Transglutaminase regulation of cell function.

Transglutaminases (TGs) are multifunctional proteins having enzymatic and scaffolding functions that participate in regulation of cell fate in a wide range of cellular systems and are implicated to have roles in development of disease. This review highlights the mechanism of action of these proteins with respect to their structure, impact on cell differentiation and survival, role in cancer development and progression, and function in signal transduction. We also discuss the mechanisms whereby TG level is controlled and how TGs control downstream targets. The studies described herein begin to clarify the physiological roles of TGs in both normal biology and disease states.

Wnt16 attenuates TGFß-induced chondrogenic transformation in vascular smooth muscle.

OBJECTIVE: Phenotypic plasticity of vascular smooth muscle cells (VSMCs) contributes to cardiovascular disease. Chondrocyte-like transformation of VSMCs associates with vascular calcification and underlies the formation of aortic cartilaginous metaplasia induced in mice by genetic loss of matrix Gla protein (MGP). Previous microarray analysis identified a dramatic downregulation of Wnt16 in calcified MGP-null aortae, suggesting an antagonistic role for Wnt16 in the chondrogenic transformation of VSMCs. APPROACH AND RESULTS: Wnt16 is significantly downregulated in MGP-null aortae, before the histological appearance of cartilaginous metaplasia, and in primary MGP-null VSMCs. In contrast, intrinsic TGFß is activated in MGP-null VSMCs and is necessary for spontaneous chondrogenesis of these cells in high-density micromass cultures. TGFß3-induced chondrogenic transformation in wild-type VSMCs associates with Smad2/3-dependent Wnt16 downregulation, but Wnt16 does not suppress TGFß3-induced Smad activation. In addition, TGFß3 inhibits Notch signaling in wild-type VSMCs, and this pathway is downregulated in MGP-null aortae. Exogenous Wnt16 stimulates Notch activity and attenuates TGFß3-induced downregulation of Notch in wild-type VSMCs, prevents chondrogenesis in MGP-null and TGFß3-treated wild-type VSMCs, and stabilizes expression of contractile markers of differentiated VSMCs. CONCLUSIONS: We describe a novel TGFß-Wnt16-Notch signaling conduit in the chondrocyte-like transformation of VSMCs and identify endogenous TGFß activity in MGP-null VSMCs as a critical mediator of chondrogenesis. Our proposed model suggests that the activated TGFß pathway inhibits expression of Wnt16, which is a positive regulator of Notch signaling and a stabilizer of VSMC phenotype. These data advance the comprehensive mechanistic understanding of VSMC transformation and may identify a novel potential therapeutic target in vascular calcification.

Effects of dietary quercetin on female fertility in mice: implication of transglutaminase 2.

Use of the dietary supplement quercetin is on the rise. Because previous studies imply an inhibitory effect of quercetin on male fertility, we explored the effects of this flavonoid on fertility in female mice. Birth outcomes, and ovarian morphology in 4-week-old offspring, were assessed in mice receiving dietary quercetin (5mgkg-1day-1) for 9 months during two breeding periods: from 2 to 6 months (prime reproductive age) and 8 to11 months of age. Quercetin increased birth spacing, leading to a 60% reduction in the number of litters, but enhanced folliculogenesis in ovaries of female offspring. While in young females quercetin caused an almost 70% increase in litter size, in older animals this effect was reversed. Consistent with the inhibitory activity of quercetin on the enzyme transglutaminase 2 (TG2), genetic ablation of TG2 in mice mirrors the effects of quercetin on birth outcomes and follicular development. Further, TG2-null mice lack responsiveness to quercetin ingestion. Our study shows for the first time that dietary quercetin can cause reduced reproductive potential in female mice and implies that TG2 may regulate ovarian ageing.

Quercetin attenuates warfarin-induced vascular calcification in vitro independently from matrix Gla protein.

Warfarin can stimulate vascular calcification in vitro via activation of ß-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, ß-catenin activity is important because inhibition of ß-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of ß-catenin using the glycogen synthase kinase-3ß (GSK-3ß) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the ß-catenin pathway. Calcification in VSMCs induced by 10 µm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new ß-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.

Two sides of MGP null arterial disease: chondrogenic lesions dependent on transglutaminase 2 and elastin fragmentation associated with induction of adipsin.

