Canonical and truncated transglutaminase-2 regulate mucin-1 expression and androgen independency in prostate cancer cell lines.

Androgen independency is associated with poor prostate cancer (PCa) survival. Here we report that silencing of transglutaminase-2 (TG2) expression by CRISPR-Cas9 is associated with upregulation of androgen receptor (AR) transcription in PCa cell lines. Knockout of TG2 reversed the migratory potential and anchorage independency of PC3 and DU145 cells and revealed a reduced level of mucin-1 (MUC1) RNA transcript through unbiased multi-omics profiling, which was restored by selective add-back of the truncated TG2 isoform (TGM2_v2). Silencing of AR resulted into increased MUC1 in TG2KO PC3 cells showing that TG2 affects transcriptional regulation of MUC1 via repressing AR expression. Treatment of PC3 WT cell line with TG2 inhibitor ZDON led to a significant increase in AR expression and decrease in MUC1. ZDON also blocked the formation of MUC1-multimers labelled with TG amine-donor substrates in reducing conditions, revealing for the first time a role for TG2, which we show to be externalised via extracellular vesicles, in MUC1 stabilisation via calcium-dependent transamidation. A specific antibody towards TGM2_v2 revealed its restricted nuclear location compared to the canonical long form of TG2 (TGM2_v1), which is predominantly cytosolic, suggesting that this form contributes to the previously suggested TG2-mediated NF-κB activation and AR transcriptional repression. As TGM2_v2 transcription was increased in biopsies of early-stage prostate adenocarcinoma (PRAD) patients compared to subjects presenting inflammatory prostatitis, and total TG2 protein expression significantly increased in PRAD versus normal tissue, the role of TG2 and its truncated form as a prostate malignancy marker is suggested. In conclusion, this investigation has provided the first unbiased discovery of a novel pathway mediated by TG2 via MUC1, which is shown to contribute to androgen insensitivity and malignancy of PCa cells and be upregulated in PCa biopsies, with potential relevance to cancer immune evasion.

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX.

Introduction: In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed "pollensomes", are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification. Methods: To do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics. Results: These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes. Discussion: The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles.

Extracellular transglutaminase-2, nude or associated with astrocytic extracellular vesicles, modulates neuronal calcium homeostasis.

We have uncovered a novel role for astrocytes-derived extracellular vesicles (EVs) in controlling intraneuronal Ca2+ concentration ([Ca2+]i) and identified transglutaminase-2 (TG2) as a surface-cargo of astrocytes-derived EVs. Incubation of hippocampal neurons with primed astrocyte-derived EVs have led to an increase in [Ca2+]i, unlike EVs from TG2-knockout astrocytes. Exposure of neurons or brain slices to extracellular TG2 promoted a [Ca2+]i rise, which was reversible upon TG2 removal and was dependent on Ca2+ influx through the plasma membrane. Patch-clamp and calcium imaging recordings revealed TG2-dependent neuronal membrane depolarization and activation of inward currents, due to the Na+/Ca2+-exchanger (NCX) operating in the reverse mode and indirect activation of L-type VOCCs, as indicated by VOCCs/NCX pharmacological inhibitors. A subunit of Na+/K+-ATPase was selected by comparative proteomics and identified as being functionally inhibited by extracellular TG2, implicating Na+/K+-ATPase inhibition in NCX reverse mode-switching leading to Ca2+ influx and higher basal [Ca2+]i. These data suggest that reactive astrocytes control intraneuronal [Ca2+]i through release of EVs with TG2 as responsible cargo, which could have a significant impact on synaptic activity in brain inflammation.

The Transglutaminase-2 Interactome in the APP23 Mouse Model of Alzheimer's Disease.

Amyloid-beta (Aß) deposition in the brain is closely linked with the development of Alzheimer's disease (AD). Unfortunately, therapies specifically targeting Aß deposition have failed to reach their primary clinical endpoints, emphasizing the need to broaden the search strategy for alternative targets/mechanisms. Transglutaminase-2 (TG2) catalyzes post-translational modifications, is present in AD lesions and interacts with AD-associated proteins. However, an unbiased overview of TG2 interactors is lacking in both control and AD brain. Here we aimed to identify these interactors using a crossbreed of the AD-mimicking APP23 mouse model with wild type and TG2 knock-out (TG2-/-) mice. We found that absence of TG2 had no (statistically) significant effect on Aß pathology, soluble brain levels of Aß1-40 and Aß1-42, and mRNA levels of TG family members compared to APP23 mice at 18 months of age. Quantitative proteomics and network analysis revealed a large cluster of TG2 interactors involved in synaptic transmission/assembly and cell adhesion in the APP23 brain typical of AD. Comparative proteomics of wild type and TG2-/- brains revealed a TG2-linked pathological proteome consistent with alterations in both pathways. Our data show that TG2 deletion leads to considerable network alterations consistent with a TG2 role in (dys)regulation of synaptic transmission and cell adhesion in APP23 brains.

Molecular profiling of urinary extracellular vesicles in chronic kidney disease and renal fibrosis.

Chronic kidney disease (CKD) is a long-term kidney damage caused by gradual loss of essential kidney functions. A global health issue, CKD affects up to 16% of the population worldwide. Symptoms are often not apparent in the early stages, and if left untreated, CKD can progress to end-stage kidney disease (ESKD), also known as kidney failure, when the only possible treatments are dialysis and kidney transplantation. The end point of nearly all forms of CKD is kidney fibrosis, a process of unsuccessful wound-healing of kidney tissue. Detection of kidney fibrosis, therefore, often means detection of CKD. Renal biopsy remains the best test for renal scarring, despite being intrinsically limited by its invasiveness and sampling bias. Urine is a desirable source of fibrosis biomarkers as it can be easily obtained in a non-invasive way and in large volumes. Besides, urine contains biomolecules filtered through the glomeruli, mirroring the pathological state. There is, however, a problem of highly abundant urinary proteins that can mask rare disease biomarkers. Urinary extracellular vesicles (uEVs), which originate from renal cells and carry proteins, nucleic acids, and lipids, are an attractive source of potential rare CKD biomarkers. Their cargo consists of low-abundant proteins but highly concentrated in a nanosize-volume, as well as molecules too large to be filtered from plasma. Combining molecular profiling data (protein and miRNAs) of uEVs, isolated from patients affected by various forms of CKD, this review considers the possible diagnostic and prognostic value of uEVs biomarkers and their potential application in the translation of new experimental antifibrotic therapeutics.

Insights into the heparan sulphate-dependent externalisation of transglutaminase-2 (TG2) in glucose-stimulated proximal-like tubular epithelial cells.

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on the heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.

Enhancement of Tomato Plant Growth and Productivity in Organic Farming by Agri-Nanotechnology Using Nanobubble Oxygation.

The development of technology to improve the mineralization of organic fertilizer and to enhance crop production is essential to achieve the transition from traditional farming to eco-friendly organic farming. Nanobubble oxygation (NB) was employed for comparison with traditional pump-aerated oxygation (AW) and a control group through both soil incubation and soil column experiments. Plant-available N and P contents in the NB treatment group were higher than those in the AW and control groups. Enzymatic activities including ß-1,4-N-acetyl-glucosaminidase, phosphatase, α-1,4-glucosidase, ß-1,4-xylosidase, peroxidase, and phenol oxidase were significantly higher in both oxygation groups compared with the control. The soil microbial biomass, activity, and diversity were also significantly improved due to the oxygation treatment. Additionally, the microbial metabolic functions were shifted in both oxygation treatments compared with the control group. The final tomato yield increase from the NB treatment group was 23%, and that from the AW treatment was 17%, compared with the control.

Search for Novel Diagnostic Biomarkers of Prostate Inflammation-Related Disorders: Role of Transglutaminase Isoforms as Potential Candidates.

Investigations on prostate inflammation-related disorders, including acute and chronic prostatitis, chronic pelvic pain syndrome, benign prostate hyperplasia (BPH), and prostate cancer (PCa), are still ongoing to find new, accurate, and noninvasive biomarkers for a differential diagnosis of those pathological conditions sharing some common macroscopic features. Moreover, an ideal biomarker should be useful for risk assessment of prostate inflammation progression to more severe disorders, like BPH or PCa, as well as for monitoring of treatment response and prognosis establishment in carcinoma cases. Recent literature evidence highlighted that changes in the expression of transglutaminases, enzymes that catalyze transamidation reactions leading to posttranslational modifications of soluble proteins, occur in prostate inflammation-related disorders. This review focuses on the role specifically played by transglutaminases 4 (TG4) and 2 (TG2) and suggests that both isoenzymes hold a potential to be included in the list of candidates as novel diagnostic biomarkers for the above-cited prostate pathological conditions.

Spotlight on the Transglutaminase 2-Heparan Sulfate Interaction.

Heparan sulfate proteoglycans (HSPGs), syndecan-4 (Sdc4) especially, have been suggested as potential partners of transglutaminase-2 (TG2) in kidney and cardiac fibrosis, metastatic cancer, neurodegeneration and coeliac disease. The proposed role for HSPGs in the trafficking of TG2 at the cell surface and in the extracellular matrix (ECM) has been linked to the fibrogenic action of TG2 in experimental models of kidney fibrosis. As the TG2-HSPG interaction is largely mediated by the heparan sulfate (HS) chains of proteoglycans, in the past few years a number of studies have investigated the affinity of TG2 for HS, and the TG2 heparin binding site has been mapped with alternative outlooks. In this review, we aim to provide a compendium of the main literature available on the interaction of TG2 with HS, with reference to the pathological processes in which extracellular TG2 plays a role.

Proteomic Profiling Reveals the Transglutaminase-2 Externalization Pathway in Kidneys after Unilateral Ureteric Obstruction.

Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance, leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of CKD, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding partners of TG2 responsible for TG2 externalization in kidneys subjected to unilateral ureteric obstruction (UUO) using TG2 knockout kidneys as negative controls. We report a robust and unbiased analysis of the membrane interactome of TG2 in fibrotic kidneys relative to the entire proteome after UUO, detected by SWATH mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD008173. Clusters of exosomal proteins in the TG2 interactome supported the hypothesis that TG2 is secreted by extracellular membrane vesicles during fibrosis progression. In established TEC lines, we found TG2 in vesicles of both endosomal (exosomes) and plasma membrane origin (microvesicles/ectosomes), and TGF-ß1 stimulated TG2 secretion. Knockout of syndecan-4 (SDC4) greatly impaired TG2 exosomal secretion. TG2 coprecipitated with SDC4 from exosome lysate but not ectosome lysate. Ex vivo, EGFP-tagged TG2 accumulated in globular elements (blebs) protruding/retracting from the plasma membrane of primary cortical TECs, and SDC4 knockout impaired bleb formation, affecting TG2 release. Through this combined in vivo and in vitro approach, we have dissected the pathway through which TG2 is secreted from TECs in CKD.

Biocatalysis by Transglutaminases: A Review of Biotechnological Applications.

The biocatalytic activity of transglutaminases (TGs) leads to the synthesis of new covalent isopeptide bonds (crosslinks) between peptide-bound glutamine and lysine residues, but also the transamidation of primary amines to glutamine residues, which ultimately can result into protein polymerisation. Operating with a cysteine/histidine/aspartic acid (Cys/His/Asp) catalytic triad, TGs induce the post-translational modification of proteins at both physiological and pathological conditions (e.g., accumulation of matrices in tissue fibrosis). Because of the disparate biotechnological applications, this large family of protein-remodelling enzymes have stimulated an escalation of interest. In the past 50 years, both mammalian and microbial TGs polymerising activity has been exploited in the food industry for the improvement of aliments' quality, texture, and nutritive value, other than to enhance the food appearance and increased marketability. At the same time, the ability of TGs to crosslink extracellular matrix proteins, like collagen, as well as synthetic biopolymers, has led to multiple applications in biomedicine, such as the production of biocompatible scaffolds and hydrogels for tissue engineering and drug delivery, or DNA-protein bio-conjugation and antibody functionalisation. Here, we summarise the most recent advances in the field, focusing on the utilisation of TGs-mediated protein multimerisation in biotechnological and bioengineering applications.

Interplay between transglutaminases and heparan sulphate in progressive renal scarring.

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-ß1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.

Syndecan-4 knockout leads to reduced extracellular transglutaminase-2 and protects against tubulointerstitial fibrosis.

Transglutaminase type 2 (TG2) is an extracellular matrix crosslinking enzyme with a pivotal role in kidney fibrosis. The interaction of TG2 with the heparan sulfate proteoglycan syndecan-4 (Sdc4) regulates the cell surface trafficking, localization, and activity of TG2 in vitro but remains unstudied in vivo. We tested the hypothesis that Sdc4 is required for cell surface targeting of TG2 and the development of kidney fibrosis in CKD. Wild-type and Sdc4-null mice were subjected to unilateral ureteric obstruction and aristolochic acid nephropathy (AAN) as experimental models of kidney fibrosis. Analysis of renal scarring by Masson trichrome staining, kidney hydroxyproline levels, and collagen immunofluorescence demonstrated progressive fibrosis associated with increases in extracellular TG2 and TG activity in the tubulointerstitium in both models. Knockout of Sdc-4 reduced these effects and prevented AAN-induced increases in total and active TGF-ß1. In wild-type mice subjected to AAN, extracellular TG2 colocalized with Sdc4 in the tubular interstitium and basement membrane, where TG2 also colocalized with heparan sulfate chains. Heparitinase I, which selectively cleaves heparan sulfate, completely abolished extracellular TG2 in normal and diseased kidney sections. In conclusion, the lack of Sdc4 heparan sulfate chains in the kidneys of Sdc4-null mice abrogates injury-induced externalization of TG2, thereby preventing profibrotic crosslinking of extracellular matrix and recruitment of large latent TGF-ß1. This finding suggests that targeting the TG2-Sdc4 interaction may provide a specific interventional strategy for the treatment of CKD.

Development of a chronic kidney disease model in C57BL/6 mice with relevance to human pathology.

BACKGROUND: Genetically modified mice are used to investigate disease and assess potential interventions. However, research into kidney fibrosis is hampered by a lack of models of chronic kidney disease (CKD) in mice. Recently, aristolochic acid nephropathy (AAN), characterised by severe tubulointerstitial fibrosis, has been identified as a cause of end stage kidney disease and proposed as a model of CKD. Published studies have used various dosing regimens, species and strains, with variable outcomes. Therefore, we aimed to develop a standardised protocol to develop tubulointerstitial fibrosis using pure aristolochic acid I (AAI) in C57BL/6 mice. METHODS: AAI dose optimisation was performed by intraperitoneal injection of AAI at varying dose, frequency and duration. Kidney function was assessed by serum creatinine. Fibrosis was quantified by hydroxyproline levels and Masson's Trichrome staining. Specific collagens were measured by immunofluorescent staining. RESULTS: Single doses of AAI of >10 mg/kg caused acute kidney failure and death. Lower doses of 2.5 mg/kg needed to be administrated more than weekly to cause significant fibrosis. 3 mg/kg once every 3 days for 6 weeks followed by a disease development time of 6 weeks after AAI led to reduced kidney weight and function. Substantial tubulointerstitial fibrosis occurred, with males more severely affected. Increased deposition of collagen I, III and IV contributed to fibrosis, with collagen III and IV higher in males. CONCLUSIONS: AAN can be induced in C57BL/6 mice. The regimen of 3 mg/kg every 3 days for 6 weeks followed by 6 weeks of disease development time gives substantial tubulointerstitial fibrosis with lesions similar to those in humans.

Transglutaminase-2 interaction with heparin: identification of a heparin binding site that regulates cell adhesion to fibronectin-transglutaminase-2 matrix.

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262-265) or KQKRK (598-602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg(19) and Arg(28) also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded "closed" protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2.

A crucial sequence for transglutaminase type 2 extracellular trafficking in renal tubular epithelial cells lies in its N-terminal beta-sandwich domain.

Transglutaminase type 2 (TG2) catalyzes the formation of an ε-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides or proteins including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlie tissue scarring and fibrosis. The extracellular trafficking of TG2 is crucial to its role in ECM homeostasis; however, the mechanism by which TG2 escapes the cell is unknown as it has no signal leader peptide and therefore cannot be transported classically. Understanding TG2 transport may highlight novel mechanisms to interfere with the extracellular function of TG2 as isoform-specific TG2 inhibitors remain elusive. Mammalian expression vectors were constructed containing domain deletions of TG2. These were transfected into three kidney tubular epithelial cell lines, and TG2 export was assessed to identify critical domains. Point mutation was then used to highlight specific sequences within the domain required for TG2 export. The removal of ß-sandwich domain prevented all TG2 export. Mutations of Asp(94) and Asp(97) within the N-terminal ß-sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain ((88)WTATVVDQQDCTLSLQLTT(106)). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence (88)WTATVVDQQDCTLSLQLTT(106) within the ß-sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin.

Heparan sulfate proteoglycans are receptors for the cell-surface trafficking and biological activity of transglutaminase-2.

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.

Fibronectin-tissue transglutaminase matrix rescues RGD-impaired cell adhesion through syndecan-4 and beta1 integrin co-signaling.

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Calpha (PKCalpha) and its subsequent interaction with beta(1) integrin since disruption of PKCalpha binding to beta(1) integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCalpha leading to its association with beta(1) integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

The cellular response to transglutaminase-cross-linked collagen.

Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts. We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression.

A novel RGD-independent cel adhesion pathway mediated by fibronectin-bound tissue transglutaminase rescues cells from anoikis.

Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies alpha5beta1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Calpha, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress.