Analysis on promotive effect of rocking culture on keratinocyte differentiation in three-dimensional reconstitution human epidermis.

A three-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by down-regulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation. A comparative study between with and without rocking was then carried out to investigate the characteristics of the recovered differentiation by morphological and biochemical analysis. In addition, transcriptome analysis revealed the expected similar pattern between air-exposure and rocking culture, including HIF-regulating transcripts. Furthermore, the promotive effect of rocking treatment was impaired under hypoxic culture conditions (1% O2). We showed that the restored promotion of differentiation by rocking culture is mainly due to the abrogation of transcriptional events by hypoxia.

Inhibition of Transglutaminase 2 Reduces Peritoneal Injury in a Chlorhexidine-Induced Peritoneal Fibrosis Model.

Long-term peritoneal dialysis (PD) is often associated with peritoneal dysfunction leading to withdrawal from PD. The characteristic pathologic features of peritoneal dysfunction are widely attributed to peritoneal fibrosis and angiogenesis. The detailed mechanisms remain unclear, and treatment targets in clinical settings have yet to be identified. We investigated transglutaminase 2 (TG2) as a possible novel therapeutic target for peritoneal injury. TG2 and fibrosis, inflammation, and angiogenesis were investigated in a chlorhexidine gluconate (CG)-induced model of peritoneal inflammation and fibrosis, representing a noninfectious model of PD-related peritonitis. Transforming growth factor (TGF)-ß type I receptor (TGFßR-I) inhibitor and TG2-knockout mice were used for TGF-ß and TG2 inhibition studies, respectively. Double immunostaining was performed to identify cells expressing TG2 and endothelial-mesenchymal transition (EndMT). In the rat CG model of peritoneal fibrosis, in situ TG2 activity and protein expression increased during the development of peritoneal fibrosis, as well as increases in peritoneal thickness and numbers of blood vessels and macrophages. TGFßR-I inhibitor suppressed TG2 activity and protein expression, as well as peritoneal fibrosis and angiogenesis. TGF-ß1 expression, peritoneal fibrosis, and angiogenesis were suppressed in TG2-knockout mice. TG2 activity was detected by α-smooth muscle actin-positive myofibroblasts, CD31-positive endothelial cells, and ED-1-positive macrophages. CD31-positive endothelial cells in the CG model were α-smooth muscle actin-positive, vimentin-positive, and vascular endothelial-cadherin-negative, suggesting EndMT. In the CG model, EndMT was suppressed in TG2-knockout mice. TG2 was involved in the interactive regulation of TGF-ß. As inhibition of TG2 reduced peritoneal fibrosis, angiogenesis, and inflammation associated with TGF-ß and vascular endothelial growth factor-A suppression, TG2 may provide a new therapeutic target for ameliorating peritoneal injuries in PD.

Novel function of transglutaminase 2 in extracellular histone-induced acute lung injury.

Extracellular histones induce endothelial damage, resulting in lung haemorrhage; however, the underlying mechanism remains unclear. Factor XIII, as a Ca2+-dependent cross-linking enzyme in blood, mediates fibrin deposition. As another isozyme, transglutaminase 2 (TG2) has a catalytic activity distributing in most tissues. Herein, we investigated whether TG2 promotes fibrin deposition and mediates the adhesion of platelets to ECs in histone-induced acute lung injury (ALI). We evaluated the lung histology and the adhesion of platelets to endothelial cells (ECs) after injecting histones to wild-type (WT) C57BL/6J and TG2 knockout (TG2-/-) mice, and administered a TG2 inhibitor (NC9) to WT mice. Pulmonary haemorrhage was more severe in TG2-/- mice than that in WT mice. The area of fibrin deposition and the proportion of CD41+CD31+ cells were lower in TG2-/- mice than in WT mice. Pre-treatment of NC9 decreased the area of fibrin deposition and the proportion of CD41+CD31+ cells in WT mice. These results suggest that TG2 prevents from pulmonary haemorrhage in ALI by promoting the adhesion of platelets to ECs and the fibrin deposition.

Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases.

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease. To elucidate TGs' mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG's physiological and pathological functions and in developing compounds that modulate TG activity.

Spatially Resolved Identification of Transglutaminase Substrates by Proteomics in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated. Here, we clarified the distributions of TG2 protein/activity and conducted quantitative proteomics analyses of possible substrates crosslinked by TG2 on unfixed lung sections in a mouse pulmonary fibrosis model. We identified 126 possible substrates as markedly TG2-dependently increased in fibrotic lung. Gene ontology analysis revealed that these identified proteins were mostly enriched in the lipid metabolic process, immune system process, and protein transport. In addition, these proteins were enriched in 21 pathways, including phagosome, lipid metabolism, several immune responses, and protein processing in endoplasmic reticulum. Furthermore, the network analyses screened out the six clusters and top 20 hub proteins with higher scores, which are related to endoplasmic reticulum stress and peroxisome proliferator-activated receptor signals. Several enriched pathways and categories were identified, some of which were the same terms based on transcription analysis in IPF. Our results provide novel pathological molecular networks driven by protein crosslinking via TG2, which can lead to the development of new therapeutic targets for IPF.

Investigation of mouse amniotic fluid for stimulating ability of keratinocyte differentiation depending on the fetal stage.

During fetal development, the barrier function of the fetal skin is developed under specific conditions for epidermis formation. In keratinocyte differentiation, the well-orchestrated production and modification of various structural proteins are induced. We assessed the epidermal barrier function in different fetal stages by evaluating the enzymatic activity of cross-linking proteins, transglutaminases, and the permeation of fluorescence dye in the stained epidermal sections. During days 15.5-17.5 in gestation, the enzymatic activities in the epidermis appeared to increase significantly; meanwhile, dye permeation was substantially decreased, suggesting the formation of a protective barrier. For the fetal epidermis formation in the earlier stage, unclarified stimulating factors in the amniotic fluid (AF) are possible to promote barrier function by stimulating keratinocyte differentiation. Thus, we performed proteomic spectrometric (MS) analysis on the components in the AF at different fetal stages. Also, we investigated the promotive ability of the components using a cultured keratinocyte differentiation system. According to the MS analysis, the AF components appeared to exhibit stage-specific variations, where possible unique functions have been identified. We also found that adding the AF from each stage to the medium for cultured keratinocytes specifically enhanced the levels of the differentiation markers. These results provide information on the possible role of AF that contains regulatory factors on keratinocyte differentiation.

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation.

At the last stage of the blood coagulation cascade, thrombin plays a central role in the processing of fibrinogen for the polymerization and in the additional activation of Factor XIII for the stable cross-linking of fibrin. In addition, thrombin carries out possible multiple roles via processing or interaction with various functional proteins. Several studies conducted in order to elucidate additional physiological significance are ongoing. To clarify further significance of thrombin and to establish an associated disease model, we characterized the orthologue gene for medaka (Oryzias latipes), a research model fish. Tissue distribution of medaka prothrombin has been immunotechnically analyzed. Furthermore, thrombin-deficient medaka mutants were viably established by utilizing a genome-editing method. The established gene-deficient mutants exhibited retarded blood coagulation even in the heterozygous fish. Taking advantage of their ease of handling, this specific model is useful for further investigation in medical research areas on human coagulation diseases.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

Identification and characterization of substrates crosslinked by transglutaminases in liver and kidney fibrosis.

The transglutaminase (TGase) family consists of eight isozymes that catalyze Ca2+-dependent crosslink formation between glutamine and lysine residues of proteins. In the pathogenesis of various chronic diseases, among the TGase isozymes, TG2 in particular is upregulated and contributes to a critical role in fibrosis development and progression via the stabilization of extracellular matrix proteins and activation of TGF-ß. Although TG2 has been considered a key enzyme in fibrosis, the causative role of TG2 and involvement of other isozymes remain unclear. We have recently developed a comprehensive analysis method targeting the isozyme-specific substrates of TGase in liver and kidney fibrosis. In this review article, we introduce a previously developed method for determining the activity and tissue distribution of TGase and for the detecting and identification of TGase substrates in an isozyme-specific manner. Using our comprehensive analysis method, we newly characterized the overlapping profile data regarding potential substrates of TG1 and TG2 that have been identified in liver and kidney fibrosis to date. Our results obtained by comparing the specificity and similarity of potential TGase substrates between different tissue fibrosis models provide a deeper understanding regarding the specific and common pathways in disease pathogenesis and progression.

Analysis of the expression of transglutaminases in the reconstructed human epidermis using a three-dimensional cell culture.

The skin epidermis functions as a barrier to various external stresses. In the outermost layer, the terminally differentiated keratinocytes result in cornification with a tough structure by formation of a cornified envelope beneath the plasma membrane. To complete the formation of the cornified envelope, several structural proteins are cross-linked via the catalytic action of transglutaminases (TG1, TG3, TG5, and TG6). The expression and activation of these enzymes are regulated in a tightly coordinated manner during keratinocyte differentiation. We here show the system detecting the activity of the TGases using specific glutamine-donor substrate peptides in a three-dimensional culture system of keratinocytes. In this review, we summarize the roles of the epidermal enzymes and introduce a detection method that will provide a system for evaluating the skin barrier function.

Transglutaminase orthologues in medaka fish - biochemical characterization and establishment of gene-deficient mutants.

By genome analysis, seven homologous genes (orthologues) of human transglutaminases (TGases) have been identified in medaka fish (Oryzias latipes), some of which clearly corresponded to Factor XIII, TG1, and TG2. The enzymatically active-recombinant proteins for these medaka TGases have been successfully produced in bacteria or baculovirus-infected insect cell systems. Specific antibodies have been prepared and used in immunohistochemical analyses to reveal tissue distribution. Furthermore, gene-deficient medaka mutants for the genes encoding Factor XIII and TG1 have been established together with analysis of their phenotypes. Retarded cross-linking of fibrin and higher sensitivity to osmolality are observed when each gene is knocked-out. In this review, we summarize these biochemical features and the phenotypes of these gene-deficient fish.

Tschimganine and its derivatives extend the chronological life span of yeast via activation of the Sty1 pathway.

Most antiaging factors or life span extenders are associated with calorie restriction (CR). Very few of these factors function independently of, or additively with, CR. In this study, we focused on tschimganine, a compound that was reported to extend chronological life span (CLS). Although tschimganine led to the extension of CLS, it also inhibited yeast cell growth. We acquired a Schizosaccharomyces pombe mutant with a tolerance for tschimganine due to the gene crm1. The resulting Crm1 protein appears to export the stress-activated protein kinase Sty1 from the nucleus to the cytosol even under stressful conditions. Furthermore, we synthesized two derivative compounds of tschimganine, α-hibitakanine and ß-hibitakanine; these derivatives did not inhibit cell growth, as seen with tschimganine. α-hibitakanine extended the CLS, not only in S. pombe but also in Saccharomyces cerevisiae, indicating the possibility that life span regulation by tschimganine derivative may be conserved across various yeast species. We found that the longevity induced by tschimganine was dependent on the Sty1 pathway. Based on our results, we propose that tschimganine and its derivatives extend CLS by activating the Sty1 pathway in fission yeast, and CR extends CLS via two distinct pathways, one Sty1-dependent and the other Sty1-independent. These findings provide the potential for creating an additive life span extension effect when combined with CR, as well as a better understanding of the mechanism of CLS.

Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli.

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

Higher susceptibility to osmolality of the medaka (Oryzias latipes) mutants in orthologue genes of mammalian skin transglutaminases.

