Transglutaminases Research Articles

Novel compound heterozygous mutations of TGM1 gene identified in a Chinese collodion baby

To identify potential mutations in a Chinese collodion baby. The patient was investigated clinically. DNA was extracted from peripheral blood of the baby and his parents. All coding exons(exons 2-15) and splicing sites of transglutaminase 1(TGM1) were amplified by polymerase chain reaction (PCR). Mutation detection was performed by directed sequencing of the PCR products. A total of 100 healthy unrelated subjects were used as controls. Haplotypes were constructed with microsatellites flanking the locus, and TGM1 genotypes of the family were used to determine parental origins of the mutations. CLUSTAL X (1.81) was employed to analyze cross-species conservation of the mutant protein sequence. The boy was found to be a compound heterozygote for two novel mutations: c.420A>G (I140M) from his father and c.832G>A (G278R) from his mother, with the former occurring in the transglutaminase N domain and the latter between transglutaminase N and transglutaminase-like domains. Both mutations were absent from the control subjects. The boy's condition was caused by two novel compound heterozygous mutations of c.420A>G and c.832G>A of TGM1. Author's results may provide new clues for molecular diagnosis of this disease.

Knocking-in the R142C mutation in transglutaminase 1 disrupts the stratum corneum barrier and postnatal survival of mice

Mutations in the gene encoding transglutaminase 1 (TG1) are responsible for various types of autosomal recessive congenital ichthyosis (ARCI), such as lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) and some minor variants of ARCI. A point mutation of R143C in the β-sandwich domain of TG1 has been often identified in patients with LI or CIE. To elucidate the effect of that point mutation on skin barrier structures and functions, we generated mice with a point mutation of R142C, which corresponds to the R143C mutation in human TG1. A mouse line with the R142C point mutation in TG1 was established using a gene targeting technique and the Cre-loxP system. The skin phenotypes were analyzed in homozygous mutant Tgm1(R142C/R142C) mice. In the skin of Tgm1(R142C/R142C) mice, expression of the mutant transcripts was comparable with wild-type or Tgm1(+/R142C) mice. However, the amount of mutated protein in the skin was markedly decreased in Tgm1(R142C/R142C) mice, and the TG1 activity of Tgm1(R142C/R142C) keratinocytes was almost lost. Tgm1(R142C/R142C) mice exhibited morphological and functional skin barrier defects and neonatal lethality. The stratum corneum of those mice lacked cornified envelopes, and loricrin, the major structural component, failed to assemble at the corneocyte cell periphery. Tgm1(R142C/R142C) mice showed a marked increase in transepidermal water loss and their skin was easily permeable to toluidine blue dye. The intercellular lipid lamellar structures of the stratum corneum were irregular and the 13-nm periodic X-ray diffractions from the stratum corneum lipid molecules were lost in vivo. From these results, we suggest that the R142C mutation of TG1 reduces the enzyme stability which is indispensable for development of the stratum corneum and skin barrier function and for postnatal survival of mice.

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 as a biomarker of osteoarthritis: an update

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca(2+)-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-β seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.

Novel roles for biogenic monoamines: from monoamines in transglutaminase‐mediated post‐translational protein modification to monoaminylation deregulation diseases

Functional protein serotonylation is a newly recognized post-translational modification with the primary biogenic monoamine (PBMA) serotonin (5-HT). This covalent protein modification is catalyzed by transglutaminases (TGs) and, for example, acts in the constitutive activation of small GTPases. Multiple physiological roles have been identified since its description in 2003 and, importantly, deregulated serotonylation was shown in the etiology of bleeding disorders, primary pulmonary hypertension and diabetes. The PBMAs 5-HT, histamine, dopamine, and norepinephrine all act as neurotransmitters in the nervous system and as hormones in non-neuronal tissues, which points out their physiological importance. In analogy to serotonylation we have found that also the other PBMAs act through the TG-catalyzed mechanisms of 'histaminylation', 'dopaminylation' and 'norepinephrinylation'. Therefore, PBMAs deploy a considerable portion of their effects via protein monoaminylation in addition to their canonical receptor-mediated signaling. Here, the implications of these newly identified post-translational modifications are presented and discussed. Furthermore, the potential regulatory roles of protein monoaminylation in small GTPase, heterotrimeric G-protein and lipid signaling, as well as in modulating metabolic enzymes and nuclear processes, are critically assessed.

Identification of human salivary transglutaminases

Transglutaminases (TGs) expression and enzymatic activities in human saliva were investigated. Specific antibodies showed the co-existence of TG1, TG2, TG3 and TG4. TG2 and TG3 were found in native and multiple proteolytic forms. Our data indicate that TG1 and TG2 isoenzymes are highly active with the major activity attributed to TG1. These findings pave the way for future studies on the physiological role of TG in the oral cavity and the potential impact of their deregulation in TG-associated oral diseases.

Transglutaminase 1 and its regulator tazarotene‐induced gene 3 localize to neuronal tau inclusions in tauopathies

Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and Pick's disease (PiD) are commonly known as tauopathies. Neurodegeneration observed in these diseases is linked to neuronal fibrillary hyperphosphorylated tau protein inclusions. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular cross-links. Both transglutaminase 1 (TG1) and transglutaminase 2 (TG2) are abundantly expressed in the brain and are associated with fibrillary hyperphosphorylated tau protein inclusions in neurons of AD, so-called neurofibrillary tangles (NFTs). However, other data obtained by our group suggested that tau pathology in the brain may be primarily related to TG1 and not to TG2 activity. To obtain more information on this issue, we set out to investigate the association of TG1, TG2, and TG-catalysed cross-links with fibrillary hyperphosphorylated tau inclusions in tauopathies other than AD, using immunohistochemistry. We found strong TG1 and TG-catalysed cross-link staining in neuronal tau inclusions characteristic of PSP, FTDP-17 with mutations in the tau gene (FTDP-17T), and PiD brain, whereas, in contrast to AD, TG2 was only rarely observed in these inclusions. Furthermore, using a biochemical approach, we demonstrated that tau is a substrate for TG1-mediated cross-linking. Interestingly, we found co-localization of the TG1 activator, tazarotene-induced gene 3 (TIG3), in the neuronal tau inclusions of PSP, FTDP-17T, and PiD, but not in NFTs of AD cases, indicating that these tau-containing protein aggregates are not identical. We conclude that TG1-catalysed cross-linking, regulated by TIG3, might play an important role in the formation of neuronal tau inclusions in PSP, FTDP-17T, and PiD brain.

Transglutaminase 6: a protein associated with central nervous system development and motor function

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second β-barrel domain in man and a variant with truncated β-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca2+ and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca2+ and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-011-1091-z) contains supplementary material, which is available to authorized users.

Evidences for a role of protein cross-links in transglutaminase-related disease

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. Considerable and intense progress has been made in the understanding of the chemistry, molecular biology and cell biology of TGs. The knowledge that very different physiological and pathological processes are dependent on the presence of adequate levels of these cross-linking enzymes and on the amount of both free and protein-conjugated polyamines by TG, has generated an incredible amount of original research and review articles. It is clear that TG-mediated reactions are essential for some biological processes, such as blood coagulation, skin barrier formation and extracellular matrix assembly, but may also be involved in pathogenetic mechanisms responsible for several human diseases, such as cancer, AIDS, neurodegenerative disorders, celiac disease, and eye lens opacification. We present here a comprehensive review of recent insights into the pathophysiology of TGs related to their protein cross-linking activity.

Concomitant kidney diseases of ichthyosis

Congenital and acquired ichthyosis and kidney diseases are common diseases in clinic.The mutation or deletion of genes encoding filaggrin,keratin,steroid sulfatase(STS) and transglutaminase-1(TGM-1) will result in ichthyosis.STS gene deletion leads to accumulation of cholesterol sulfate in the glomerular visceral epithelial cells,induces TGM-1 dysfunction,thus causing dysfunction of slit diaphragm of the glomerular visceral epithelial cells and proteinuria,and finally results in the occurrence and development of kidney diseases.The concomitant kidney diseases of ichthyosis may be manifested as proteinuria,steroid-resistant nephrotic syndrome,chronic pyelonephritis,renal failure,or even renal carcinoma.

Transglutaminases (TGs) in Ocular and Periocular Tissues: Effect of Muscarinic Agents on TGs in Scleral Fibroblasts

ObjectiveTo investigate the expression of transglutaminases (TGs) in the ocular surface, the eyelid margin and associated glands and to determine effect of muscarinic agents on TGs in scleral fibroblasts (SF).Materials and MethodsPrimary SFs cultured from mouse and human sclera were treated with atropine and carbachol for 5 days. Lysed cell RNA was used for real-time PCR, protein was used for Western blot analysis and TG-2 transamidase activity was measured by ELISA. Immunohistochemistry was done to determine the expression of TGases.ResultsImmunohistochemistry and western blot confirmed the expression of TGs-1, 2, 3 and 5 proteins in cultured SFs and eye tissues. Real time PCR showed TG-1, 2, 5 transcript levels to be down regulated 3 fold (p<0.05) in cultured human and mouse SFs after incubation with atropine and this was reversed by carbachol. However, TG-3 expression was increased with atropine and decreased with carbachol at all concentrations. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. TGs-1, 3, 5 were localized in the entire mouse corneal epithelium, stroma and endothelium but TG-2 was present only in the corneal subepithelium and stroma. All TGs were localized in mouse Meibomian glands however TG-2 had a weak expression.ConclusionsOur results confirm that TGs-1, 2, 3 and 5 are expressed in human SF and murine ocular tissues, eyelid and associated Meibomian glands. Real-time PCR and Western blot results showed that muscarinic antagonist down-regulates TGs-1, 2 and 5 in both cultured human and mouse SFs and upregulates TG-3. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. These results suggest that manipulation of TGs by way of muscarinic receptor acting drugs may be a plausible method of intervention in wound healing and scleral remodeling.

Beneficial effects of treatment with transglutaminase inhibitor cystamine on the severity of inflammation in a rat model of inflammatory bowel disease

Inflammatory bowel disease (IBD) represents a socially and clinically relevant disorder, characterized by intestinal chronic inflammation. Cystamine (CysN) is a multipotent molecule with healthy effects and, moreover, it is an inhibitor of transglutaminases (TGs), including the TG type 2 (TG2), an enzyme with pleiotropic functions, involved in different pathways of inflammation and central in the pathogenesis of some human disorders as the IBD. Our aim was to evaluate the effect of CysN in an IBD rat model. A total of 30 rats were divided into 4 groups: controls without treatment (CTR; n=7); receiving the 2,4,6-trinitrobenzene sulfonic acid enema (TNBS group; n=8); treated with TNBS enema plus oral CysN (TNBS–CysN group; n=8); treated with CysN (CysN group; n=7). After killing, bowel inflammation was evaluated applying specific scores. TG activity, TG2 and isopeptide bond immunohistochemical expression, and tumor necrosis factor-α (TNF-α) were evaluated in the colonic tissue, such as interleukin-6 (IL-6) serological levels (ELISA). TG2 was also evaluated on the luminal side of the colon by immunoautoradiography. Colonic samples from IBD patients were compared with animal results. TNBS–CysN group developed a less severe colitis compared with the TNBS group (macroscopic score 0.43±0.78 vs 3.28±0.95, microscopic score 6.62±12.01 vs 19.25±6.04, P<0.05, respectively) associated with a decrease of TG activity, TG2 and isopeptide bond immunohistochemical expression, TNF-α and IL-6 levels. No statistically significant differences were found between CysN and CTR groups. The colonic immunolocalization of TG2 was comparable in humans affected by IBD and TNBS-administered animals. This is the first demonstration that treatment with a CysN has an anti-inflammatory effect, reducing severity of colitis in a rat model. CysN could be tested as a possible treatment or co-treatment in IBD therapeutic trials.

A report of two cases of TGM1 mutations in Iranian patients with lamellar ichthyosis.

Objective Autosomal Recessive Congenital Ichthyosis (ARCI) is a rare, heterogenous keratinization disorder of the skin, classically divided into two clinical subtypes, Lamellar Ichthyosis (LI) and Nonbullous Congenital Ichthyosi-formis Erythroderma (NCIE). Lamellar Ichtyosis is caused by mutations in the TGM1 gene that encodes transglutaminase 1 enzyme, which is critical for the assembly of the cornified cell envelope in terminally differentiating keratinocytes. TGM1 is a complex enzyme existing as both cytosolic and membrane-bound forms. Moreover, TGM1 is proteolytically processed, and the major functionally active form consists of a membrane-bound 67/33/10-kDa complex with a myristoylated and palmitoylated amino-terminal 10-kDa membrane anchorage fragment. In this study, all 14 coding exons of TGM1 gene were investigated using PCRsequencing method in three Iranian patients with different phenotypes which are often caused by homozygote or compound heterozygote mutations and a homozygote mutation (G218S) in exon 4 and three heterozygote mutations (R37K, D58N, D86N) in exon 2 were observed. The mutation (D86N) was seen in two patients simultaneously. Key words: TGM1gene, mutation, ARCI, lamellar, ichthyosis, sequencing.

Early increases in transglutaminase activity and polyamine levels in a Mallory-Denk body mouse model

Rodents treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) are a model of two hepatic toxic manifestations: porphyria and the appearance of hepatic cytoplasmic protein aggregates (Mallory-Denk Bodies, MDBs). MDBs are induced after long-term DDC feeding, consist primarily of keratins 8 and 18, and contain glutamine-lysine cross-links generated by transglutaminases (TGs). TGs are Ca 2+-dependent enzymes which catalyze the formation of covalent bonds between proteins and between proteins and polyamines. The aim of the current study was to investigate the time-course of TG hepatic activity in CF1 male mice either acutely or chronically treated with DDC and to correlate this activity with polyamine and porphyrin levels. On day 3 of the treatment, statistically significant increases in TG activity (75%), porphyrin content (6740%) and spermidine levels (73%) were observed. Although not statistically significant, at this time point putrescine levels showed an increase of 52%. The highest TG activity was observed on day 30 (522%), while porphyrin levels were still gradually increasing by day 45 (37,000%). From day 7 of the treatment and until the end of the experiment, putrescine levels remained increased (781%). Spermine levels were not affected by the treatment. The DDC-induced increases in putrescine and spermidine levels herein reported seem to be an early event contributing to the stimulation of liver TG activity, and thus to the promotion of cross-linking reactions between keratin proteins. This in turn would contribute to the formation of protein aggregates, which would lead to the appearance of MDBs. Due to the pro-oxidant and antioxidant properties of polyamines, it is possible to speculate that putrescine and spermidine may also participate at several levels in the oxidative stress processes associated with MDB formation.

Déjà vu with a twist: transglutaminases in bioenergetics and transcriptional dysfunction in Huntington's disease

The article by McConoughey et al in the current issue of EMBO Molecular Medicine examines the contribution of transglutaminase 2 (TG2) to Huntington's disease (HD) pathogenesis. The authors find that TG2 inhibition can ameliorate HD neurodegeneration, and thereby elevate the status of transglutaminases (TGs) to a major therapeutic target—not because of their well-known activity in mutant protein aggregation, but instead based upon their ability to epigenetically modulate transcription and energy production. While the reintroduction of TG inhibition as a therapy for HD may evoke feelings of déjà vu, the outcome this time around could go in a dramatically different direction.

Phenotype, genotype and ultrastructural findings in a pedigree with autosomal recessive congenital ichthyosis

Objective To investigate the clinical phenotype, genotype and ultrastructural features in a pedigree with autosomal recessive congenital ichthyosis. Methods Patients were examined for clinical manifestation. PCR was carried out to amplify all the 15 exons and adjacent splice sites of TGM1 gene followed by bidirectional sequencing. Skin samples were taken by biopsy from the back of the proband, fixed in 3% glu-taraldehyde for transmission electron microscopy. Results The proband presented an intermediate clinical phenotype between lamellar ichthyosis (LI) and non-bullous congenital ichthyosiform erythroderma (NCIE), while his brother manifested as a collodion baby. A C551T heterozygous mutation which located in the third exon of TGM1 gene and resulted in a substitution of arginine by cysteine at codon 143 (R143C), was detected in the proband, his brother and father. Meanwhile, another heterozygous C-to-T transition at position 759 causing a substitution of serine by phenylalanine at codon 212 (S212F), was noted in the proband, his brother and mother. Electron microscopy revealed not only features of ichthyosis congenital type Ⅲ but also those of ichthyosis congenital type Ⅱ in lesions of the proband. Conclusions The patients in this pedigree carry compound heterozygous mutations, i.e. R143C, a hot missense mutation, as well as a de novo mutation S212F. The proband, who harbors mutations in the TGM1 gene, shows electron microscopic features characteristic not only of ichthyosis congenital type Ⅱ but also of ichthyosis congenital type Ⅲ. Key words: Ichthyosis; Chromosome disorders; Phenotype; Genotype; Microscopy, electron

Bcl‐2 overexpression in hepatic stellate cell line CFSC‐2G, induces a pro‐fibrotic state

Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro- and anti-apoptotic molecules. Since Bcl-2 overexpression preserves viability against OS, our objective was to address the effect of Bcl-2 overexpression in the hepatic stellate cells (HSC) cell-line CFSC-2G under acetaldehyde and H(2)O(2) challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. To induce Bcl-2 overexpression, HSC cell line CFSC-2G was transfected by lipofection technique. Green fluorescent protein-only CFSC-2G cells were used as a control. Cell survival after H(2)O(2) treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation-rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue-inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a-actin (alpha-SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor-beta (TGF-beta) mRNA. Cells overexpressing Bcl-2 survived approximately 20% more than control cells when exposed to H(2)O(2) and approximately 35% proteins were protected from oxidation, but Bcl-2 did not slow proliferation or induced senescence. Bcl-2 overexpression did not change alpha-SMA levels, but it increased TIMP-1 (55%), tissue transglutaminases (tTG) (25%) and TGF-beta mRNA (49%), when exposed to acetaldehyde, while MMP-13 content decreased (47%). Bcl-2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP-1, tTG and TGF-beta mRNA levels and decreased MMP-13 content, suggesting that Bcl-2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.

Over-expression of transglutaminase in the Drosophila eye imaginal disc induces a rough eye phenotype

Transglutaminases (TGs) catalyze the cross-linking of proteins and are involved in various biological processes in mammals. In invertebrates, except for the involvement in the hemolymph clotting, the functions of TG have not been revealed. Drosophila has a single TG gene (CG7356), from which two kinds of mRNAs (dTG-RA and dTG-RB) are formed. RT-PCR analyses indicated that both dTGs-RA and -RB are synthesized in all the developmental stages tested. To reveal the roles of dTG during the development, we examined a phenotype induced through the ectopic expression of dTG by using a GAL4-UAS targeted expression system. Over-expression of dTG-A in the eye imaginal disc of larva induced a rough eye phenotype in adult compound eyes. Co-expression of P35, an inhibitor of apoptosis, suppressed the rough eye phenotype, suggesting that the rough eye phenotype induced by the over-expression of dTG-A in the eye imaginal disc is due to the occurrence of apoptosis. The rough eye phenotype induced by the over-expression of dTG-A was suppressed by the crossing with mutant fly lines lacking Drosophila JNK gene basket (bsk) or Drosophila JNKK gene hemipterous. FLP-out experiments using an enhancer trap line showed that the over-expression of dTG-A in the eye imaginal disc increased the puckered enhancer activity, a reporter of Bsk activity. These results suggested that the rough eye phenotype induced by the over-expression of dTG-A is related to an enhancement of JNK signaling pathway.

Abstract 3802: PPAR gamma mediated induction of transglutaminase in aerodigestive preneoplasia in vitro

Abstract Currently there are no validated biomarkers or therapies for human aerodigestive preneoplasia. There is increasing interest in the utilization of PPAR gamma mediated therapy for oral leukoplakia reversal based on preliminary clinical trial results. Transglutaminases (TGMs) are associated with squamous differentiation, and therefore might be hypothesized to be upregulated with PPAR gamma differentiation therapies. In the present study we sought to examine the feasibility of utilizing transglutaminases as a downstream readout for PPAR gamma activator treatment of oral leukoplakia. First we examined our Affymetrix database of human head and neck tumors for the presence or absence of transglutaminases 1-3 in 41 tumors compared to 13 normal oral mucosa samples. We discovered that TGM 1 and 3 were significantly upregulated in normal tissues compared to tumors (p&amp;lt;0.0001). Next, we examined whether PPAR gamma activators could activate TGM1 reporter genes in cell lines. We found that TGM1 luciferase reporters were significantly activated by ciglitazone (0-5uM) and pioglitazone (0-10 uM) in MSK leuk and HOK oral preneoplastic cell lines, as well as CA-9-22 oral cancer cells in several experiments. Next we examined pioglitazone activation of transglutaminases in these cell lines by real time PCR. We found that pioglitazone significantly activated TGM 1 and 2 in MSK leuk and CA-9-22 oral cancer cells on qRT-PCR. We conclude that the clinically relevant PPAR gamma activator, pioglitazone, can activate transglutaminases in aerodigetsive cancer cell lines and may be useful in upregulating differentiation in oral preneoplasia in clinical trials. We also conclude that transglutaminases may provide a family of proteins for PPAR gamma effects and should be included as exploratory biomarkers in leukoplakia clinical trials with PPAR gamma activators. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3802.

Transglutaminases locate to calcified areas in advanced human atherosclerotic lesions

We investigated the involvement of the two major transglutaminases (TGs), tissue transglutaminase (TG2) and factor XIIIa (FXIIIA), in human atherosclerotic calcification.MethodsCross‐sections of human carotid atherosclerotic plaques were stained for TG2, FXIIIA, the TG‐induced cross‐link and macrophage marker CD68. The correlation of these staining patterns to calcified areas throughout the specimens was tested by multi‐scale 3D mapping of the microscopic images to μCT images.ResultsThe TG‐induced cross‐link was found in 64 ± 8% (mean ± SEM) of the analyzed areas inside the plaques. Calcified areas correlated with this cross‐link (p&lt;0.001). FXIIIA was found to be the dominant transglutaminase (56 ± 10% of the cross‐link‐positive areas), as compared to 11 ± 3% for TG2. Both transglutaminases correlated with the cross‐link (p=&lt;0.001 and p=0.002 respectively). Staining for FXIIIA correlated with CD68 (p&lt;0.001) on tissue level and also co‐localized with CD68 positive cells as shown by double immunofluorescent staining.ConclusionAreas of calcification locate to the presence and activity of transglutaminases in human atherosclerotic arteries. FXIIIA is dominantly present over TG2 in these plaques and may be derived from local macrophages.Supported by the Netherlands Heart Foundation (NHS 2001T038).