👤 Hirobumi Saitoh

Snail is a zinc finger transcription factor encoded by the SNAI1 gene and triggers a cellular process termed epithelial-mesenchymal transition (EMT) upon its increased expression and/or functional activation. Snail expression and activity are regulated by various extracellular stimuli, including cytokines and environmental factors. Transforming growth factor-β (TGF-β) is a Snail inducer that functions via Smad3-mediated transcriptional activation. In the present study, we identified a distal enhancer that modulates TGF-β-induced SNAI1 expression. ChIP-seq and Hi-C analyses showed that the enhancer is located 46 kb downstream of the SNAI1 gene; in TGF-β-stimulated cells, it associates with Smad3 and interacts with the SNAI1 proximal promoter. Inhibiting the activity of the enhancer using CRISPRi attenuated TGF-β-induced SNAI1 expression, stress fiber formation, and cell motility enhancement, suggesting that the enhancer mediates TGF-β-induced EMT. The enhancer contains a Smad-binding CAGA motif and an activator protein-1 (AP-1) binding motif that function in transcriptional activation. Ras-responsive element binding protein 1 (RREB1), a transcription factor required for TGF-β-induced Snail expression, regulated the basal activity of the enhancer but not its inducibility by TGF-β. In contrast to the enhancer, the association of Smad3 with the proximal promoter was not evident. These findings suggest that the proximal promoter and the distal enhancer respond to distinct signaling cues, integrate them, and cooperatively function to drive SNAI1 expression. Show less

Computational modeling indicated that pathological high shear stress (HSS; 100 dyn/cm We used the Ibidi perfusion system to determine whether HSS applied to human PA endothelial cells (ECs) induces EndMT when compared with physiological laminar shear stress (15 dyn/cm EndMT, a feature of PAH not previously attributed to HSS, was observed. HSS did not alter the induction of transcription factors KLF (Krüppel-like factor) 2/4, but an ERG (ETS-family transcription factor) was reduced, as were histone H3 lysine 27 acetylation enhancer-promoter peaks containing ERG motifs. Consequently, there was reduced interaction between ERG and KLF2/4, a feature important in tethering KLF and the chromatin remodeling complex to DNA. In PA ECs under laminar shear stress, reducing ERG by siRNA caused EndMT associated with decreased BMPR2 (bone morphogenetic protein receptor 2), CDH5 (cadherin 5), and PECAM1 (platelet and EC adhesion molecule 1) and increased SNAI1/2 (Snail/Slug) and ACTA2 (smooth muscle α2 actin). In PA ECs under HSS, transfection of ERG prevented EndMT. HSS was then induced in mice by an aortocaval shunt, causing progressive PAH over 8 weeks. An adeno-associated viral vector (AAV2-ESGHGYF) was used to replenish ERG selectively in PA ECs. Elevated PA pressure, EndMT, and vascular remodeling (muscularization of peripheral arteries) in the aortocaval shunt mice were markedly reduced by ERG delivery. Pathological HSS reduced lung EC ERG, resulting in EndMT and PAH. Agents that upregulate ERG could reverse HSS-mediated PAH and occlusive vascular remodeling resulting from high flow or narrowed PAs. Show less

Cancer cells acquire malignant phenotypes through an epithelial-mesenchymal transition, which is induced by environmental factors or extracellular signaling molecules, including transforming growth factor-β (TGF-β). Among epithelial-mesenchymal transition-associated cell responses, cell morphological changes and cell motility are closely associated with remodeling of the actin stress fibers. Here, we examined the TGF-β signaling pathways leading to these cell responses. Through knockdown experiments in A549 lung adenocarcinoma cells, we found that Smad3-mediated induction of Snail, but not that of Slug, is indispensable for morphological changes, stress fiber formation, and enhanced motility in cells stimulated with TGF-β. Ectopic expression of Snail in SMAD3-knockout cells rescued the defect in morphological changes and stress fiber formation by TGF-β, indicating that the role of Smad3 in these responses is to upregulate Snail expression. Mechanistically, Snail is required for TGF-β-induced upregulation of Wnt5b, which in turn activates RhoA and subsequent stress fiber formation in cooperation with phosphoinositide 3-kinase. However, ectopic expression of Snail in SMAD3-knockout cells failed to rescue the defect in cell motility enhancement by TGF-β, indicating that activation of the Smad3/Snail/Wnt5b axis is indispensable but not sufficient for enhancing cell motility; a Smad3-dependent but Snail-independent pathway to activate Rac1 is additionally required. Therefore, the Smad3-dependent pathway leading to enhanced cell motility has two branches: a Snail-dependent branch to activate RhoA and a Snail-independent branch to activate Rac1. Coordinated activation of these branches, together with activation of non-Smad signaling pathways, mediates enhanced cell motility induced by TGF-β. Show less

A gain-of-function mutation in germline ABL1 causes a syndrome including congenital heart defects. However, the molecular mechanisms of this syndrome remain unknown. In this study, we found a novel ABL1 mutation in a Japanese family with ventricular septal defect, finger contracture, skin abnormalities and failure to thrive, and the molecular mechanisms of these phenotypes were investigated. Whole-exome sequencing on several family members revealed a novel mutation (c.1522A > C, p.I508L) in the tyrosine kinase domain of ABL1, and complete co-segregation with clinical presentations was confirmed in all members. Wild-type and mutant ABL1 were transfected into human embryonic kidney 293 cells for functional analysis. Western blotting confirmed that tyrosine phosphorylation in STAT5, a substrate of ABL1, was enhanced, and the novel mutation was proved to be a gain-of-function mutation. Since this novel mutation in ABL1 enhances tyrosine kinase activity, phosphorylated proteome analysis was used to elucidate the molecular pathology. The proteome analysis showed that phosphorylation in proteins such as UFD1, AXIN1, ATRX, which may be involved in the phenotypes, was enhanced in the mutant group. The onset of congenital heart defects associated with this syndrome appears to involve a mechanism caused by UFD1 common to 22q.11.2 deletion syndrome. On the other hand, AXIN1 and ATRX may be important in elucidating the mechanisms of other phenotypes, such as finger contracture and failure to thrive. Verification of these hypotheses would lead to further understanding of the pathophysiology and the development of treatment methods. Show less

Fabry disease is caused by mutations in the α-galactosidase A (GLA) gene, which is located in X-chromosome coding for the lysosomal enzyme of GLA. Among many gene mutations, E66Q mutation is under discussion for its pathogenicity because there is no clinical report showing pathological evidence of Fabry disease with E66Q mutation. A 65-year-old Japanese female was referred to our hospital for chest discomfort on effort. Transthoracic echocardiography showed severe left ventricular (LV) hypertrophy with LV outflow obstruction. Maximum LV outflow pressure gradient was 87 mmHg, and Valsalva maneuver increased the pressure gradient up to 98 mmHg. According to medical interview, one of her younger sister and a nephew died suddenly at age 42 and 36, respectively. Another younger sister also presented LV hypertrophy with outflow obstruction. Maximum LV outflow pressure gradient was 100 mmHg, and the E66Q mutation was detected similar to the case. Endomyocardial biopsy specimens presented vacuolation of cardiomyocytes, in which zebra bodies were detected by electron microscopic examination. Although the enzymatic activity of GLA was within normal range, the c. 196G>C nucleotide change, which lead to the E66Q mutation of GLA gene, was detected. We initially diagnosed her as cardiac Fabry disease based on the findings of zebra body. However, immunostaining showed few deposition of globotriaosylceramide in left ventricular myocardium, and gene mutations in the disease genes for hypertrophic cardiomyopathy (HCM), MYBPC3 and MYH6, were detected. Although the pathogenicity of the E66Q mutation cannot be ruled out, hypertrophic obstructive cardiomyopathy (HOCM) was more reasonable to explain the pathophysiology in the case. This is the confusable case of HOCM with Fabry disease with the GLA E66Q mutation. We have to take into consideration the possibility that some patients with the E66Q mutation may have similar histological findings of Fabry disease, and should be examed the possibility for harboring gene mutations associated with HCM. Show less

Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1beta and HP1gamma. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing. Show less

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software. We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells. There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin beta, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin epsilon 1, N-myristoyltransferase 1, hemoglobin alpha 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin beta 1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicin-resistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis. Show less