may be at least partially responsible for the deposition of FN within injured glomeruli in experimental FSGS.     

Tissue transglutaminase: An emerging target for therapy and imaging

Tissue transglutaminase (transglutaminase 2) is a multifunctional enzyme with many interesting properties resulting in versatile roles in both physiology and pathophysiology. Herein, the particular involvement of the enzyme in human diseases will be outlined with special emphasis on its role in cancer and in tissue interactions with biomaterials. Despite recent progress in unraveling the different cellular functions of transglutaminase 2, several questions remain. Transglutaminase 2 features in both confirmed and some still ambiguous roles within pathological conditions, raising interest in developing inhibitors and imaging probes which target this enzyme. One important prerequisite for identifying and characterizing such molecular tools are reliable assay methods to measure the enzymatic activity. This digest Letter will provide clarification about the various assay methods described to date, accompanied by a discussion of recent progress in the development of inhibitors and imaging probes targeting transglutaminase 2.     

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

Tissue 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.     

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

The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr¹²¹⁴ and its downstream effectors Akt and ERK1/2, and importantly its association with β1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity.     

Mucosal tissue transglutaminase expression in celiac disease

Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that ( i ) tTG is equally expressed in CD at different stages of disease; ( ii ) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD.     

Expression in Escherichia coli and purification of hexahistidine-tagged human tissue transglutaminase

Recent evidence suggests that aberrant transglutaminase activity is associated with a wide variety of diseases. Tissue transglutaminase is the most widely distributed of the six well-characterized transglutaminases in humans. We describe a method for expressing hexahistidine-tagged human tissue transglutaminase in Escherichia coli BL21(DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with CelLytic B Bacterial Cell Lysis/Extraction Reagent was accomplished by immobilized metal (Ni2+) affinity column chromatography. The procedure typically yields highly purified and highly active recombinant human tissue transglutaminase in about 1 day (about 0.6 mg/from a 1-liter culture).     

Aging differentially alters the expression of angiogenic genes in a tissue-dependent manner

Organ functions are altered and impaired during aging, thereby resulting in increased morbidity of age-related diseases such as Alzheimer’s disease, diabetes, and heart failure in the elderly. Angiogenesis plays a crucial role in the maintenance of tissue homeostasis, and aging is known to reduce the angiogenic capacity in many tissues. Here, we report the differential effects of aging on the expression of angiogenic factors in different tissues, representing a potentially causes for age-related metabolic disorders. PCR-array analysis revealed that many of angiogenic genes were down-regulated in the white adipose tissue (WAT) of aged mice, whereas they were largely up-regulated in the skeletal muscle (SM) of aged mice compared to that in young mice. Consistently, blood vessel density was substantially reduced and hypoxia was exacerbated in WAT of aged mice compared to that in young mice. In contrast, blood vessel density in SM of aged mice was well preserved and was not different from that in young mice. Moreover, we identified that endoplasmic reticulum (ER) stress was strongly induced in both WAT and SM during aging in vivo. We also found that ER stress significantly reduced the expression of angiogenic genes in 3T3-L1 adipocytes, whereas it increased their expression in C2C12 myotubes in vitro. These results collectively indicate that aging differentially affects the expression of angiogenic genes in different tissues, and that aging-associated down-regulation of angiogenic genes in WAT, at least in part through ER stress, is potentially involved in the age-related adipose tissue dysfunction.     

Tissue transglutaminase activation modulates inflammation in cystic fibrosis via PPARgamma down-regulation

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.     

Tissue transglutaminase and the stress response

Summary. The expression of the protein crosslinking enzyme tissue transglutaminase (TG2, tTG), the ubiquitous member of transglutaminase family, can be regulated by multiple factors. Although it has been suggested that TG2 can be involved in apoptotic cell death, high levels of enzyme have also been associated with cell survival in response to different stimuli. Furthermore, evidence indicates that increases in TG2 production cause enzyme translocation to cell membrane. Cell stress can also lead to TG2 accumulation on the cell surface and in the extracellular matrix resulting in changes in cell-matrix interactions. Here, we discuss the underlying mechanisms of TG2 up-regulation induced by various stimuli including glutamate exposure, calcium influx, oxidative stress, UV, and inflammatory cytokines. These findings agree with a postulated role for transglutaminases in molecular mechanisms involved in several diseases suggesting that cross-linking reactions could be a relevant part of the biochemical changes observed in pathological conditions.     

Tissue transglutaminase acylation: Proposed role of conserved active site Tyr and Trp residues revealed by molecular modeling of peptide substrate binding

Transglutaminases (TGases) catalyze the cross-linking of peptides and proteins by the formation of gamma-glutamyl-epsilon-lysyl bonds. Given the implication of tissue TGase in various physiological disorders, development of specific tissue TGase inhibitors is of current interest. To aid in the design of peptide-based inhibitors, a better understanding of the mode of binding of model peptide substrates to the enzyme is required. Using a combined kinetic/molecular modeling approach, we have generated a model for the binding of small acyl-donor peptide substrates to tissue TGase from red sea bream. Kinetic analysis of various N-terminally derivatized Gln-Xaa peptides has demonstrated that many CBz-Gln-Xaa peptides are typical in vitro substrates with K(M) values between 1.9 mM and 9.4 mM, whereas Boc-Gln-Gly is not a substrate, demonstrating the importance of the CBz group for recognition. Our binding model of CBz-Gln-Gly on tissue TGase has allowed us to propose the following steps in the acylation of tissue TGase. First, the active site is opened by displacement of conserved W329. Second, the substrate Gln side chain enters the active site and is stabilized by hydrophobic interaction with conserved residue W236. Third, a hydrogen bond network is formed between the substrate Gln side chain and conserved residues Y515 and the acid-base catalyst H332 that helps to orient and activate the gamma-carboxamide group for nucleophilic attack by the catalytic sulphur atom. Finally, an H-bond with Y515 stabilizes the oxyanion formed during the reaction.