Transglutaminase (TG) is an essential enzyme to catalyze cross-linking reactions of epidermal proteins. Recently, we biochemically characterized human skin TG orthologues for medaka (Oryzias latipes), a model fish. By genome editing, gene-modified fishes for the two orthologues were obtained, both of which lack the ordinal enzymes. These fish appeared to exhibit higher susceptibility to osmolality at the period of larvae.

FRET-based detection of isozyme-specific activities of transglutaminases.

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

Biochemical characterization of the medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2).

Transglutaminase is an enzyme family responsible for post-translational modification such as protein cross-linking and the attachment of primary amine and/or deamidation of glutamine-residue in proteins. Medaka (Oryzias latipes), a recently established model fish, has similar functional proteins to those characterized in mammals. Previously, we found the apparent orthologues that correspond to human transglutaminases in medaka. In this study, regarding the medaka orthologue of human tissue-type transglutaminase (OlTGT), recombinant protein was expressed in an active form in bacteria cultured at low temperature. Using the recombinant protein, we biochemically characterized the enzymatic activity and also obtained a monoclonal antibody that specifically recognized OlTGT. Immunochemical analysis revealed that OlTGT was not expressed ubiquitously, unlike its mammalian orthologue, but in primarily limited tissues such as the eye, brain, spinal cord, and gas gland.

Analysis on transglutaminase 1 and its substrates using specific substrate peptide in cultured keratinocytes.

Transglutaminase (TGase) catalyzes protein cross-linking reactions essential for several biological processes. In differentiating keratinocytes, TG1 (keratinocyte-type) is crucial for the cross-linking of substrate proteins required for the complete formation of the cornified envelop, a proteinaceous supermolecule located in the outermost layer of the epidermis. TG1 expressions and its substrate were induced in cultured keratinocytes at differentiation-stage specific manner. In the cultured keratinocytes, we used the TG1-specific substrate peptide, which enables the specific detection of enzymatic activity to investigate its induction patterns. As a further application of the substrate peptide, several substrate candidates of TG1 that may be essential for cornified envelope formation were identified and characterized.

Distribution of transglutaminase family members in mouse whole body sections.

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Eight enzymes have been identified and designated as factor XIII (FXIII) and TG1-7. Expression studies of four major members, i.e., FXIII, TG1, TG2, and TG3, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. The structural and biochemical characteristics of these individual isozymes and expression analyses of TG family in some tissue extracts have been reported, but there have been no simultaneous comparative analyses of both their mRNA and protein expression patterns in tissues distributions. Thus, we developed novel experimental systems for in situ hybridization using cryofilm attached to whole body sections of neonatal mice, thereby obtaining data regarding the tissue distributions of the major TG isozymes. In this study, we performed the first detailed comparative analysis of the mRNA and protein distribution studies of TG family members in a wide range of mouse tissues. These data will be helpful for elucidating the unknown physiological and pathological functions of TGs.

Early response as shown by enhancement of transglutaminase 1 expression after cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) is caused by drugs and other stimuli, which limits the use of several therapeutic approaches. The AKI mouse model generated by intraperitoneal administration with cisplatin, one of the most widely used anti-cancer drugs, is generally applied to study on this disease. Transglutaminases are posttranslational modifying enzymes that catalyze irreversible cross-linking reactions between proteins in several biological events such as skin formation and blood coagulation. In this study, we found an increase in the expression level of transglutaminase (TG1) in the kidney of mice which had been injected with cisplatin and underwent progressive nephrotoxicity. Before the appearance of the tentative symptoms of renal failure, which is apparent by morphological damage in the kidney and increases in blood creatinine levels, both the expression level and activity of TG1 rapidly increased mainly at the proximal tubule. On the other hand, the protein expression level of another major isozyme (TG2) remained mostly unaltered. This investigation will provide a possible basal-level biomarker and also information on progression of renal failure from the aspect of the protein-modifying enzyme, transglutaminase.

Pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1) gene is suppressed by transglutaminase 2 activation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress.