Cross-linking Of Extracellular Matrix Proteins Research Articles

Modulating extracellular matrix stiffness: a strategic approach to boost cancer immunotherapy.

The interplay between extracellular matrix (ECM) stiffness and the tumor microenvironment is increasingly recognized as a critical factor in cancer progression and the efficacy of immunotherapy. This review comprehensively discusses the key factors regulating ECM remodeling, including the activation of cancer-associated fibroblasts and the accumulation and crosslinking of ECM proteins. Furthermore, it provides a detailed exploration of how ECM stiffness influences the behaviors of both tumor and immune cells. Significantly, the impact of ECM stiffness on the response to various immunotherapy strategies, such as immune checkpoint blockade, adoptive cell therapy, oncolytic virus therapy, and therapeutic cancer vaccines, is thoroughly examined. The review also addresses the challenges in translating research findings into clinical practice, highlighting the need for more precise biomaterials that accurately mimic the ECM and the development of novel therapeutic strategies. The insights offered aim to guide future research, with the potential to enhance the effectiveness of cancer immunotherapy modalities.

The upregulation of TGM2 is associated with poor prognosis and the shaping of the inflammatory tumor microenvironment in lung squamous cell carcinoma.

Tissue transglutaminase (TGM2) is a member of the glutamine transferase superfamily, located within cells and their membranes. When secreted, it catalyzes the cross-linking of extracellular matrix proteins and promotes the formation of extracellular matrix scaffolds. To determine the function of TGM2 in the tumorigenesis of lung squamous cell carcinoma (LUSC), we conducted a comprehensive bioinformatics analysis of TGM2. Our findings indicate that high expression of TGM2 in LUSC was associated with a poorer prognosis. Additionally, we found that high expression of TGM2 is closely related to tumor-promoting inflammation and may increase sensitivity to immunotherapy. We further confirmed the cancer-promoting effect of TGM2 in LUSC through in vitro overexpression and knockdown experiments and showed that TGM2 primarily affects cancer cell proliferation, apoptosis, and invasion. In summary, TGM2 promoted the progression of LUSC, and targeting TGM2 is expected to become a new therapeutic approach for LUSC treatment.

Discovery and Characterization of PROTACs Targeting Tissue Transglutaminase (TG2).

Tissue transglutaminase (TG2) is a multifunctional enzyme involved in the cross-linking of extracellular matrix proteins, formation of complexes with fibronectin (FN) and integrins, and GTP hydrolysis. TG2 is activated in several pathological conditions, including cancer. We recently described a novel series of ligands that bind to TG2 and inhibit its interaction with FN. Because TG2 acts via multiple mechanisms, we set out to pursue a targeted protein degradation strategy to abolish TG2's myriad functions. Here, we report the synthesis and characterization of a series of VHL-based degraders that reduce TG2 in ovarian cancer cells in a proteasome-dependent manner. Degradation of TG2 resulted in significantly reduced cancer cell adhesion and migration in vitro in scratch-wound and migration assays. These results strongly indicate that further development of more potent and in vivo efficient TG2 degraders could be a new strategy for reducing the dissemination of ovarian and other cancers.

Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2.

Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed.

Metformin prevents hypoxia-induced podocyte injury by regulating the ZEB2/TG2 axis.

