Tissue transglutaminase in normal and abnormal wound healing: review article

A complex series of events involving inflammation, cell migration and proliferation, ECM stabilisation and remodelling, neovascularisation and apoptosis are crucial to the tissue response to injury. Wound healing involves the dynamic interactions of multiple cells types with components of the extracellular matrix (ECM) and growth factors. Impaired wound healing as a consequence of aging, injury or disease may lead to serious disabilities and poor quality of life. Abnormal wound healing may also lead to inflammatory and fibrotic conditions (such as renal and pulmonary fibrosis). Therefore identification of the molecular events underlying wound repair is essential to develop new effective treatments in support to patients and the wound care sector. Recent advances in the understating of the physiological functions of tissue transglutaminase a multi functional protein cross-linking enzyme which stabilises tissues have demonstrated that its biological activities interrelate with wound healing phases at multiple levels. This review describes our view of the function of tissue trasnglutaminase in wound repair under normal and pathological situations and highlights its potential as a strategic therapeutic target in the development of new treatments to improve wound healing and prevent scarring

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 -( -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 -sandwich domain prevented all TG2 export. Mutations of Asp94 and Asp97 within the N-terminal -sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain (88WTATVVDQQDCTLSLQLTT106). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence 88WTATVVDQQDCTLSLQLTT106 within the -sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin

Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme β-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal β-sandwich domain and that this interaction is crucial for cell surface association of tTG

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-b1. 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-b1. This finding suggests that targeting the TG2- Sdc4 interaction may provide a specific interventional strategy for the treatment of CKD

Microvesicles: Novel Biomarkers for Neurological Disorders

Microvesicles (MVs) are released by most cell types in physiological conditions, but their number is often increased upon cellular activation or neoplastic transformation. This suggests that their detection may be helpful in pathological conditions to have information on activated cell types and, possibly, on the nature of the activation. This could be of paramount importance in districts and tissues that are not accessible to direct examination, such as the central nervous system. Increased release of MVs has been described to be associated to the acute or active phase of several neurological disorders. While the subcellular origin of MVs (exosome or ectosomes) is basically never addressed in these studies because of technical limitations, the cell of origin is always identified. Endothelium- or platelet-derived MVs, detected in plasma or serum, are linked to neurological pathologies with a vascular or ischemic pathogenic component, and may represent a very useful marker to support therapeutic choices in stroke. In neuroinflammatory disorders, such as multiple sclerosis, MVs of oligodendroglial, or microglial origin have been described in the cerebrospinal fluid and may carry, in perspective, additional information on the biological alterations in their cell of origin. Little specific evidence is available in neurodegenerative disorders and, specifically, MVs of neural origin have never been investigated in these pathologies. Few data have been reported for neuroinfection and brain trauma. In brain tumors, despite the limited number of studies performed, results are very promising and potentially close to clinical translation. We here review all currently available data on the detection of MVs in neurological diseases, limiting our search to exclusively human studies. Current literature and our own data indicate that MVs detection may represent a very promising strategy to gain pathogenic information, identify therapeutic targets, and select specific biomarkers for neurological disorders

Analysis of tissue transglutaminase function in the migration of swiss 3T3 fibroblasts - the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme’s mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr274 (Y274A), the proposed site for the cis- ,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix

GENETIC DISSECTION OF DEVELOPMENTAL TRAITS IN BARLEY (HORDEUM VULGARE)

Barley (Hordeum vulgare) ranks in fourth place among cultivated cereals for worldwide production and is a recognized model organism for genetic and genomic studies in the Triticeae tribe, which includes wheats (Triticum species) and rye (Secale cereale). Root and shoot architecture traits are key factors in plant performance, competition with weeds, adaptation and stress responses thus having an important impact on yield and yield stability. Breeders have proposed hypothetical optimal morphological parameters to improve production in relation to different environmental conditions. Leaf size and orientation are determinants of canopy transpiration and radiation interception e.g. in dry and sunny Mediterranean environments reduced size and erect orientation of the leaves can reduce water loss by transpiration and allow deeper light penetration into the canopy. Tillering influences crop performance, biomass and grain production, e.g. a reduction in tillering compensated by an increase dimension and number of kernels per spike could be a strategy of adaptation to dry climates. A reduction in plant height and an augment in stem thickness is connected to lodging resistance. Root system extension is connected to the ability of the plant to reach water. The objectives of this project were to dissect genetic variability for shoot and root morphological traits in barley, identifying genomic regions and characterizing genes controlling these traits, and exploring how different traits influence each other. To this end, two approaches were undertaken depending on the trait(s) under study: \u2022 the first exploited natural variation in a panel of modern and old European barley cultivars to carry out association mapping of flowering date, stem diameter, spike fertility, leaf dimension, plant height, tillering and root extension (Chapters 2 and 3); \u2022 the second was to characterize the ontogenetic basis of increased tillering using as a case study the many-noded dwarf6.6 (mnd6.6) high tillering barley mutant (Chapter 4). In the first approach, we focused on winter barley because of its agronomic interest in the Mediterranean area, where genetic improvement of drought tolerance is particularly important. We analyzed a panel of 142 European winter barley cultivars (67 two-rowed and 75 six-rowed) with a view to conduct a genome wide association scan (GWAS) for shoot and root architecture traits in two separate sets of experiments. To this end, genotyping data for 4,083 SNPs were available from previous projects of which 2,521 mapped on the POPSEQ barley reference map. PCoA results indicated the existence of two major sub-populations in our germplasm panel, corresponding to two-rowed and six-rowed barley cultivars. In order to study shoot developmental traits (Chapter 2) the panel was phenotyped during the growing season 2012-2013 in a field trial at Fiorenzuola d\u2019Arda, Piacenza, Italy. The experimental scheme consisted in 3 replicates (each being a plot of 24 well spaced plants) in randomized blocks. For selected traits data were integrated and analyzed together with those coming from a parallel field trial that was carried out at the University of Shiraz, Iran (data courtesy of Dr. Elahe Tavakol). Flowering date (FD) and leaf width (LW) were measured in both Italy and Iran, leaf length (LL) was measured only in Iran, plant height (PH), spike length (SL), number of fertile rachis node per spike (NFRN) tillering (T) and (SD) were measured only in Italy. Best Linear Unbiased Estimators (BLUEs) of FD, LW were calculated as the phenotypic values estimated for each genotype in a mixed linear model, where genotypes were set as fixed factor and location, location-genotype interaction and replicates as random factors. For BLUEs calculation of all other traits only replicates were used as random factors. BLUEs were subjected to GWAS analyses, using a mixed linear model (MLM) correcting for population structure with a Q matrix (PCA first three coordinate) and for individuals co-ancestry using a K matrix (a pair-wise matrix defining the degree of genetic covariance among individuals). Significance of marker-trait associations was evaluated based on false discovery rate (FDR)-adjusted p-values (threshold value for significant association was set at 0.05). All traits except tillering exhibited good heritability. Few QTLs were detected in GWAS (five for FD, two for LW, three for LL, one for PH, two for SL, two for NFRN, one for tillering, no one for SM). Flowering date exhibited significant correlation with leaf dimension and spike length and six markers designed on Photoperiod-H1 (Ppd-H1) gene (the major determinant for photoperiod response in barley) were the most significantly associated to FD, LW, LL and SL. In particular the recessive ppd- H1 allele causing reduced photoperiod sensitivity, delayed flowering date and increased leaf dimension and spike length compared to the Ppd-H1 allele. Three markers diagnostic for the HvCEN gene (which regulates flowering date independently from photoperiod) were significantly associated to FD and SL. These results suggested that genes for flowering date could have pleiotropic effects on other morphological traits that may mask other genetic effects. For this reason we tested a novel approach repeating GWAS for LW, LL and SL using flowering date as a cofactor (fixed effect) in further analyses. For SL and LW no new significant associations were found with this method, while new significant associations were uncovered for LL, including two markers on chromosome 5H mapped in a region where narrow leaf dwarf 1a (nld-1a) mutant had previously been previously mapped. Tillering and NFRN were only associated to markers diagnostic INTERMEDIUM-C (INT-C), one of the two main genes controlling row type: in our panel two-rowed genotypes had a significantly higher number of tillers and NFRN compared to six- rowed varieties, confirming the known pleiotropic effects of row-type genes on tillering and NFRN and the balancing of patterns of development by breeding practice for the particular row-type. Based on these results, we run GWAS for NFRN and tillering using row-type as covariate. With this model, we found six markers associated with NFRN on chromosome 5H, in the region hosting HvCO12, HvCO13, HvCO15, XvCCA-1, HvLHY, genes involved in control of flowering date. These same markers, were associated to the duration of the phase between awn primordia formation and tipping (awn arising from flag leaf) in a recently published GWAS study. Together, results from Chapter 2 provide the first evidence of the involvement of the Ppd-H1 gene in control of leaf size and spike length. Thus few QTLs were detected that explain the phenotypic variation for our morphological traits, with some major genes having strong pleiotropic effects that mask minor genetic effects. The use of traits that appear to influence others measures as covariates in GWAS models seems to be a promising approach, although the statistical power of this strategy is still to be evaluated. Germplasm collections with uniform growth habit and row-type are an attractive alternative to prevent confounding effects and allow additional loci to be detected. In Chapter 3, we explored natural genetic variation in root extension using the same winter barley panel as Chapter 2 in growth chamber experiments. In order to evaluate root growth we built 50 cm deep cylindrical pots (called rhizotrons) and used digital scans of the root system to measure total root extension with the winRHIZO software. Based on a series of preliminary tests, we used siliceous sand supplemented with controlled release fertilizer to analyze 4th leaf stage plants from 31 genotypes (9 plants per genotype). Root extension per se exhibited 75% heritability, while normalizing root extension on shoot dry weight resulted in low variability (22%) likely due to low heritability of shoot dry weight in our system. These results support the validity of our protocol for evaluation of genetic variation in root extension in barley and other cereals and indicate significant variation exists in our germplasm panel. Thus, the already collected material will be analysed to phenotype the entire panel. In the future, more variability may be uncovered by exploring wild barleys (Hordeum vulgare spp. spontaneum) or landraces. Tillering is a plastic trait affected by the complex interplay of genetic and hormonal factors with environmental conditions such as plant density/light quality and nutrient availability, which likely complicated genetic dissection of this trait in our field experiment on the winter barley panel (Chapter 2). To circumvent the limited power of the GWAS approach for this trait and understand more about the mechanisms subtending tiller formation, we decided to use the mnd6.6 mutant as a case study to investigate the ontogenetic basis of high tillering in barley and its relation to leaf development. Mutant and wild-type plants were grown in growth in a controlled chamber under long day conditions, and dissected weekly from the emergence to anthesis, registering the development of axillary buds, leaves and tillers together with internode elongation, in relation to shoot apical meristem (SAM) stage. Results show that the mutant is not altered in timing of apical meristem development and differentiation to spike, but has a shorter phyllochron that leads to an increment in the number of leaves per vegetative axis. This in turn results in a higher number of axillary buds and a higher number of tillers. The HvMND6 gene was recently identified and our results are consistent with the activity of the previously characterized rice homologue PLASTOCHRON1, indicating an evolutionarily conserved link between plastochron/phyllochron duration and tillering. Concluding, while significant genetic variation was identified for various traits within the gene pool of our winter barley collection, variability of morphological traits as leaf dimension was subordinated to the length of vegetative period. Indeed, flowering date is one of the major factors on which breeding practice has worked to adapt barley to different environments. Beyond modern European varieties, barley breeding for new ideotypes should explore wider genetic resources as Hordeum spp. spontaneum or landraces. In any case, the existence of correlations between different phenotypes calls for careful evaluation of sources of traits to avoid undesired effects on other traits, e.g. due to the relation between tillering and phyllochrone, breeding for early plant vigour through shortening phyllochron, may have pleiotropic effects and result in increased tillering whose benefits would have to be evaluated

FCI: an R-based algorithm for evaluating uncertainty of absolute real-time PCR quantification

Background: FCI is an R code for analyzing data from real-time PCR experiments. This algorithm estimates standard curve features as well as nucleic acid concentrations and confidence intervals according to Fieller's theorem. Results: In order to describe the features of FCI four situations were selected from real data collected during an international external quality assessment program for quantitative assays based on real-time PCR. The code generates a diagnostic figure suitable for assessing the quality of the quantification process. Conclusion: We have provided a freeware programme using this algorithm specifically designed to increase the information content of the real-time PCR assay. \ua9 2008 Verderio et al; licensee BioMed Central Ltd

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-β1 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