Mutations in matrix Gla protein (MGP) have been correlated with vascular calcification. In the mouse model, MGP null vascular disease presents as calcifying cartilaginous lesions and mineral deposition along elastin lamellae (elastocalcinosis). Here we examined the mechanisms underlying both of these manifestations. Genetic ablation of enzyme transglutaminase 2 (TG2) in Mgp(-/-) mice dramatically reduced the size of cartilaginous lesions in the aortic media, attenuated calcium accrual more than 2-fold, and doubled longevity as compared with control Mgp(-/-) animals. Nonetheless, the Mgp(-/-);Tgm2(-/-) mice still died prematurely as compared with wild-type and retained the elastocalcinosis phenotype. This pathology in Mgp(-/-) animals was developmentally preceded by extensive fragmentation of elastic lamellae and associated with elevated serine elastase activity in aortic tissue and vascular smooth muscle cells. Systematic gene expression analysis followed by an immunoprecipitation study identified adipsin as the major elastase that is induced in the Mgp(-/-) vascular smooth muscle even in the TG2 null background. These results reveal a central role for TG2 in chondrogenic transformation of vascular smooth muscle and implicate adipsin in elastin fragmentation and ensuing elastocalcinosis. The importance of elastin calcification in MGP null vascular disease is highlighted by significant residual vascular calcification and mortality in Mgp(-/-);Tgm2(-/-) mice with reduced cartilaginous lesions. Our studies identify two potential therapeutic targets in vascular calcification associated with MGP dysfunction and emphasize the need for a comprehensive approach to this multifaceted disorder.

Map of open and closed chromatin domains in Drosophila genome.

BACKGROUND: Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification. RESULTS: We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin "states", individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse "exceptions" from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin. CONCLUSIONS: These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.

Transglutaminase 2 promotes PDGF-mediated activation of PDGFR/Akt1 and ß-catenin signaling in vascular smooth muscle cells and supports neointima formation.

BACKGROUND: Phenotypic switch of vascular smooth muscle cells (VSMCs) accompanies neointima formation and associates with vascular diseases. Platelet-derived growth factor (PDGF)-induced activation of PDGFR/Akt1 and ß-catenin signaling pathways in VSMCs has been implicated in vessel occlusion. Transglutaminase 2 (TG2) regulates these pathways and its levels are increased in the neointima. OBJECTIVE: The aim of this study was to evaluate the role of TG2 in PDGF/ß-catenin signaling cross-talk and assess its contribution to neointima. METHODS: Aortic VSMCs from wild-type and TG2 knockout mice were tested in vitro for levels of VSMC markers, proliferation, migration and PDGF-induced activation of PDGFR/Akt1 and ß-catenin pathways. Neointima in these mice was studied ex vivo in coronary vessels using a heart slice model and in vivo using a carotid artery ligation model. RESULTS: Genetic deletion of TG2 attenuated the PDGF-induced phenotypic switch of aortic VSMCs, reduced their proliferation and migration rates, and inhibited PDGF-induced activation of PDGFR/Akt1 and ß-catenin pathways in both ex vivo and in vivo neointima models. Importantly, genetic deletion of TG2 also markedly attenuated vessel occlusion. CONCLUSIONS: TG2 promotes neointima formation by mediating the PDGF-induced activation of the PDGFR/Akt1 and ß-catenin pathways in VSMCs. This study identifies TG2 as a potential therapeutic target for blocking neointima in blood vessels.

Transglutaminase inhibitors attenuate vascular calcification in a preclinical model.

OBJECTIVE: In vitro, transglutaminase-2 (TG2)-mediated activation of the ß-catenin signaling pathway is central in warfarin-induced calcification, warranting inquiry into the importance of this signaling axis as a target for preventive therapy of vascular calcification in vivo. METHODS AND RESULTS: The adverse effects of warfarin-induced elastocalcinosis in a rat model include calcification of the aortic media, loss of the cellular component in the vessel wall, and isolated systolic hypertension, associated with accumulation and activation of TG2 and activation of ß-catenin signaling. These effects of warfarin can be completely reversed by intraperitoneal administration of the TG2-specific inhibitor KCC-009 or dietary supplementation with the bioflavonoid quercetin, known to inhibit ß-catenin signaling. Our study also uncovers a previously uncharacterized ability of quercetin to inhibit TG2. Quercetin reversed the warfarin-induced increase in systolic pressure, underlying the functional consequence of this treatment. Molecular analysis shows that quercetin diet stabilizes the phenotype of smooth muscle and prevents its transformation into osteoblastic cells. CONCLUSIONS: Inhibition of the TG2/ß-catenin signaling axis seems to prevent warfarin-induced elastocalcinosis and to control isolated systolic hypertension.

BMP2 cross-linked by transglutaminase 2 to collagen-plla scaffold promotes osteogenic differentiation in mesenchymal stem cells.

Transglutaminase 2 (TG2) was used to attach biologically-active BMP2 to collagen type I-coated poly-L-lactic acid (PLLA) nanofibrous scaffolds. Irreversibly cross-linked BMP2 retained its activity and induced Smad-dependent gene expression in cells seeded on PLLA-BMP2 scaffolds. These modified scaffolds promote osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) cultured in low-serum and growth factor free medium and support deposition of the calcified matrix and induction of the molecular osteogenic markers Runx2, osteopontin, osteonectin and bone sialoprotein. Importantly, the PLLA-BMP2 scaffolds did not support chondrogenic differentiation in hBMSCs as there was no expression of chondrogenic markers aggrecan, Sox 9, and collagen type II, and no deposition of cartilaginous glycosaminoglycan-rich matrix. Thus, TG2-mediated cross-linking of BMP2 to a scaffold is a novel approach to induce osteoblast-specific programming of hBMSCs in a spatially controlled manner.

Tissue-specific responses to loss of transglutaminase 2.

Of the eight catalytic transglutaminases (TGs), transglutaminase 2 (TG2) has been the most comprehensively studied due to its ubiquitous expression in multiple cell types. Despite the observed critical role for this enzyme in multiple biological processes in vitro, TG2 knockout mouse models have shown no severe developmental phenotypes, suggesting compensation by other TGs. To begin characterization of the compensating mechanisms, we analyzed total transamidating activity and expression patterns of all catalytically active TGs in seven different tissues/organs from wild-type and TG2 knockout mice. Inhibitory analysis with TG2-specific inhibitor KCC-009 suggests that relative contribution of TG2 in total transamidating activity differs in various tissues. Accordingly, our data indicate tissue-specific mechanisms of compensation for the loss of TG2, including transcriptional compensation in heart and liver versus functional compensation in aorta, kidney and skeletal/cartiagenous tissues. On the contrary, no compensation has been detected in skeletal muscle, suggesting a limited role for the TG2-mediated transamidation in normal development of this tissue.

Transglutaminase 2-mediated activation of ß-catenin signaling has a critical role in warfarin-induced vascular calcification.

OBJECTIVE: Accumulating experimental evidence implicates ß-catenin signaling and enzyme transglutaminase 2 (TG2) in the progression of vascular calcification, and our previous studies have shown that TG2 can activate ß-catenin signaling in vascular smooth muscle cells (VSMCs). Here we investigated the role of the TG2/ß-catenin signaling axis in vascular calcification induced by warfarin. METHODS AND RESULTS: Warfarin-induced calcification in rat A10 VSMCs is associated with the activation of ß-catenin signaling and is independent of oxidative stress. The canonical ß-catenin inhibitor Dkk1, but not the Wnt antagonist Wif-1, prevents warfarin-induced activation of ß-catenin, calcification, and osteogenic transdifferentiation in VSMCs. TG2 expression and activity are increased in warfarin-treated cells, in contrast to canonical Wnt ligands. Vascular cells with genetically or pharmacologically reduced TG2 activity fail to activate ß-catenin in response to warfarin. Moreover, warfarin-induced calcification is significantly reduced on the background of attenuated TG2 both in vitro and in vivo. CONCLUSIONS: TG2 is a critical mediator of warfarin-induced vascular calcification that acts through the activation of ß-catenin signaling in VSMCs. Inhibition of canonical ß-catenin pathway or TG2 activity prevents warfarin-regulated calcification, identifying the TG2/ß-catenin axis as a novel therapeutic target in vascular calcification.

Induction of chondrogenic differentiation in mesenchymal stem cells by TGF-beta cross-linked to collagen-PLLA [poly(L-lactic acid)] scaffold by transglutaminase 2.

Transglutaminase-mediated cross-linking has been employed to optimize the mechanical properties and stability of tissue scaffolds. We have characterized tissue transglutaminase (TG2)-mediated cross-linking as a useful tool to deliver biologically-active TGF to mesenchymal stem cells (MSCs) and direct their differentiation towards a chondrogenic lineage. TGF-ß3 is irreversibly cross-linked by TG2 to collagen type II-coated poly(L-lactic acid) nanofibrous scaffolds and activates Smad phosphorylation and Smad-dependent expression of a luciferase reporter. Human bone marrow-derived MSCs cultured on these scaffolds deposit cartilaginous matrix after 14 days of culture at 50 % efficiency compared to chondrogenesis in the presence of soluble TGF-ß3. These findings are significant because they suggest a novel approach for the programming of MSCs in a spatially controlled manner by immobilizing biologically active TGF-ß3 via cross-linking to a collagen-coated polymeric scaffold.

Tissue transglutaminase regulates chondrogenesis in mesenchymal stem cells on collagen type XI matrices.

Tissue transglutaminase (tTG) is a multifunctional enzyme with a plethora of potential applications in regenerative medicine and tissue bioengineering. In this study, we examined the role of tTG as a regulator of chondrogenesis in human mesenchymal stem cells (MSC) using nanofibrous scaffolds coated with collagen type XI. Transient treatment of collagen type XI films and 3D scaffolds with tTG results in enhanced attachment of MSC and supports rounded cell morphology compared to the untreated matrices or those incubated in the continuous presence of tTG. Accordingly, enhanced cell aggregation and augmented chondrogenic differentiation have been observed on the collagen type XI-coated poly-(L-lactide) nanofibrous scaffolds treated with tTG prior to cell seeding. These changes implicate that MSC chondrogenesis is enhanced by the tTG-mediated modifications of the collagen matrix. For example, exogenous tTG increases resistance to collagenolysis in collagen type XI matrices by catalyzing intermolecular cross-linking, detected by a shift in the denaturation temperature. In addition, tTG auto-crosslinks to collagen type XI as detected by western blot and immunofluorescent analysis. This study identifies tTG as a novel regulator of MSC chondrogenesis further contributing to the expanding use of these cells in cartilage bioengineering.

Characterization of the transglutaminase gene family in zebrafish and in vivo analysis of transglutaminase-dependent bone mineralization.

We have characterized the protein cross-linking enzyme transglutaminase (TGs) genes in zebrafish, Danio rerio, based on the analysis of their genomic organization and phylogenetics. Thirteen zebrafish TG genes (zTGs) have been identified, of which 11 show high homology to only 3 mammalian enzymes: TG1, TG2 and FXIIIa. No zebrafish homologues were identified for mammalian TGs 3-7. Real-time PCR analysis demonstrated distinct temporal expression profiles for zTGs in larvae and adult fish. Analysis by in situ hybridization revealed restricted expression of zTG2b and zFXIIIa in skeletal elements, resembling expression of their mammalian homologues in osteo-chondrogenic cells. Mammalian TG2 and FXIIIa have been implicated in promoting osteoblast differentiation and bone mineralization in vitro, however, mouse models lacking either gene have no skeletal phenotype likely due to a compensation effect. We show in this study that mineralization of the newly formed vertebrae is significantly reduced in fish grown for 5 days in the presence of TG inhibitor KCC-009 added at 3-5 days post fertilization. This treatment reduces average vertebrae mineralization by 30%, with complete inhibition in some fish, and no effect on the overall growth and vertebrae number. This is the first in vivo demonstration of the crucial requirement for the TG-catalyzed cross-linking activity in bone mineralization.

Phosphorylation regulates the ferritoid-ferritin interaction and nuclear transport.

Ferritin is an iron-sequestering protein that is generally cytoplasmic; however, our previous studies have shown that in avian corneal epithelial (CE) cells ferritin is nuclear. We have also observed that this nuclear localization involves a tissue-specific nuclear transporter that we have termed ferritoid, and that nuclear ferritin protects DNA from oxidative damage. Recently we have determined that ferritoid functions not only as a nuclear transporter, but also, within the nucleus, it remains associated with ferritin as a heteropolymeric complex. This ferritoid-ferritin complex has unique properties such as being half the size of a typical ferritin molecule and showing preferential binding to DNA. It is likely that the association between ferritoid and ferritin is involved both in the nuclear transport of ferritin and in determining certain of the properties of the complex; therefore, we have been examining the mechanisms involved in regulating the association of these two components. As the ferritoid sequence contains six putative phosphorylation sites, we have examined here whether phosphorylation is one such mechanism. We have determined that ferritoid in the nuclear ferritoid-ferritin complexes is phosphorylated, and that inhibition of this phosphorylation, using inhibitors of PKC, prevents its interaction with ferritin. Furthermore, in an experimental model system in which the nuclear transport of ferritin normally occurs (i.e., the co-transfection of COS-1 cells with full length constructs for ferritin and ferritoid), when phosphorylation sites in ferritoid are mutated, the interaction between ferritoid and ferritin is inhibited, as is the nuclear transport of ferritin.

Extracellular transglutaminase 2 activates beta-catenin signaling in calcifying vascular smooth muscle cells.

Accumulation of transglutaminase 2 (TG2) is often associated with mineral deposits in vasculature. Here, we demonstrate that purified TG2 stimulated a 3-fold increase in matrix mineralization and up-regulation of osteoblastic markers in cultured primary vascular smooth muscle cells (VSMCs). Extracellular TG2 interacts with the low density lipoprotein related-protein 5 receptor and activates beta-catenin signaling in VSMCs. These results suggest that TG2 may promote vascular calcification by activating the beta-catenin signaling pathway.

Transglutaminases in mineralized tissues.

Bone development and formation during embryogenesis as well as postnatally during bone remodeling is a complex process controlled systemically and locally by hormones, growth factors and matrix molecules. Transglutaminases (TGases) are the protein cross-linking enzymes, which have long been implicated in bone development and formation. Two members of TGase family, TG2 (also called tissue transglutaminase) and FXIIIA (the enzymatic A subunit of coagulation factor XIII), are expressed in chondrocytes and osteoblasts. The results of analyses in vivo and in vitro accumulated to date indicate an important role of these enzymes in promoting chondrocyte and osteoblast differentiation and matrix mineralization. These effects could be mediated by protein cross-linking activity of TGases, by GTPase activity of TG2 or via non-catalytic signaling effects. The aim of this review is to summarize the available data regarding the expression, localization and activity of TG2 and FXIIIA in mineralizing tissues and to discuss a number of mechanisms by which TGases could exert their promineralizing effects.

New therapeutic approaches to arterial calcification via inhibition of transglutaminase and ß-catenin signaling.

Arterial calcification (AC) is a hallmark of many serious diseases, including atherosclerosis, chronic kidney disease, and diabetes. AC may also develop as a side-effect of therapy with anticoagulants, such as warfarin which is widely used for prophylaxis of thrombosis. In our studies, we established the relation between warfarin-induced AC and activation of enzyme transglutaminase 2 (TG2) and ß-catenin signaling. We showed that TG2-specific inhibitor KCC-009 significantly attenuated the damaging effects of warfarin on arterial tissue. A similar protective effect was also achieved with a dietary bioflavonoid quercetin that inhibits TG2 and ß-catenin signaling. We have shown that quercetin intercepts the chondrogenic transformation of vascular smooth muscle and also drastically attenuates calcifying cartilaginous metaplasia in another model of AC caused by genetic loss of matrix gla protein (MGP). These findings suggest that quercetin may be considered as a promising anti-AC therapeutic in the clinical settings of warfarin supplementation and MGP dysfunction. Further studies are required to test the efficacy of quercetin on other types of AC.

Binding of pro-migratory serum factors to electrospun PLLA nano-fibers.

Architecture of the poly(l-lactic acid) (PLLA) scaffolds is known to affect protein affinity and binding strength. Here, we demonstrate that nanofibrous electrospun PLLA scaffolds reversibly absorb the pro-migratory serum factors that stimulate migration of vascular smooth muscle via an NFkB-dependent mechanism. Further, we demonstrate that mesenchymal stem cells seeded on the PLLA scaffolds do not enhance muscle migration but may maintain the ability of induced cells to migrate in an NFkB-independent manner. These findings further support the promising application of PLLA scaffolds for therapeutic angiogenesis and vascular graft engineering.

Attenuation of chondrogenic transformation in vascular smooth muscle by dietary quercetin in the MGP-deficient mouse model.

RATIONALE: Cartilaginous metaplasia of vascular smooth muscle (VSM) is characteristic for arterial calcification in diabetes and uremia and in the background of genetic alterations in matrix Gla protein (MGP). A better understanding of the molecular details of this process is critical for the development of novel therapeutic approaches to VSM transformation and arterial calcification. OBJECTIVE: This study aimed to identify the effects of bioflavonoid quercetin on chondrogenic transformation and calcification of VSM in the MGP-null mouse model and upon TGF-ß3 stimulation in vitro, and to characterize the associated alterations in cell signaling. METHODS AND RESULTS: Molecular analysis revealed activation of ß-catenin signaling in cartilaginous metaplasia in Mgp-/- aortae in vivo and during chondrogenic transformation of VSMCs in vitro. Quercetin intercepted chondrogenic transformation of VSM and blocked activation of ß-catenin both in vivo and in vitro. Although dietary quercetin drastically attenuated calcifying cartilaginous metaplasia in Mgp-/- animals, approximately one-half of total vascular calcium mineral remained as depositions along elastic lamellae. CONCLUSION: Quercetin is potent in preventing VSM chondrogenic transformation caused by diverse stimuli. Combined with the demonstrated efficiency of dietary quercetin in preventing ectopic chondrogenesis in the MGP-null vasculature, these findings indicate a potentially broad therapeutic applicability of this safe for human consumption bioflavonoid in the therapy of cardiovascular conditions linked to cartilaginous metaplasia of VSM. Elastocalcinosis is a major component of MGP-null vascular disease and is controlled by a mechanism different from chondrogenic transformation of VSM and not sensitive to quercetin.