Podocytes, a vital component of the glomerular filtration barrier, are vulnerable to various noxious stimuli, including Hypoxic. HIF1α that transduces hypoxic adaptations induces Transglutaminase 2 (TG2), which catalyses cross-linking of extracellular matrix proteins. In this study, we investigated the mechanism of regulation of TG2 by HIF1α. HIF1α was induced in podocytes by treating with FG4592 (Roxadustat) or hypoxia (1% oxygen) and in mice by treating with FG4592. Gene expression and protein analysis of ZEB2, TRPC6 and TG2 were performed in both experimental models. Histological and kidney function analyses were performed in mice. Data mining revealed co-expression of HIF1α, ZEB2, TRPC6 and TG2 in the chronic kidney diseases (CKD)-validated dataset. We observed elevated expression of ZEB2, TRPC6 and TG2 in FG4592-treated podocytes. Ectopic expression of ZEB2 resulted in high TRPC6 expression, elevated intracellular calcium levels and increased TG2 activity. Blocking the TRPC6 channel or inhibiting its expression partially attenuated FG4592-induced TG2 activity, whereas suppression of ZEB2 expression significantly abolished TG2 activity. Furthermore, we noticed the induction of the ZEB2/TRPC6/TG2 axis in podocytes in mice administered with FG-4592. Metformin ameliorated the HIF1α-induced podocyte injury and proteinuria in mice administered with FG-4592. This study demonstrates that HIF1α stimulates both TG2 expression and activity via ZEB2/TRPC6 axis, whereas abrogation of HIF1α by metformin prevented hypoxia-induced glomerular injury. Metformin could be explored to treat proteinuric diseases such as CKD, sleep apnea and renal Ischemia-reperfusion-injury, where hypoxia is considered a risk factor.

Respiratory syncytial virus infection induces the release of transglutaminase 2 from human airway epithelial cells.

Respiratory syncytial virus (RSV) is an important human pathogen that causes severe lower respiratory tract infections in young children, the elderly, and the immunocompromised, yet no effective treatments or vaccines are available. The precise mechanism underlying RSV-induced acute airway disease and associated sequelae are not fully understood; however, early lung inflammatory and immune events are thought to play a major role in the outcome of the disease. Moreover, oxidative stress responses in the airways play a key role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium (Ca2+) levels, which in turn activate Ca2+-dependent enzymes, including transglutaminase 2 (TG2). Transglutaminase 2 is a multifunctional cross-linking enzyme implicated in various physiological and pathological conditions; however, its involvement in respiratory virus-induced airway inflammation is largely unknown. In this study, we demonstrated that RSV-induced oxidative stress promotes enhanced activation and release of TG2 from human lung epithelial cells as a result of its translocation from the cytoplasm and subsequent release into the extracellular space, which was mediated by Toll-like receptor (TLR)-4 and NF-κB pathways. Antioxidant treatment significantly inhibited RSV-induced TG2 extracellular release and activation via blocking viral replication. Also, treatment of RSV-infected lung epithelial cells with TG2 inhibitor significantly reduced RSV-induced matrix metalloprotease activities. These results suggested that RSV-induced oxidative stress activates innate immune receptors in the airways, such as TLRs, that can activate TG2 via the NF-κB pathway to promote cross-linking of extracellular matrix proteins, resulting in enhanced inflammation.

Engineering and Monitoring 3D Cell Constructs with Time-Evolving Viscoelasticity for the Study of Liver Fibrosis In Vitro.

Liver fibrosis is generally associated with an over-production and crosslinking of extracellular matrix proteins, causing a progressive increase in both the elastic and viscous properties of the hepatic tissue. We describe a strategy for mimicking and monitoring the mechano-dynamics of the 3D microenvironment associated with liver fibrosis. Cell-laden gelatin hydrogels were crosslinked with microbial transglutaminase using a purpose-designed cytocompatible two-step protocol, which allows for the exposure of cells to a mechanically changing environment during culturing. A bioreactor was re-engineered to monitor the mechanical properties of cell constructs over time. The results showed a shift towards a more elastic (i.e., solid-like) behaviour, which is likely related to an increase in cell stress. The method effectively mimics the time-evolving mechanical microenvironment associated with liver fibrosis and could provide novel insights into pathophysiological processes in which both elastic and viscous properties of tissues change over time.

Methylsulfonylmethane increases osteogenesis and regulates the mineralization of the matrix by transglutaminase 2 in SHED cells.

Methylsulfonylmethane (MSM) is a naturally occurring, sulfate-containing, organic compound. It has been shown to stimulate the differentiation of mesenchymal stem cells into osteoblast-like cells and bone formation. In this study, we investigated whether MSM influences the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into osteoblast-like cells and their osteogenic potential. Here, we report that MSM induced osteogenic differentiation through the expression of osteogenic markers such as osterix, osteopontin, and RUNX2, at both mRNA and protein levels in SHED cells. An increase in the activity of alkaline phosphatase and mineralization confirmed the osteogenic potential of MSM. These MSM-induced effects were observed in cells grown in basal medium but not osteogenic medium. MSM induced transglutaminase-2 (TG2), which may be responsible for the cross-linking of extracellular matrix proteins (collagen or osteopontin), and the mineralization process. Inhibition of TG2 ensued a significant decrease in the differentiation of SHED cells and cross-linking of matrix proteins. A comparison of mineralization with the use of mineralized and demineralized bone particles in the presence of MSM revealed that mineralization is higher with mineralized bone particles than with demineralized bone particles. In conclusion, these results indicated that MSM could promote differentiation and osteogenic potential of SHED cells. This osteogenic property is more in the presence of mineralized bone particles. TG2 is a likely cue in the regulation of differentiation and mineral deposition of SHED cells in response to MSM.

Metformin Suppresses Expression of Pre-Fibrotic Enzyme Lysyl-Oxidase Levels through Inhibition of UPR in MPN Cells

The endoplasmic reticulum (ER) is a cytoplasmic organelle required for proper protein folding and sorting. When protein load exceeds the capacity of the ER, the accumulation of the misfolded proteins triggers a quality control response known as the unfolded protein response (UPR). Recent studies have revealed that increased activation of the UPR is one of the hallmarks of hematological malignancies. In addition, excess UPR is associated with pro-fibrotic conditions, i.e., pulmonary fibrosis, renal fibrosis and kidney fibrosis.Lysyl-oxidase (LOX) is an enzyme that regulates the crosslinking of extracellular matrix proteins, such as collagen, and enhances a fibrotic phenotype. Recent studies revealed that the genes of LOX family members are upregulated in myeloproliferative neoplasms (MPN). Furthermore, megakaryocytes and platelets derived from MPN patients had increased LOX protein concentrations in comparison with healthy subjects. It is also reported that the UPR is one of the important regulators of LOX. Based on this knowledge, we speculated that the enhanced UPR might be involved in the elevation of LOX in JAK2V617F harboring MPN and may contribute to the development of myelofibrosis. To study this hypothesis, we initially confirmed that JAK2V617F-positive cell lines(HEL and SET-2) show enhanced activation of UPR, including phosphorylation of eIF2-alpha, nuclear localization of ATF6 and XBP1s and upregulation of glucose response protein 78 (GRP78). We also confirmed the presence of elevated levels of LOX in these cells. Treatment of the cells with a chemical chaperone, namely, tauroursodeoxycholic acid (TUDCA), suppressed UPR and decreased expression of LOX, suggesting that UPR is responsible for elevation of LOX in MPN cells. To analyze whether JAK2V617F is responsible for the enhanced UPR and expression of LOX in MPN cells, we treated the cells with JAK1/JAK2 inhibitor ruxolitinib. Unexpectedly, ruxolitinib did not block UPR nor expression of LOX. Previously, we reported that metformin, which is a member of the biguanide family, inhibited growth of MPN cells through activation of AMPK and PP2A (Kawashima et al. Exp.Hematol, 2016). Furthermore, a number of studies demonstrated that metformin suppressed UPR in several cancer cells through activation of AMPK. Therefore, we investigated whether metformin could block UPR and LOX levels in these cells. As expected, metformin reduced the phosphorylation levels of eIF2alpha and nuclear localization of XBP1 and ATF6. Expression of GRP78 was also blocked by metformin. These results clearly indicated that metformin suppressed UPR in MPN cells. Metformin also blocked the activation of the molecules required for proteins synthesis including p70S6kinase, mTOR and 4E-PB1. These results indicated that suggested that metformin blocked aberrant UPR through inhibition of enhanced protein synthesis. Importantly, we also confirmed that LOX expression was also suppressed by metformin in MPN cells.Our observations indicated that the UPR is enhanced in MPN cells irrespective of JAK2V617F, and metformin could block this process leading to suppression of LOX levels. Combination of ruxolitinib with metformin might be a new and attractive method to suppress myelofibrosis in MPN. DisclosuresKirito:Novartis Pharma KK: Honoraria.

Differentiation of fibrotic liver tissue using laser-induced breakdown spectroscopy.

Hepatic cirrhosis is a major cause of morbidity and mortality worldwide due to hepatitis C, alcoholism and fatty liver disease associated with obesity. Assessment of hepatic fibrosis relies in qualitative histological evaluation of biopsy samples. This method is time-consuming and depends on the histopathologists' interpretation. In the last decades, non-invasive techniques were developed to detect and monitor hepatic fibrosis. Laser-induced breakdown spectroscopy (LIBS) is a good candidate for a real-time, independent and fast technique to diagnose hepatic fibrosis. In this work LIBS was employed to characterize rat liver tissues with different stages of fibrosis. Depth profiling measurements were carried out by using a nanosecond Nd:YAG laser operated at the fundamental wavelength and an echelle spectrometer coupled with an ICCD camera. Due to the soft nature of the samples, plasma conditions largely change between consecutives shots. Thus, a theoretically supported procedure to correct the spectral line intensities was implemented. This procedure allows the reduction of the intensities' dispersion from 67% to 12%. After the correction, the LIBS signal shows an enhancement in calcium intensity by a factor of three as the fibrosis progressed. Calcium is known to increase crosslinking of extracellular matrix proteins in the fibrous septa. Therefore, our result singles it out as a key participant in the hepatic fibrosis.

The effect of 1 year of Alagebrium and moderate-intensity exercise training on left ventricular function during exercise in seniors: a randomized controlled trial.

Sedentary aging leads to left ventricular (LV) and vascular stiffening due in part to advanced glycation end-products (AGEs) cross-linking of extracellular matrix proteins. Vigorous lifelong exercise ameliorates age-related cardiovascular (CV) stiffening and enhances exercise LV function, although this effect is limited when exercise is initiated later in life. We hypothesized that exercise training might be more effective at improving the impact of age-related CV stiffening during exercise when combined with an AGE cross-link breaker (Alagebrium). Sixty-two seniors (≥60 yr) were randomized into four groups: sedentary + placebo, sedentary + Alagebrium, exercise + placebo, and exercise + Alagebrium for 1 yr. Moderate-intensity aerobic exercise was performed 3-4 sessions/wk; controls underwent similar frequency of yoga/balance training. Twenty-four similarly-aged, lifelong exercisers (4-5 sessions/wk) served as a comparator for the effect of lifelong exercise on exercising LV function. Oxygen uptake (Douglas bags), stroke index (SI; acetylene rebreathing), and effective arterial elastance (Ea) were collected at rest and submaximal and maximal exercise. Maximum O2 uptake (23 ± 5 to 25 ± 6 ml·kg(-1)·min(-1)) increased, while SI (35 ± 11 to 39 ± 12 ml/m(2)) and Ea (4.0 ± 1.1 to 3.7 ± 1.2 mmHg·ml(-1)·m(-2)) were improved across all conditions with exercise, but remained unchanged in controls (exercise × time, P ≤ 0.018). SI or Ea were not affected by Alagebrium (medication × time, P ≥ 0.468) or its combination with exercise (interaction P ≥ 0.252). After 1 yr of exercise plus Alagebrium, exercise SI and Ea remained substantially below that of lifelong exercisers (15-24 and 9-22%, respectively, P ≤ 0.415). In conclusion, Alagebrium plus exercise had no synergistic effect on exercise LV function and failed to achieve levels associated with lifelong exercise, despite a similar exercise frequency.

Two functional loci in the promoter of EPAS1 gene involved in high-altitude adaptation of Tibetans.

EPAS1 involves in the hypoxic response and is suggested to be responsible for the genetic adaptation of high-altitude hypoxia in Tibetans. However, the detailed molecular mechanism remains unknown. In this study, a single nucleotide polymorphism rs56721780:G>C and an insertion/deletion (indel) polymorphism −742 indel in the promoter region showed divergence between Tibetans and non-Tibetan lowlanders. rs56721780:G>C regulated the transcription of EPAS1 by IKAROS family zinc finger 1 (IKZF1), which was identified as a new transcriptional repressor for EPAS1 gene. It demonstrated that the C allele of rs56721780:G>C decreased the binding of IKZF1, leading to the attenuated transcriptional repression of EPAS1 gene. The insertion at −742 indel provided a new binding site for Sp1 and was related to the activation of EPAS1 promoter. Further functional analysis revealed that lysyl oxidase (LOX) gene, which was reported to be responsible for extracellular matrix protein cross-linking of amnion previously, was a direct target of EPAS1. The CC genotype at rs56721780:G>C and the insertion genotype at −742 indel were found associated with higher EPAS1 and LOX expression levels in amnion, as well as higher birth weight of Tibetan newborns, suggesting that EPAS1 gene might play important roles in the development of amnion, fetus growth and high-altitude adaptation of Tibetans.

Association of the G473A polymorphism and expression of lysyl oxidase with breast cancer risk and survival in European women: a hospital-based case-control study.

BackgroundLysyl oxidase (LOX) is an extracellular enzyme essential for the covalent crosslinking of extracellular matrix proteins and may also have additional functions. LOX expression can be both up- and downregulated in cancer and is associated both with tumour suppression and metastasis progression. The G473A polymorphism (rs1800449) results in the Arg158Gln amino acid substitution in the LOX propeptide, compromises its tumour suppressive activity, and was associated with an increased breast cancer risk in a Chinese Han population. In the first hospital-based case-control study in European women, we aimed at investigating the association of LOX expression and the G473A polymorphism with breast cancer risk and survival in unselected and estrogen receptor (ER) negative patients.Methodology/Principal FindingsThe G473A polymorphism was genotyped in 386 breast cancer patients and 243 female controls. Moreover, LOX mRNA expression was quantified in the tumors of 105 patients by qRT-PCR. We found that the minor A-allele of this polymorphism is associated with a later age at breast cancer onset, a trend towards a decreased disease-free and metastasis-free survival, but not with an increased breast cancer risk. LOX mRNA expression was significantly elevated in tumours of patients older than 55 years, postmenopausal patients, estrogen receptor positive tumours, and p53 negative tumours, but was unaffected by G473A genotype in tumours and breast cancer cell lines. High LOX expression was associated with a poor disease-free and metastasis-free survival in ER negative but not ER positive patients. LOX expression was an independent prognostic parameter in multivariate analysis, whereas G473A genotype was not. A small, distinct subgroup of the ER negative patients was identified which exhibited a considerably elevated LOX expression and a very poor disease-free (p = 0.001) and metastasis-free survival (p = 0.0003).Conclusions/SignificanceThis newly identified ER negative/LOX high subgroup may be a suitable collective for future individualized breast cancer diagnosis and therapy.

Emerging role of advanced glycation-end products (AGEs) in the pathobiology of eye diseases.

Advanced glycation end products (AGEs) have been implicated in vision loss associated with macula degeneration, cataract formation, diabetic retinopathy and glaucoma. This pathogenic potential is mainly attributed to their accumulation in ocular tissues where they mediate aberrant crosslinking of extracellular matrix proteins and disruption of endothelial junctional complexes that affects cell permeability, mediates angiogenesis and breakdown of the inner blood-retinal barrier. Furthermore, AGEs severely affect cellular metabolism by disrupting ATP production, enhancing oxidative stress and modulating gene expression of anti-angiogenic and anti-inflammatory genes. Elucidation of AGE-induced mechanisms of action in different eye compartments will help in the understanding of the complex cellular and molecular processes associated with eye diseases. Several pharmaceutical agents with anti-glycating and anti-oxidant properties as well as AGE crosslink 'breakers' have been currently applied to eye diseases. The role of diet and the beneficial effects of certain nutriceuticals provide an alternative way to manage chronic visual disorders that affect the quality of life of millions of people.

Inhibition of Transglutaminase 2, a Novel Target for Pulmonary Fibrosis, by Two Small Electrophilic Molecules

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibrotic destruction of normal lung architecture. Due to a lack of effective treatment options, new treatment approaches are needed. We previously identified transglutaminase (TG)2, a multifunctional protein expressed by human lung fibroblasts (HLFs), as a positive driver of fibrosis. TG2 catalyzes crosslinking of extracellular matrix proteins, enhances cell binding to fibronectin and integrin, and promotes fibronectin expression. We investigated whether the small electrophilic molecules 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2) inhibit the expression and profibrotic functions of TG2. CDDO and 15d-PGJ2 reduced expression of TG2 mRNA and protein in primary HLFs from control donors and donors with IPF. CDDO and 15d-PGJ2 also decreased the in vitro profibrotic effector functions of HLFs including collagen gel contraction and cell migration. The decrease in TG2 expression did not occur through activation of the peroxisome proliferator activated receptor γ or generation of reactive oxidative species. CDDO and 15d-PGJ2 inhibited the extracellular signal-regulated kinase pathway, resulting in the suppression of TG2 expression. This is the first study to show that small electrophilic compounds inhibit the expression and profibrotic effector functions of TG2, a key promoter of fibrosis. These studies identify new and important antifibrotic activities of these two small molecules, which could lead to new treatments for fibrotic lung disease.

Influence of copper on early development: Prenatal and postnatal considerations

Copper (Cu) is an essential nutrient whose requirement is increased during pregnancy and lactation. These represent times of critical growth and development, and the fetus and neonate are particularly vulnerable to deficiencies of this nutrient. Genetic mutations that predispose the offspring to inadequate stores of Cu can be life threatening as is observed in children with Menkes disease. During the last decade, severe Cu deficiency, once thought to be a rare condition, has been reported in the literature at an increasing frequency. Secondary Cu deficiencies can be induced by a variety of ways such as excessive zinc or iron intake, certain drugs, and bariatric surgery. Premature and low birth weight infants can be born with low Cu stores. A number of mechanisms can contribute to the teratogenicity of Cu including decreased activity of select cuproenzymes, increased oxidative stress, decreased nitric oxide availability, altered iron metabolism, abnormal extracellular matrix protein crosslinking, decreased angiogenesis and altered cell signaling among others. The brain, heart, and vessels as well as tissues such as lung, skin and hair, and systems including the skeletal, immune, and blood systems, are negatively affected by suboptimal Cu during development. Additionally, persistent structural, biochemical, and functional adverse effects in the offspring are noted even when Cu supplementation is initiated after birth, supporting the concept that adequate Cu nutriture during pregnancy and lactation is critical for normal development. Although Cu-containing IUDs are an effective method for increasing intrauterine Cu concentrations and for reducing the risk of pregnancy, high amounts of dietary Cu are not thought to represent a direct developmental risk.

The role of “semicarbazide-sensitive amine oxidase” in the arterial wall

Summary Semicarbazide-sensitive amine (SSAO) metabolizes primary amines into aldehydes, hydrogen peroxide and ammonia and is largely expressed in adipocytes, lymphatic endothelial cells and vascular smooth muscle cells (VSMCs). In vitro SSAO expression and activity increase during VSMC differentiation and SSAO participates in glucose uptake but its exact role in the vascular wall is unclear. Nevertheless, we think that SSAO could modulate VSMC differentiation, as it does in adipocytes. Previous work suggested that SSAO could contribute to vascular remodelling observed in age-related pathologies as plasma levels increase in several cardiovascular diseases and in diabetes. In such cases, SSAO may play a detrimental role by inducing unwanted cross-linking of extracellular matrix proteins and could thus be an interesting target to modulate arterial stiffening. It may also participate in LDL oxidation and inflammation. A physiological role for SSAO in vascular development and cross-linking of elastin has been proposed, but is not yet established. Our studies on SSAO knockout mice revealed no anomalies in insoluble elastin content, arterial elasticity or vasomotor tone. In growing rats treated with semicarbazide (SCZ), an SSAO inhibitor, or with β-aminopropionitrile (BAPN), a lysyl oxidase (LOX) inhibitor, we confirmed that LOX was the most important enzyme implicated in elastin cross-linking during arterial maturation, but in absence of specific inhibitors, we cannot formally exclude a minor role for SSAO in this process. Specific effects of SSAO inhibition could be an increase in the production of various extracellular matrix proteins, possibly related to the effect of SSAO on VSMC differentiation.

Microbial transglutaminases generate T cell stimulatory epitopes involved in celiac disease

Celiac disease (CD) is a permanent intolerance to gluten. In CD patients, gluten peptides cause an inflammation in the small intestine leading to tissue damage. Tissue transglutaminase (tTG) is an enzyme involved in the repair of damaged tissue by crosslinking of extracellular matrix proteins. Under certain conditions, tTG can deamidate glutamine into glutamic acid. Compared to native gluten, deamidated gluten elicits a more powerful inflammatory response. To improve the quality and texture of food products microbial transglutaminases (mTG) are used in the food industry. In this study, we investigated whether deamidation of gluten by mTG enhances the immunogenic nature of gluten. We found that mTG have a broader substrate specificity than tTG and deamidate both synthetic and natural gluten peptides which were recognized by gluten-specific T cells. Therefore mTG can enhance the immunogenicity of gluten and should not be used in food products intended for consumption by CD patients.

Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures

Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and beta-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms--TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin-collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.

Antisense inhibition of transglutaminase 2 affects development of mouse embryo submandibular gland in organ culture

Tissue transglutaminase (TGase 2) has been implicated in numerous cellular functions, i.e., apoptosis, differentiation, extracellular matrix protein cross-linking and organogenesis. Earlier report of the strong transient expression of TGase 2 localized at the anchoring sites of muscle bundles of human embryo and recent findings of a similar transient expression of the TGase 2 in the salivary myoepithelial cells of mouse embryo indicated a definitive role of TGase 2 in the cytodifferentiation of myoepithelial cells. To understand functional role(s) of TGase 2 in the organogenesis of salivary gland, antisense inhibition of TGase 2 expression was performed in the organ culture of mouse embryo submandibular gland. The antisense of TGase 2 transfection tested using oral keratinocyte cell line, KB cells, elicited significant inhibition of cellular transglutaminase expression. The same antisense treatment of submandibular glands in organ culture also resulted in the suppression of cellular TGase 2 expression as indicated by weak immunoreaction against anti-TGase 2 in the myoepithelial cells of submandibular glands in contrast to strong reaction in those of the normal and sense-treated glands. Antisense to TGase 2 treatment induced retarded growth of salivary epithelium in 1 week and severe aberrant growth of salivary ducts and acini in 2 weeks and also expression of apoptotic inducer, Bax specifically localized in the myoepithelial cells, suggesting apoptotic state of myoepithelial cells. These data suggest that the antisense inhibition of TGase 2 expression affects the cytodifferentiation of ductal cells and myoepithelial cells, and resulted in severe retardation of tubuloalveolar structure formation of salivary gland.