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Epithelial-mesenchymal transition (EMT) is a major contributor to metastatic progression and is prominently regulated by TGF-β signalling. Both EMT and TGF-β pathway components are tightly controlled by non-coding RNAs - including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) - that collectively have major impacts on gene expression and resulting cellular states. While miRNAs are the best characterised regulators of EMT and TGF-β signaling and the miR-200-ZEB1/2 feedback loop plays a central role, important functions for lncRNAs and circRNAs are also now emerging. This review will summarise our current understanding of the roles of non-coding RNAs in EMT and TGF-β signaling with a focus on their functions in cancer progression. [ABSTRACT FROM AUTHOR]

Epithelial to mesenchymal transition (EMT) is a physiological process during development where epithelial cells transform to acquire mesenchymal characteristics, which allows them to migrate and colonize secondary tissues. Many cellular signaling pathways and master transcriptional factors exert a myriad of controls to fine tune this vital process to meet various developmental and physiological needs. Adding to the complexity of this network are post-transcriptional and post-translational regulations. Among them, alternative splicing has been shown to play important roles to drive EMT-associated phenotypic changes, including actin cytoskeleton remodeling, cell-cell junction changes, cell motility and invasiveness. In advanced cancers, transforming growth factor-β (TGF-β) is a major inducer of EMT and is associated with tumor cell metastasis, cancer stem cell self-renewal, and drug resistance. This review aims to provide an overview of recent discoveries regarding alternative splicing events and the involvement of splicing factors in the EMT and TGF-β signaling. It will emphasize the importance of various splicing factors involved in EMT and explore their regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) signaling regulates cell-specific programs involved in embryonic development, wound-healing, and immune homeostasis. Yet, during tumor progression, these TGF-β-mediated programs are altered, leading to epithelial cell plasticity and a reprogramming of epithelial cells into mesenchymal lineages through epithelial-to-mesenchymal transition (EMT), a critical developmental program in morphogenesis and organogenesis. These changes, in turn, lead to enhanced carcinoma cell invasion, metastasis, immune cell differentiation, immune evasion, and chemotherapy resistance. Here, we discuss EMT as one of the critical programs associated with carcinoma cell plasticity and the influence exerted by TGF-β on carcinoma status and function. We further explore the composition of carcinoma and other cell populations within the tumor microenvironment, and consider the relevant outcomes related to the programs associated with cancer treatment resistance. [ABSTRACT FROM AUTHOR]

Tumor cells evolve in tumor microenvironment composed of multiple cell types. Among these, endothelial cells (ECs) are the major players in tumor angiogenesis, which is a driver of tumor progression and metastasis. Increasing evidence suggests that ECs also contribute to tumor progression and metastasis as they modify their phenotypes to differentiate into mesenchymal cells through a process known as endothelial-mesenchymal transition (EndoMT). This plasticity of ECs is mediated by various cytokines, including transforming growth factor-β (TGF-β), and modulated by other stimuli depending on the cellular contexts. Recent lines of evidence have shown that EndoMT is involved in various steps of tumor progression, including tumor angiogenesis, intravasation and extravasation of cancer cells, formation of cancer-associated fibroblasts, and cancer therapy resistance. In this review, we summarize current updates on EndoMT, highlight the roles of EndoMT in tumor progression and metastasis, and underline targeting EndoMT as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Calcific aortic valve disease affects the aortic side of the valve, exposed to low magnitude multidirectional ("disturbed) blood flow, more than it affects the ventricular side, exposed to high magnitude uniaxial flow. Overt disease is preceded by endothelial dysfunction and inflammation. Here we investigate the potential role of the transforming growth factor-β (TGF-β) receptor ALK5 in this process. Although ECs are always subject to shear stress due to blood flow, and their responses to shear stress are important in healthy valve development and homeostasis, low magnitude multidirectional flow can induce pathophysiological changes. Previous work has shown ALK5 to be an important mechanosensor. ALK5 transduces mechanically sensed signals via the activation of the SMAD2/3 transcriptional modulators. However, it is currently unclear precisely how ALK5-mediated shear stress responses translate into pathological changes under conditions of chronically disturbed flow. Here, we demonstrate that ALK5 mechanosensory signalling influences flow-induced endothelial leukocyte adhesion and paracellular permeability. Low magnitude multidirectional flow resulted in downregulation of the receptor, accompanied by increased SMAD2 phosphorylation, in human umbilical vein endothelial cell (HUVEC) monolayers. These changes correlated with elevated monocyte adhesion and significantly increased transendothelial transport of an albumin-sized tracer. These effects were abolished by inhibition of ALK5 kinase activity. Analysis of ALK5 expression patterns in porcine aortic valve tissue corroborated the findings from cell-based experiments. Together, these results suggest that ALK5 has a role in shear stress-associated cardiovascular disease pathology, emphasising the importance of further mechanistic investigations and supporting it as a potential therapeutic target. • Low magnitude multidirectional flow (LMMF) downregulate ALK5 expression in endothelial cells (EC). • LMMF increase SMAD2 phosphorylation in EC. • Inhibition of ALK5 abolishes LMMF-induced monocyte adhesion and transendothelial transport of an albumin-sized tracer. • ALK5 expression is higher in EC of the ventricularis of porcine aortic valve (compared with fibrosa). [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 21 (FGF21), a well-known regulator of metabolic disorders, exhibits the potential to prevent renal fibrosis by negatively regulating the transforming growth factor β (TGF-β)/Smad3 signaling pathway. Gemigliptin and other dipeptidyl peptidase-4 inhibitors are frequently used for the management of patients with type 2 diabetes. However, the protective effect of gemigliptin against renal fibrosis, particularly its potential to upregulate the expression of FGF21, remains incompletely understood. This study assessed the renoprotective effects of gemigliptin against TGF-β-induced renal fibrosis by enhancing the expression of FGF21 in the cultured human proximal tubular epithelial cell line HK-2. Treatment with FGF21 effectively prevented TGF-β-induced renal fibrosis by attenuating the TGF-β/Smad3 signaling pathway. Similarly, gemigliptin exhibited protective effects against TGF-β-induced renal fibrosis by mitigating TGF-β/Smad3 signaling through the upregulation of FGF21 expression. However, the protective effects of gemigliptin were blocked when FGF21 expression was knocked down in TGF-β-treated HK-2 cells. These results indicate that gemegliptin has the potential to exhibit protective effects against TGF-β-induced renal fibrosis by elevating FGF21 expression levels in cultured human proximal tubular epithelial cells. • FGF21 inhibits renal fibrosis by inhibiting the TGF-β/Smad3 pathway. • Gemigliptin potentially increases FGF21 expression. • Gemigliptin protects against TGF-β-induced renal fibrosis by enhancing FGF21. [ABSTRACT FROM AUTHOR]

Vascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-β) activation is critical. Understanding TGF-β activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-β activation and vascular fibrosis development. In TAK patients, PCSK5 and TGF-β expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-β in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients. Patients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-β and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-β (pro-TGF-β) to the mature form by binding the pro-TGF-β cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-β binding, decreasing TGF-β activation and ECM expression, which was also partially validated in leflunomide-treated patients. The findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-β and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-β binding, presenting a new TAK treatment approach. • Vascular fibrosis leads to vascular thickening and narrowing and further organ ischemia in TAK. • TGF-β overexpression and activation plays an important role in the progression of vascular fibrosis. • GWAS linked the PCSK5 gene locus rs6560480 to TAK. • PCSK5 promotes vascular fibrosis in TAK by inducing TGF-β expression and activation. [ABSTRACT FROM AUTHOR]

[Display omitted] • New quinazoline-sulfonamides with variable acetamide tails 3a-n were synthesized. • Compounds 3d and 3n showed antibacterial effects against eight resistant strains. • Compound 3n was extensively potent against MRSA with MIC 50 μg/mL. • Impact safety of compound 3n after in-vitro and in-vivo tests. • Compound 3n showed anti-COX-2 activity & mitigated lung TGF-β1, IL-6 and serum CRP. A library of new quinazoline pharmacophores bearing benzenesulfonamide moiety was designed and synthesized. Compounds 3a–n were screened for their in vitro antimicrobial activity against eight multidrug-resistant clinical isolates. Compounds 3d and 3n exhibited prominent antibacterial activity, specifically against MRSA. After exhibiting relative in vitro and in vivo safety, compound 3n was selected to assess its anti-inflammatory activity displaying promising COX-2 inhibitory activity compared to Ibuprofen. In vivo experimental MRSA pneumonia model was conducted on immunodeficient (irradiated) mice to reveal the antimicrobial and anti-inflammatory responses of compound 3n compared to azithromycin (AZ). Treatment with compound 3n (10 and 20 mg/kg) as well as AZ resulted in a significant decrease in bacterial counts in lung tissues, suppression of serum C-reactive protein (CRP), lung interleukin-6 (IL-6), myeloperoxidase activity (MPO) and transforming growth factor-β (TGF-β). Compound 3n showed a non-significant deviation of lung TGF-β1 from normal values which in turn controlled the lung inflammatory status and impacted the histopathological results. Molecular docking of 3n showed promising interactions inside the active sites of TGF-β and COX-2. Our findings present a new dual-target quinazoline benzenesulfonamide derivative 3n , which possesses significant potential for treating MRSA-induced pneumonia in an immunocompromised state. [ABSTRACT FROM AUTHOR]

• TGF-β is the only pathway that the largest proportion of components have opportunity recognized as a sex determination gene. • Functions and transduction of ligands, receptors and SMADs of TGF-β signaling are fully discussion. • The regulatory networks of TGF-β superfamily sex-determination genes are highly complexity and plasticity. Sex determination and differentiation in fish has always been a hot topic in genetic breeding of aquatic animals. With the advances in next-generation sequencing (NGS) in recent years, sex chromosomes and sex determining genes can be efficiently identified in teleosts. To date, master sex determination genes have been elucidated in 114 species, of which 72 species have sex determination genes belonging to TGF-β superfamily. TGF-β is the only signaling pathway that the largest proportion of components, which including ligands (amhy , gsdfy , gdf6), receptors (amhr , bmpr), and regulator (id2bby), have opportunity recognized as a sex determination gene. In this review, we focus on the recent studies about teleost sex-determination genes within TGF-β superfamily and propose several hypotheses on how these genes regulate sex determination process. Differing from other reviews, our review specifically devotes significant attention to all members of the TGF-β signal pathway, not solely the sex determination genes within the TGF-β superfamily. However, the functions of the paralogous genes of TGF superfamily are still needed ongoing research. Further studies are required to more accurately interpret the molecular mechanism of TGF-β superfamily sex determination genes. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) has been identified as a driver of therapy resistance, particularly in esophageal adenocarcinoma (EAC), where transforming growth factor beta (TGF-β) can induce this process. Inhibitors of TGF-β may counteract the occurrence of mesenchymal, resistant tumor cell populations following chemo(radio)therapy and improve treatment outcomes in EAC. Here, we aimed to identify predictive biomarkers for the response to TGF-β targeting. In vitro approximations of neoadjuvant treatment were applied to publicly available primary EAC cell lines. TGF-β inhibitors fresolimumab and A83–01 were employed to inhibit EMT, and mesenchymal markers were quantified via flow cytometry to assess efficacy. Our results demonstrated a robust induction of mesenchymal cell states following chemoradiation, with TGF-β inhibition leading to variable reductions in mesenchymal markers. The cell lines were clustered into responders and non-responders. Genomic expression profiles were obtained through RNA-seq analysis. Differentially expressed gene (DEG) analysis identified 10 positively- and 23 negatively-associated hub genes, which were bioinformatically identified. Furthermore, the correlation of DEGs with response to TGF-β inhibition was examined using public pharmacogenomic databases, revealing 9 positively associated and 11 negatively associated DEGs. Among these, ERBB2, EFNB1, and TNS4 were the most promising candidates. Our findings reveal a distinct gene expression pattern associated with the response to TGF-β inhibition in chemo(radiated) EAC. The identified DEGs and predictive markers may assist patient selection in clinical studies investigating TGF-β targeting. [Display omitted] [ABSTRACT FROM AUTHOR]

Tooth germ development involves continuous and sequential steps with reciprocal interactions between odontogenic epithelium and the adjacent mesenchyme. Several growth factors, including Wnt, are essential for tooth germ development. Molecular mechanisms underlying Wnt/β-catenin-regulated tooth germ development are poorly understood. In tooth germ rudiments culture, we recently demonstrated that Semaphorin 3A (Sema3A), an axonal guidance factor, stimulation reversed Wnt/β-catenin signaling-dependent decreased cell proliferation but did not completely rescue the morphological anomalies of tooth germ, suggesting that an uncharacterized signaling pathway may be essential in Wnt/β-catenin signaling-dependent tooth germ development. Herein, an enrichment analysis using DNA microarray data, which was obtained in our previous research, revealed that Wnt/β-catenin signaling negatively regulates YAP1 and/or TGF-β signalings. In odontogenic epithelial cells and tooth germ rudiments, Wnt/β-catenin signaling activation reduced YAP1 expression, thereby suppressing YAP1 and TGF-β signalings sequentially. Additionally, YAP1 signaling induced TGF-β2 expression to promote TGF-β signaling in the cells. Finally, Wnt/β-catenin signaling-dependent disorganized tooth germ development, in which YAP1 signaling was suppressed, was reversed by TGF-β stimulation. These results suggest that Wnt/β-catenin signaling contributes to the tooth germ development through YAP1-TGF-β signaling. • Wnt/β-catenin signaling suppressed YAP1 signaling through a reduction in YAP1 expression in odontogenic epithelial cells. • Wnt/β-catenin signaling suppressed TGF-β signaling through YAP1 singling-dependent TGF-β2 expression. • Wnt/β-catenin signaling regulates tooth germ development through TGF-β signaling in the tooth germ rudiments culture. [ABSTRACT FROM AUTHOR]

TGF-β plays a prominent role as an inducer of epithelial-mesenchymal transitions (EMTs) during development and wound healing and in disease conditions such as fibrosis and cancer. During these processes EMT occurs together with changes in cell proliferation, differentiation, communication, and extracellular matrix remodeling that are orchestrated by multiple signaling inputs besides TGF-β. Chief among these inputs is RAS-MAPK signaling, which is frequently required for EMT induction by TGF-β. Recent work elucidated the molecular basis for the cooperation between the TGF-β-SMAD and RAS-MAPK pathways in the induction of EMT in embryonic, adult and carcinoma epithelial cells. These studies also provided direct mechanistic links between EMT and progenitor cell differentiation during gastrulation or intra-tumoral fibrosis during cancer metastasis. These insights illuminate the nature of TGF-β driven EMTs as part of broader processes during development, fibrogenesis and metastasis. [ABSTRACT FROM AUTHOR]

The ascites ecosystem in ovarian cancer is inhabited by complex cell types and is bathed in an environment rich in cytokines, chemokines, and growth factors that directly and indirectly impact metabolism of cancer cells and tumor associated cells. This milieu of malignant ascites, provides a 'rich' environment for the disease to thrive, contributing to every aspect of advanced ovarian cancer, a devastating gynecological cancer with a significant gap in targeted therapeutics. In this perspective we focus our discussions on the 'acellular' constituents of this liquid malignant tumor microenvironment, and how they influence metabolic pathways. Growth factors, chemokines and cytokines are known modulators of metabolism and have been shown to impact nutrient uptake and metabolic flexibility of tumors, yet few studies have explored how their enrichment in malignant ascites of ovarian cancer patients contributes to the metabolic requirements of ascites-resident cells. We focus here on TGF-βs, VEGF and ILs, which are frequently elevated in ovarian cancer ascites and have all been described to have direct or indirect effects on metabolism, often through gene regulation of metabolic enzymes. We summarize what is known, describe gaps in knowledge, and provide examples from other tumor types to infer potential unexplored roles and mechanisms for ovarian cancer. The distribution and variation in acellular ascites components between patients poses both a challenge and opportunity to further understand how the ascites may contribute to disease heterogeneity. The review also highlights opportunities for studies on ascites-derived factors in regulating the ascites metabolic environment that could act as a unique signature in aiding clinical decisions in the future. [ABSTRACT FROM AUTHOR]

TGF-βs regulate macrophage responses, by activating Smad2/3. We have previously demonstrated that macrophage-specific Smad3 stimulates phagocytosis and mediates anti-inflammatory macrophage transition in the infarcted heart. However, the role of macrophage Smad2 signaling in myocardial infarction remains unknown. We studied the role of macrophage-specific Smad2 signaling in healing mouse infarcts, and we explored the basis for the distinct effects of Smad2 and Smad3. In infarct macrophages, Smad3 activation preceded Smad2 activation. In contrast to the effects of Smad3 loss, myeloid cell-specific Smad2 disruption had no effects on mortality, ventricular dysfunction and adverse remodeling, after myocardial infarction. Macrophage Smad2 loss modestly, but transiently increased myofibroblast density in the infarct, but did not affect phagocytic removal of dead cells, macrophage infiltration, collagen deposition, and scar remodeling. In isolated macrophages, TGF-β1, −β2 and -β3, activated both Smad2 and Smad3, whereas BMP6 triggered only Smad3 activation. Smad2 and Smad3 had similar patterns of nuclear translocation in response to TGF-β1. RNA-sequencing showed that Smad3, and not Smad2, was the main mediator of transcriptional effects of TGF-β on macrophages. Smad3 loss resulted in differential expression of genes associated with RAR/RXR signaling, cholesterol biosynthesis and lipid metabolism. In both isolated bone marrow-derived macrophages and in infarct macrophages, Smad3 mediated synthesis of Nr1d2 and Rara , two genes encoding nuclear receptors, that may be involved in regulation of their phagocytic and anti-inflammatory properties. In conclusion, the in vivo and in vitro effects of TGF-β on macrophage function involve Smad3, and not Smad2. The relative role of Smad2 and Smad3 signaling in regulation of macrophage phenotype in myocardial infarction. A: Smad3 exhibits early activation in infarct macrophages and is involved in activation of a phagocytic program and in anti-inflammatory transition. In contrast, macrophage Smad2 is activated later and does not play a central role in repair and remodeling of the infarcted heart. All 3 TGF-β isoforms activate both Smad2 and Smad3. Macrophage Smad3, but not Smad2, is also activated by BMP6 stimulation. Phagocytosis also activates Smad3 through actions that may be TGF-independent. Although distinct patterns of binding to anchor proteins, and the more promiscuous interactions of Smad3 with nuclear import proteins have been previously suggested as mechanisms that may account for the higher impact of Smad3 vs. Smad2 in other cell types, our experiments showed similar patterns of activation and nuclear translocation of Smad2 and Smad3 in isolated macrophages. Differences in the regulatory sequences targeted by Smad2 and Smad3 and distinct patterns of co-operation with transcriptional regulators are the most likely basis for the differential actions. Our RNA-sequencing analysis shows that Smad3, and not Smad2, mediates TGF-β1-induced transcription in macrophages, and suggests that Smad3 may modulate the RAR/RXR axis. Transcription of the nuclear receptors Nr1d2 and Rara in macrophages is dependent on Smad3. The proteins encoded by these 2 genes may be involved in the anti-inflammatory and phagocytosis-activating effects of Smad3. ITG, integrin; PS, phosphatidylserine; MFGE8, milk fat globule-EGF factor 8. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) [Display omitted] • TGF-βs trigger rapid Smad2/Smad3 activation and nuclear translocation in macrophages. • Smad3 activation precedes Smad2 activation in infarct macrophages. • Macrophage Smad2 does not play a critical role in cardiac repair and remodeling. • In vitro, Smad3 and not Smad2 mediates the transcriptional effects of TGF-β1. • Smad3 loss is associated with modulation of the RAR/RXR axis. [ABSTRACT FROM AUTHOR]

• The importance of myostatin and other members of the TGF-β signaling pathway in muscle atrophy conditions such as muscular dystrophy, sarcopenia, and cachexia. • The role of myostatin in regulating the balance between muscle protein synthesis and degradation. • Potential therapeutic strategies for muscle atrophy treatment targeting TGF-β signaling. Skeletal muscle, comprising a significant proportion (40 to 50 percent) of total body weight in humans, plays a critical role in maintaining normal physiological conditions. Muscle atrophy occurs when the rate of protein degradation exceeds protein synthesis. Sarcopenia refers to age-related muscle atrophy, while cachexia represents a more complex form of muscle wasting associated with various diseases such as cancer, heart failure, and AIDS. Recent research has highlighted the involvement of signaling pathways, including IGF1-Akt-mTOR, MuRF1-MAFbx, and FOXO, in regulating the delicate balance between muscle protein synthesis and breakdown. Myostatin, a member of the TGF-β superfamily, negatively regulates muscle growth and promotes muscle atrophy by activating Smad2 and Smad3. It also interacts with other signaling pathways in cachexia and sarcopenia. Inhibition of myostatin has emerged as a promising therapeutic approach for sarcopenia and cachexia. Additionally, other TGF-β family members, such as TGF-β1, activin A, and GDF11, have been implicated in the regulation of skeletal muscle mass. Furthermore, myostatin cooperates with these family members to impair muscle differentiation and contribute to muscle loss. This review provides an overview of the significance of myostatin and other TGF-β signaling pathway members in muscular dystrophy, sarcopenia, and cachexia. It also discusses potential novel therapeutic strategies targeting myostatin and TGF-β signaling for the treatment of muscle atrophy. [ABSTRACT FROM AUTHOR]

[Display omitted] Activin receptor‑like kinase-5 (ALK5) is an outstanding member of the transforming growth factor-β (TGF-β) family. (TGF-β) signaling pathway integrates pleiotropic proteins that regulate various cellular processes such as growth, proliferation, and differentiation. Dysregulation within the signaling pathway can cause variety of diseases, such as fibrosis, cardiovascular disease, and especially cancer, rendering ALK5 a potential drug target. Hence, various small molecules have been designed and synthesized as potent ALK5 inhibitors. In this review, we shed light on the current ATP-competitive inhibitors of ALK5 through diverse heterocyclic based scaffolds that are in clinical or pre-clinical phases of development. Moreover, we focused on the binding interactions of the compounds to the ATP binding site and the structure–activity relationship (SAR) of each scaffold, revealing new scopes for designing novel candidates with enhanced selectivity and metabolic profiles. [ABSTRACT FROM AUTHOR]

Nephronophthisis (NPHP) is a kind of ciliopathy. Interstitial fibrosis occurs at the early stage of the disease. TGF-β/Smad is a key signaling pathway in regulating interstitial fibrosis and epithelial-mesenchymal transition (EMT). In this study, we explored the activation of the TGF-β/Smad signaling pathway and EMT in NPHP1-defective MDCK cells to further understand the pathogenesis of NPHP. NPHP1 -knockdown (NPHP1KD) MDCK cells were constructed by recombinant lentiviral short hairpin RNA, and NPHP1-knockout (NPHP1 KO) MDCK cells were constructed by using the CRISPR/Cas9 technique. The morphology and migration ability were observed under a microscope. Western blotting was used to detect the expression of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), fibroblast-specific protein-1(FSP1), TGF-β1, Smad2, Smad3, p-Smad3, Smad4 and Smad7. The localization of Smad3 was determined by immunofluorescence assay. NPHP1 KD and NPHP1KO MDCK cells were spindle-shaped and presented EMT-like changes. E-cadherin and β-catenin expression decreased, while α-SMA and FSP1 expression increased; the TGF-β/Smad signaling pathway was activated, Smad2, Smad3, p-Smad3 and Smad4 expression increased, Smad3 translocated to nuclear and Smad7 expression decreased compared with those in wild type MDCK cells. Overexpression of Smad7 reversed these changes to different degrees. Our results indicate that NPHP1 defects induce the activation of the TGF-β/Smad signaling pathway and EMT in MDCK cells. These factors may be implicated in the pathogenesis of interstitial fibrosis in NPHP. • NPHP1 defects induce the activation of the TGF-β/Smad signaling pathway. • NPHP1 defects induce EMT in MDCK cells. • TGF-β/Smad signaling may be related with the pathogenesis of interstitial fibrosis in NPHP. [ABSTRACT FROM AUTHOR]

Nickel compounds are environmental toxicants, prevalent in the atmosphere due to their widespread use in several industrial processes, extensive consumption of nickel containing products, as well as burning of fossil fuels. Exposure to nickel is associated with a multitude of chronic inflammatory lung diseases including asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. In addition, nickel exposure is implicated in the development of nasal and lung cancers. Interestingly, a common pathogenic mechanism underlying the development of diseases associated with nickel exposure is epithelial-mesenchymal transition (EMT). EMT is a process by which the epithelial cells lose their junctions and polarity and acquire mesenchymal traits, including increased ability to migrate and invade. EMT is a normal and essential physiological process involved in differentiation, development and wound healing. However, EMT also contributes to a number of pathological conditions, including fibrosis, cancer and metastasis. Growing evidence suggest that EMT induction could be an important outcome of nickel exposure. In this review, we discuss the role of EMT in nickel-induced lung diseases and the mechanisms associated with EMT induction by nickel exposure. [ABSTRACT FROM AUTHOR]

Fibroblast migration is closely regulated by the mechanical characteristics in surrounding microenvironment. While increased interstitial hydrostatic pressure (HP) is a hallmark in many pathological and physiological conditions, little is known about how the HP affects fibroblast motility. Using cell-culture chips with elevated HP conditions, we showed that 20 cmH 2 O HP significantly accelerated fibroblast migration. The HP-induced migration acceleration was dependent on the augmentation of transforming growth factor-β 1 , and correlated with the activation of filamin A via the phosphorylation of p38 mitogen-activated protein kinase. Our results suggest that interstitial HP elevation associated with various pathological states could significantly regulate fibroblast migration. • Increased hydrostatic pressure (HP) accelerates fibroblast migration. • The increased HP-induced migration acceleration is dependent on the augmentation of transforming growth factor-β 1. • The HP-induced migration acceleration correlates with the p38 MAPK phosphorylation-dependent activation of filamin-A. [ABSTRACT FROM AUTHOR]

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths because of its strong metastatic ability. It is important to understand NSCLC's molecular mechanisms of metastasis. RhoJ, a protein that belongs to the Rho family of small GTPases, regulates endothelial motility, angiogenesis, and adipogenesis. Recently, bioinformatics analysis showed that NSCLC patients with lower RhoJ expression had a worse survival outcome than those with high RhoJ expression. However, little is known about RhoJ's role in NSCLC. In the present study, we demonstrated that RhoJ knockdown accelerated TGF-βmediated epithelial-to-mesenchymal transition (EMT), an important cancer metastasis process, in A549 and PC-9 cells. Furthermore, using Matrigel-coated transwell chambers, we showed that RhoJ knockdown enhanced the invasion capacity of A549 cells that had undergone EMT. Also, reduced RhoJ expression increased Smad3 phosphorylation and Snail expression during the EMT process. Our results provide the first evidence of a potential novel role for RhoJ in the inhibition of EMT via modulation of the TGF-β–Smad signaling pathway, and shed new light on the mechanisms underlying EMT in NSCLC. • RhoJ knockdown promotes TGF-β1-mediated epithelial-to-mesenchymal transition (EMT) in human non-small-cell lung cancer cells. • RhoJ knockdown enhances the invasion capacity of A549 cells that had undergone EMT. • Reduced RhoJ expression enhances Smad3 phosphorylation and Snail expression during TGF-β1-mediated EMT in A549 cells. [ABSTRACT FROM AUTHOR]

Posterior subcapsular cataract (PSC) frequently develops as a complication in patients with retinitis pigmentosa (RP). Despite numerous scientific investigations, the intricate pathomechanisms underlying cataract formation in individuals affected by RP remain elusive. Therefore, our study aims to elucidate the potential pathogenesis of cataracts in an RP model using splicing factor subunit 3b (sf3b4) mutant zebrafish. By analyzing our previously published transcriptome dataset, we identified that, in addition to RP, cataract was listed as the second condition in our transcriptomic analysis. Furthermore, we confirmed the presence of nucleus retention in the lens fiber cells, along with abnormal cytoskeleton expression in both the lens fiber cells and lens epithelial cells in sf3b4 -depleted fish. Upon closer examination, we identified 20 differentially expressed genes (DEGs) that played a role in cataract formation, with 95 % of them related to the downregulation of structural lens proteins. Additionally, we also identified that among all the DEGs, 13 % were associated with fibrotic processes. It seems that the significant upregulation of inflammatory mediators, in conjunction with TGF-β signaling, plays a central role in the cellular biology of PSC and posterior capsular opacification (PCO) in sf3b4 mutant fish. In summary, our study provides valuable insights into cataract formation in the RP model of sf3b4 mutants, highlighting its complexity driven by changes in structural lens proteins and increased cytokines/growth factors. [ABSTRACT FROM AUTHOR]

Ruditapes philippinarum is an economically important marine shellfish aquaculture species, and it has the ability to regenerate its siphons. To gain a greater understanding of the molecular mechanisms at work during siphon regeneration and to provide evidence for morphological regeneration, we examined transcriptome responses of siphon tissue of R. philippinarum during regeneration and observed regenerative siphons under the stereomicroscope. The overall process of siphon regeneration was dissected based on the morphological changes of siphon and the identification of up-regulated key differentially expressed genes (DEGs). The protein biosynthesis and metabolism played important roles in wound healing and siphon regeneration of R. philippinarum. Transcriptomic analysis identified the Wnt and TGF-β signaling pathways by focusing on the function and expression pattern of genes in these pathways during siphon regeneration. In addition, we carried out a genome-wide identification and phylogenetic analysis of TGF-β superfamily in R. philippinarum. The expression profiles of the TGF-β superfamily genes were analyzed in eight adult tissues (adductor muscle, mantle, foot, gill, siphon, digestive gland, gonad, and labial palp) and regenerative siphon. This study shed new light on the process of morphological regeneration and regenerative mechanism of siphon of R. philippinarum. • Morphological observation of siphon regeneration under the SEM showed the process of siphon regeneration. • Transcriptomic analysis identified the functional DEGs during siphon regeneration. • Wnt signaling pathway plays an important role in wound healing and the blastemal formation. • The siphon regeneration of Ruditapes philippinarum showed the cell proliferation-dependent and blastemal-based regeneration. • The first identification and gene structure analysis of TGF-β superfamily were conducted in Ruditapes philippinarum. [ABSTRACT FROM AUTHOR]

Myocardial ischemia-reperfusion (MIR) represents critical challenge for the treatment of acute myocardial infarction diseases. Presently, identifying the molecular basis revealing MIR progression is scientifically essential and may provide effective therapeutic strategies. Phosphoglycerate mutase 1 (PGAM1) is a key aerobic glycolysis enzyme, and exhibits critical role in mediating several biological events, such as energy production and inflammation. However, whether PGAM1 can affect MIR is unknown. Here we showed that PGAM1 levels were increased in murine ischemic hearts. Mice with cardiac knockout of PGAM1 were resistant to MIR-induced heart injury, evidenced by the markedly reduced infarct volume, improved cardiac function and histological alterations in cardiac sections. In addition, inflammatory response, apoptosis and fibrosis in hearts of mice with MIR operation were significantly alleviated by the cardiac deletion of PGAM1. Mechanistically, the activation of nuclear transcription factor κB (NF-κB), p38, c-Jun NH2-terminal kinase (JNK) and transforming growth factor β (TGF-β) signaling pathways were effectively abrogated in MI-operated mice with specific knockout of PGAM1 in hearts. The potential of PGAM1 suppression to inhibit inflammatory response, apoptosis and fibrosis were verified in the isolated cardiomyocytes and fibroblasts treated with oxygen-glucose deprivation reperfusion (OGDR) and TGF-β, respectively. Importantly, PGAM1 directly interacted with TGF-β to subsequently mediate inflammation, apoptosis and collagen accumulation, thereby achieving its anti-MIR action. Collectively, these findings demonstrated that PGAM1 was a positive regulator of myocardial infarction remodeling due to its promotional modulation of TGF-β signaling, indicating that PGAM1 may be a promising therapeutic target for MIR treatment. • Cardiac PGAM1 knockout alleviates heart failure in mice after MI. • Cardiac PGAM1 deletion attenuates inflammation and fibrosis after MI in vivo. • PGAM1 interacts with TGF-β to modulate inflammation, apoptosis and fibrosis in vitro. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is one of the greatest health burdens with an increasing global prevalence. Renal fibrosis (RF) is the hallmark of all forms of CKD which shows a strong positive correlation with severity of the disease. However, there are no therapeutic options available for treatment of RF. In the present study, we used an animal model based on unilateral ureteral obstruction (UUO), for renal injury and fibrosis. The UUO animals were treated with soluble guanylyl cyclase (sGC) stimulator, riociguat (RIO) (1, 3 and 10 mg/kg) to investigate its possible renoprotective effects. Kidneys of animals treated with RIO were found to show less abnormalities as compared to UUO control. Further, the levels of proinflammatory cytokines were reduced in RIO treated group. Furthermore, administration of RIO reduced expression of collagen-1, TGF-β, CTGF, α-SMA, vimentin along with transcription factors including Snail and Slug. The results of the present study provided strong evidence to support the antifibrotic activity of RIO. • Riociguat, a soluble guanylyl cyclase stimulator ameliorates renal injury. • Riociguat shows potent antioxidant and anti-inflammatory activities. • The Riociguat inhibited master regulators of EMT, Snail and Slug. [ABSTRACT FROM AUTHOR]

Fibrosis is attributed to excess deposition of extracellular matrix (ECM) proteins including collagen and is associated with various organ dysfunction. This excessive ECM is produced by myofibroblasts, which are differentiated from various cells by a variety of stimuli, represented by TGF-β. However, molecular mechanisms for the regulation of ECM production in myofibroblasts remain obscure. In this study, we demonstrate that the expression of drebrin, which binds to and increases the stability of actin filament in neurons, is increased in mouse hearts and lungs upon fibrosis. Drebrin is mainly expressed in myofibroblasts in the fibrotic hearts and lungs and promotes the expression of fibrosis-related genes, such as Acta2 and Col1a1. Taken together, our study identifies drebrin as a molecule that promotes the production of fibrosis-related genes in myofibroblasts. • Dbn1 expression is increased in infarcted mouse hearts. • Dbn1 is predominantly expressed in myofibroblasts in infarcted mouse hearts. • Knockdown of Dbn1 attenuates fibrosis-related gene production in myofibroblasts. • Dbn1 induced during differentiation of lung myofibroblasts promotes lung fibrosis. [ABSTRACT FROM AUTHOR]

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone, is involved in multiple cellular functions. We previously demonstrated that Hsc70 is required for TGF-β-induced Smad signaling in mesenchymal-like NRK-49F cells. In the present study, to compare the Hsc70 functions in TGF-β-related signaling between epithelial and mesenchymal cells, we examined the effect of Hsc70 downregulation on TGF-β-induced signaling in epithelial-like NRK-52E cells. TGF-β-induced Smad signaling was suppressed in cells treated with small interfering RNA (siRNA) for Hsc70. Interestingly, despite interference with TGF-β signaling, TGF-β-induced suppression of E-cadherin expression was not affected by Hsc70 knockdown. Instead, Hsc70 knockdown itself caused the suppression of E-cadherin expression at the transcription level in cells treated with Hsc70 siRNA. We also examined the effects of Hsc70 knockdown on the level of E-cadherin-gene repressors, such as Snail1, Slug, Zeb1, Zeb2, and Twist1, and found that transcription of the repressors was upregulated after 24- or 36-h treatment with Hsc70 siRNA. Collectively, these results indicate that, in addition to a supportive role in TGF-β-induced signaling, Hsc70 supports E-cadherin expression through downregulation of the E-cadherin-gene repressors in NRK-52E cells, suggesting that Hsc70 plays a functional role to maintain the epithelial cell phenotype. • Hsc70 knockdown suppresses TGF-β-induced Smad signaling in epithelial NRK-52E cells. • Hsc70 knockdown downregulates E-cadherin, regardless of the effect on TGF-β signaling. • Hsc70 knockdown upregulates E-cadherin-gene repressors. • Hsc70 is required for the maintenance of E-cadherin expression in NRK-52E cells. [ABSTRACT FROM AUTHOR]

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-β. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1 , Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-β. Surprisingly, addition of TGF-β induced a set of genes, including Slc28a2 , Tubb2a, and Cpe , that were expressed preferentially in fetal IECs. Moreover, TGF-β significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs. • A wound repair model of untransformed intestinal epithelial cells was established. • TGF-b significantly promoted wound repair response and fetal-like gene expression. • TGF-b significantly enhanced the peak velocity of migrating epithelial cells. [ABSTRACT FROM AUTHOR]

Kidney is one of the main target organs in Fabry disease, a lysosomal X-linked genetic disorder. Renal involvement is characterized by proteinuria and progressive chronic kidney disease leading to end-stage renal disease. Pathogenic mechanisms in the progression of renal damage in Fabry disease are not thoroughly known yet. The lysosomal Gb3 deposition is the first step of complex pathological pathways resulting in renal sclerosis/fibrosis. Our hypothesis is that Fabry disease associated cellular alterations in tubular cells induce the production of TGF-β1, which mediate transdifferentiation of renal cells into myofibroblasts resulting in fibrosis of renal tissue. The aim of this work is to study the mechanisms leading to fibrosis in kidney from human Fabry patients. Fifteen renal biopsies from naïve Fabry patients were included. Histological and immunohistochemical analysis was carried out. Positive staining for TGF-β1 was found in tubular epithelial cells in biopsies from Fabry patients. Apoptosis was determined by active caspase 3 staining in tubular and mesangial glomerular cells. Due to TGF-β1 is the main profibrotic cytokine and induces accumulation of myofibroblasts, we performed a study for its marker α-smooth muscle actin (α-SMA). This study revealed expression of α-SMA on pericytes surrounding peritubular capillaries, smooth muscle cells of blood vessels, mesangial cells and periglomerular zone. TGF-β1 is produced mainly by tubular cells in Fabry kidney biopsies probably inducing cellular trans-differentiation of renal cells into myofibroblasts. A positive staining for a marker of myofibroblasts was present, affirming the presence of those profibrotic cells. These results show for the first time that TGF-β1 is expressed in human renal tissue from Fabry patients, and that this profibrotic cytokine is mainly produced by proximal tubular cells. In addition both, peritubular interstitium and glomeruli, presented cells positive for myofibroblasts markers. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer (EOC) is usually diagnosed at advanced stages with highly variable clinical outcomes, even among patients with similar clinical characteristics and treatments. Host immune system plays a pivotal role in EOC pathogenesis and progression. Here, we assessed the clinical significance of 192 single nucleotide polymorphisms (SNPs) on 34 immune-system related genes in EOC patients. Two hundred and thirty advanced EOC patients treated with platinum-based chemotherapy were included. Germ-line DNA was analyzed with Illumina GoldenGate Genotyping Assay. Nineteen polymorphisms were significantly associated with overall survival (OS), 17 with progression free survival (PFS) and 20 with platinum-free interval (PFI). Of the 8 polymorphisms associated with all three outcomes, 7 SNPs belonged to genes involved in the TGF-β pathway. A genetic score was built considering the unfavourable genotypes (UGs) of these 7 polymorphisms (group 0–2 UGs: presence of 0, 1, or 2 UGs; group 3–4 UGs: 3 or 4 UGs; group 5–7: 5, 6, or 7 UGs). According to this score, OS decreased as the number of UGs increased (median OS: 0–2 UGs = not reached, 3–4 UGs = 44.6 and 5–7 UGs = 19.3 months, p < 0.0001). The same trend was observed also for PFS (median PFS: 0–2 UGs = 21.5, 3–4 UGs = 17.3 and 5–7 UGs = 11 months, p < 0.0001) and PFI (median PFI: 0–2 UGs = 16.6, 3–4 UGs = 9.8 and 5–7 UGs = 3.8 months, p < 0.0001). The score was validated by permutation analysis. The proposed TGF-β pathway score could be useful to define prognosis and platinum sensitivity of advanced EOC patients. Image 1 • Germ-line polymorphisms in immune system were significantly associated with the outcome of advanced ovarian cancer patients. • TGF-β signaling emerged as a pathway with a possible role in the definition of patient's prognosis and platinum-sensitivity. • The score based on unfavourable genotypes in TGF- β signaling was able to stratify patients according to PFI, PFS and OS. [ABSTRACT FROM AUTHOR]

The Dpp signaling, as one of the branches within the TGF-β superfamily, plays a crucial role in regulating various biological processes in insects. However, its impact on female reproduction through vitellogenesis remains unclear. In this study, the expression profiles implied that the Dpp signaling genes, including Dpp , Punt , Mad, and Medea , were up-regulated during reproductive development in the ovary of Colaphellus bowringi. Knockdown of these five Dpp signaling genes revealed significant effects of Dpp , Tkv , Mad, and Medea on ovarian development through vitellogenesis in the fat body. Our finding further indicated that Dpp signaling influences the expression of 20-hydroxyecdysone (20E) receptor and responsive genes in the fat body. Additionally, knockdown of 20E receptor EcR resulted in similar phenotypes as observed in the Dpp pathway genes knockdown, implying a regulatory role for Dpp signaling via EcR in vitellogenesis. Furthermore, knocking down Dpp , Tkv , and EcR in female adults led to a reduction in total dry weight and protein content, as well as the expression of mTOR , a factor linked to protein intake. These results suggest that the Dpp signaling pathway modulates vitellogenesis by impacting the AA/TOR-mediated 20E pathway in the fat body, providing novel insights into the network governing insect reproduction and offering potential targets for controlling female pest reproduction. [Display omitted] • Knocking down of Dpp , Tkv , Mad and Medea inhibits female vitellogenin expression in the fat body of Colaphellus bowringi. • Dpp signaling modulates vitellogenin expression by specifically targeting the 20E signaling transduction in the fat body. • The mTOR-mediated AA/TOR pathway may play a pivotal role in mediating the influence of Dpp-20E signaling on vitellogenesis. [ABSTRACT FROM AUTHOR]

The exocyst, an evolutionarily conserved octomeric protein complex, has been demonstrated as an essential component for vesicle tethering during cell exocytosis, and participates in various physiological processes in the cell. Although subunits of the exocyst complex have been reported to be involved in the regulation of TGF-β induced cancer cell migration and epithelial-mesenchymal transition (EMT), the potential function of Sec3 in these regulated processes remains unclear. Here, we show that Sec3 knockdown abolishes TGF-β stimulated A549 lung cancer cell migration in vitro and causes defects in the regulated EMT process. In addition, we find that depletion of Sec3 significantly inhibits TGF-β stimulated Akt phosphorylation in A549 cells, whereas the increase of Smad2 phosphorylation is unaffected. Furthermore, replenishment of an RNAi-resistant form of Sec3 is shown to restore the defects of TGF-β induced cell migration, EMT and Akt signaling activation. In summary, our study provides evidence that Sec3 is involved in TGF-β induced cell migration and EMT processes, presumably through the regulation of PI3K/Akt signaling activation in A549 cancer cells. • Sec3 knockdown abolishes TGF-β induced A549 cell migration. • Sec3 knockdown inhibits TGF-β stimulated Akt phosphorylation, but not that of Smad2. • Replenishment of Sec3 restores TGF-β induced cell migration and EMT. • Sec3 may have a unique function in regulation of TGF-β induced EMT. [ABSTRACT FROM AUTHOR]

Kruppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays a role in terminal differentiation of epidermal keratinocytes. There are conflicting reports regarding the role of KLF4 in tumor development, with both the tumor suppressive and/or oncogenic properties depending on different conditions and cell types. In this study, we investigated the functional importance of KLF4 in cutaneous squamous cell carcinoma (SCC). Immunohistochemistry showed that KLF4 expression was relatively low in SCC lesion compared to normal epidermis. To examine the effects of KFL4, we transduced SCC lines (SCC12 and SCC13 cells) with the KLF4-expressing recombinant adenovirus. Overexpression of KLF4 significantly decreased cell proliferation and colony forming activity. In addition, overexpression of KLF4 markedly reduced invasive potential, along with the downregulation of epithelial-mesenchymal transition (EMT)-related molecules. In a mechanistic study, KLF4 inhibited SOX2, of which expression is critical for tumor initiation and growth of SCC. Further investigations indicated that SOX2 expression is induced by TGF-β/SMAD signaling, and that overexpression of KLF4 inhibited SMAD signaling via upregulation of SMAD7, an important inhibitory SMAD molecule. Based on these data, KLF4 plays a tumor suppressive role in cutaneous SCC cells. • Overexpression of KLF4 decreased the cell proliferation, colony forming activity, invasion and EMT potential in cutaneous SCC cells. • Overexpression of KLF4 decreased SOX2 level via inhibition of SMAD2 phosphorylation. • Overexpression of KLF4 inhibited SMAD signaling via upregualtion of SMAD7, an inhibitory SMAD molecule. [ABSTRACT FROM AUTHOR]

• Cell treatment with IFNαenhances TGFβ-induced apoptosis and caspase 8 activation. • IFNα upregulates PML isoforms and TGFβ induces nuclear PML SUMOylation. • SUMOylated PML and caspase 8 were found in the nuclear matrix in response to TGF-β. • PMLIV C-terminal is needed for the recruitment of caspase 8 within PML NBs. • TGF-β promotes caspase-dependent PML degradation resulting in PML NB disruption. ProMyelocytic Leukemia (PML) protein is essential for the formation of nuclear matrix-associated organelles named PML nuclear bodies (NBs) that act as a platform for post-translational modifications and protein degradation. PML NBs harbor transiently and permanently localized proteins and are associated with the regulation of several cellular functions including apoptosis. There are seven PML isoforms, six nuclear (PMLI-VI) and one cytoplasmic (PMLVII), which are encoded by a single gene via alternative RNA splicing. It has been reported that murine PML-null primary cells are resistant to TGF-β-induced apoptosis and that cytoplasmic PML is an essential activator of TGF-β signaling. The role and the fate of interferon (IFN)-enhanced PML NBs in response to TGF-β have not been investigated. Here we show that IFNα potentiated TGF-β-mediated apoptosis in human cells. IFNα or ectopic expression of PMLIV, but not of PMLIII, enhanced TGF-β-induced caspase 8 activation. In response to TGF-β, both PMLIII and PMLIV were conjugated to SUMO and shifted from the nucleoplasm to the nuclear matrix, however only PMLIV, via its specific C-terminal region, interacted with caspase 8 and recruited it within PML NBs. This process was followed by a caspase-dependent PML degradation and PML NB disruption. Taken together, these findings highlight the role of PML NBs in the enhancement by IFN of TGF-β-induced apoptosis and caspase 8 activation. [ABSTRACT FROM AUTHOR]

Our previous study reported that cancer upregulated gene (CUG)2, a novel oncogene, induces both faster cell migration and anti-cancer drug resistance. We thus wonder whether CUG2 also induces stemness, a characteristic of cancer stem cells (CSCs) and further examine the molecular mechanism of this phenotype. To test that CUG2 induces stemness, we examined expression of stemness-related factors. Overexpression of CUG2 enhanced expression levels of stemness-related factors in human lung carcinoma A549 and immortalized bronchial BEAS-2B cells. Consequently, CUG2 increased cellular spherical cluster forming ability. Overexpression of CUG2 also induced tumor formation in xenotransplanted nude mice whereas transplantation of control cells failed to, implying that CUG2 possesses malignant tumorigenic potential. We paid attention to nucleophosmin (NPM1) for its known interaction with CUG2. Suppression of NPM1 hindered the CUG2-mediated stemness-like phenotypes and diminished TGF-β transcriptional activity and signaling. TGF-β increased stemness-like phenotypes in the control cells whereas TGF-β inhibitor blocked induction of the phenotypes, indicating that NPM1 is required for CUG2-mediated stemness-like phenotypes through TGF-β signaling. Furthermore, the suppression of Smad- and non-Smad-dependent TGF-β signaling pathways also prevented CUG2 from inducing stemness-like phenotypes. Altogether, we suggest that the novel CUG2 oncogene promotes cellular transformation and stemness, mediated by nuclear NPM1 protein and TGF-β signaling. • Overexpression of CUG2 promotes stemness-like properties. • NPM1 is involved in CUG2-mediated stemness-like phenotypes. • NPM1 utilizes TGF-β signaling for CUG2-mediated stemness-like phenotypes. • TGF-β signaling plays a crucial role in CUG2-mediated stemness-like phenotypes. [ABSTRACT FROM AUTHOR]

TGF-βs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-β1, -β2 and -β3, but not angiotensin 2 and bone morphogenetic proteins-2, −4 and −7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression. • Smad2 is activated in TGF-β-stimulated fibroblasts, and in infarct myofibroblasts. • Myofibroblast-specific Smad2 loss transiently attenuates post-infarction remodeling. • Smad3, but not Smad2 signaling, mediates formation of organized myofibroblast arrays. • Smad3, but not Smad2 mediates integrin and RhoA expression in cardiac fibroblasts. • Smad3 and Smad2 regulate expression of planar cell polarity genes. [ABSTRACT FROM AUTHOR]

To investigate the thoracic irradiation induced abscopal effect on distal testes and the underlying inflammatory factors, the rats were irradiated on right thorax with fractionated doses. It was found the testes structures were damaged including disorder of spermatogenic cell arrangement and decrease of sperm number. Moreover, the expressions of caspase-3 and caspase-8 in testis tissue were enhanced, and the concentrations of TGF-β and TNF-α in the rat serum were increased. When TM4 cells were treated with the conditioned medium (CS) collected from irradiated rat, the cellular ROS and apoptosis was significantly increased. When the CS was neutralized with anti-TGF-β, its toxic effects were reduced. These results suggest that the thoracic irradiation-induced TGF-β was involved in the above abscopal damage of testes, which reinforces the necessity of new prevention strategy development of radiotherapy in avoiding any abnormal genetic consequence. • Fractionated thoracic irradiation (FT-IR) induced distal injuries in rat testes and sperm cells. • FT-IR increased TGF-β in rat serum that increased ROS and apoptosis of Sertoli cells in vitro. • The cytotoxic effect of condition serum was diminished by anti-TGF-β treatment. • TGF-β contributes to FT-IR induced abscopal damage on rat testes. [ABSTRACT FROM AUTHOR]

To test whether platelets could increase invasion potential and initiate EMT in ovarian cancer cells via a TGF-β signaling pathway. Blood samples were collected in 69 patients with ovarian cancer, 16 patients with benign ovarian tumor and 64 healthy donors. SK-OV-3 and OVCAR-3 ovarian cancer cells were treated with platelets. Transwell assays were used to analyze the invasive capacity, and EMT was assessed by microarray analysis, quantitative real-time PCR (qPCR) and Western blotting. Activation of TGF-β pathway was examined by ELISA and Western blotting. TGF-β type I receptor (TβR I) inhibitor A83-01 was used to confirm the role of TGF-β pathway in vitro and in vivo. Clinical data showed ovarian cancer patients with elevated platelet counts had a higher incidence of advanced stages. Treatment with platelets increased the invasive properties of both cell lines. Mesenchymal markers (snail family transcriptional repressor-1, vimentin, neural cadherin, fibronectin-1 and matrix metalloproteinase-2) were up-regulated in platelet-treated cells, while the epithelial marker (epithelial cadherin) was down-regulated. Higher TGF-β level was observed in patients with elevated platelet counts when compared to the subjects. Higher levels of TGF-β were also found in culture medium treated with platelets, and cells treated with platelets also showed increased phosphorylation of Smad2. TβR I inhibitor A83–01 reversed the EMT-like alterations and inhibited platelet-induced invasion in vitro and in vivo. Platelet increased invasion potential and induced EMT in ovarian cancer cells in a TGF-β dependent pathway. Platelet-derived TGF-β may be useful as a new target treatment for ovarian cancer. • Ovarian cancer patients with elevated platelets showed higher incidence of metastasis. • Platelets increase invasive ability and induce EMT in ovarian cancer cells. • Serum TGF-β level was higher in ovarian cancer patients with elevated platelet. • TGF-β/Smad pathway was activated in platelet-treated ovarian cancer cells. • TGF-β type I receptor inhibitor A83-01 abolished platelets-induced invasion and EMT in vitro and in vivo. [ABSTRACT FROM AUTHOR]

• Ado induces the expression and secretion of TGF-β1 in CeCa cells. • A2AR and A2BR blockade inhibits TGF-β1 secretion in CeCa cells. • CD73 expression on CeCa cells is maintained by the production of TGF-β. In cancer, the adenosinergic pathway participates in the generation of an immunosuppressive microenvironment and in the promotion of tumor growth through the generation of adenosine (Ado). The present study analyzed the participation of Ado, generated through the functional activity of the cervical cancer (CeCa) pathway in CeCa cells, to induce the expression and secretion of TGF-β1, as well as the participation of this factor to maintain CD73 expression. Ado concentrations greater than 10 μM were necessary to induce an increase of over 50% in the production and expression of TGF-β1 in CeCa tumor cells. Blockade of A2AR and A2BR with the specific antagonists, ZM241385 and MRS1754, respectively, strongly reversed the production of TGF-β1. TGF-β1 produced by CeCa cells was necessary to maintain CD73 expression because the addition of anti-TGF-β neutralizing antibodies or the inhibition of TGF-βRI strongly reversed the expression of CD73 in the CeCa cells. These results suggested a feedback loop in CeCa cells that favors immunosuppressive activity through the production of TGF-β1 and Ado as well as the autocrine activity of TGF–β1 and expression of CD73. [ABSTRACT FROM AUTHOR]

Abstract Although our previous studies have showed that a novel oncogene, cancer upregulated gene (CUG)2 induced epithelial-mesenchymal transition (EMT), the detailed molecular mechanism remains unknown. Because several lines of evidence documented that Yes-Associated Protein (YAP)1 is closely associated with cancer stem cell (CSC)-like phenotypes including EMT, stemness, and drug resistance, we wondered if YAP1 is involved in CUG2-induced EMT. We herein found that the overexpression of CUG2 increased YAP1 expression at the transcriptional as well as protein levels. Chromatin immunoprecipitation assay revealed that the elevated YAP1 transcripts are attributed to c-Jun and AP2 bindings to the YAP1 promoter. Akt and MAPK kinases including ERK, JNK, and p38 MAPK enhanced the level of YAP1 protein. In spite of a close relationship between β-catenin and YAP1, not β-catenin but NEK2 played the role in increasing YAP1 expression. Silencing YAP1 inhibited CUG2-induced cell migration and invasion. N-cadherin and vimentin expressions were decreased during YAP1 knockdown. The suppression of YAP1 diminished TGF-β transcriptional activity and expression as well as phosphorylation level of Smad2 and Twist protein. Conversely, LY2109761 or Smad2 siRNA treatment reduced YAP1 protein levels, indicating a close interplay between YAP1 and TGF-β signaling. Taken together, we suggest that CUG2 induces up-regulation of YAP1 expression, leading to enhancing CUG2-induced EMT via a close crosstalk between YAP1 and TGF-β signaling. Highlights • CUG2 enhances expression of YAP1 protein. • Akt and MAPK kinases are involved in the increase of YAP1 protein expression under overexpression of CUG2. • Not β-catenin but NEK2 is involved in increase of YAP1 protein expression under overexpression of CUG2. • YAP1 is involved in CUG2-induced EMT. [ABSTRACT FROM AUTHOR]

Abstract We previously demonstrated that maternal exposure to di-n-butyl phthalate (DBP) resulted in renal fibrosis in male offspring; however, the underlying mechanism governing this effect has not been thoroughly elucidated to date. We hypothesized that DBP exposure induces TGF-β expression and abnormal activation of epithelial-mesenchymal transition (EMT) in fibrotic kidneys. Pregnant rats received DBP orally at a dose of 850 mg/kg BW/day during gestational days 14–18. In the DBP-exposed group, immunohistochemistry (IHC) staining showed increased expression of TGF-β1 and EMT markers. In rat kidney tubular epithelial cells (NRK52E), ROS production increased expression levels of TGF-β1 and subsequently contributed to the induction of Snail1-mediated EMT. Notably, DBP exposure also promoted autophagy that downregulated TGF-β1. Taken together, our findings suggest that maternal exposure to DBP promotes EMT in tubular epithelial cells via upregulation of TGF-β1. Highlights • Maternal exposure to DBP promoted TGF-β1 expression in kidney. • Autophagy regulated expression of TGF-β1 induced by DBP in renal tubular cells. • TGF-β signaling regulation contributed to DBP-induced EMT. [ABSTRACT FROM AUTHOR]

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease, and consequently, effective antifibrotic drugs are strongly desired. Although we have previously reported a validated Col1a1-Luc Tg rat model for fibrosis, there are only a few mouse models that enable the evaluation of fibrosis in a short time period and with high sensitivity. Therefore, we generated a Col1a1-internal ribosome entry site (IRES)-Luc knock-in (KI) mouse in which the IRES-luciferase gene construct was inserted into the 3′-UTR of the type I collagen alpha 1 gene (Col1a1). There was a high correlation between luciferase activity and hydroxyproline content in the KI mice, which is similar to the result that we have previously reported for the Col1a1-Luc Tg rat model. In a bleomycin (BLM)-induced lung fibrosis model, luciferase activity in the lung showed a significant increase 3 days after BLM treatment, while only a slight increase was observed in the hydroxyproline content. An ALK-5 inhibitor—R-268712—was effective in inhibiting the luciferase activity in both the in vivo BLM-induced lung fibrosis model and in vitro primary mouse lung fibroblasts. This suggests that fibroblasts are the major collagen-producing cells in lung fibrosis. In human lung fibroblasts, TGF-β stimulation induced α-smooth muscle actin as observed by immunostaining, suggesting that myofibroblast transdifferentiation (MTD) plays an important role in lung fibrosis. Together, these results indicated that ALK-5 inhibitors might affect lung fibrosis mainly via the inhibition of MTD. Thus, the Col1a1-IRES-Luc KI mouse might be useful for the evaluation of antifibrotic effects and their underlying mechanisms. [ABSTRACT FROM AUTHOR]

Abstract Graves' ophthalmopathy (GO), a complication of Graves' disease (GD), is typified by orbital inflammation, ocular tissue expansion and remodeling and, ultimately, fibrosis. Orbital fibroblasts are key effectors of GO pathogenesis exhibiting exaggerated inflammatory and fibroproliferative responses to cytokines released by infiltrating immune cells. Activated orbital fibroblasts also produce inflammatory mediators that contribute to disease progression, facilitate the orbital trafficking of monocytes and macrophages, promote differentiation of matrix-producing myofibroblasts and stimulate accumulation of a hyaluronan-rich stroma, which leads to orbital tissue edema and fibrosis. Proteomic and transcriptome profiling of the genomic response of ocular and non-ocular fibroblasts to INF-γ and TGF-β1 focused on identification of translationally-relevant therapeutic candidates. Induction of plasminogen activator inhibitor-1 (PAI-1, SERPINE1), a clade E member of the serine protease inhibitor (SERPIN) gene family and a prominent regulator of the pericellular proteolytic microenvironment, was one of the most highly up-regulated proteins in INF-γ- or TGF-β1-stimulated GO fibroblasts as well as in severe active GD compared to patients without thyroid disease. PAI-1 has multifunctional roles in inflammatory and fibrotic processes that impact tissue remodeling, immune cell trafficking and survival as well as signaling through several receptor systems. This review focuses on the pathophysiology of the GO fibroblast and possible targets for effective drug therapy. [ABSTRACT FROM AUTHOR]

Abstract Endogenous bone marrow-derived mesenchymal stem cells (BM-MSCs) are mobilized into peripheral blood and injured tissues by various growth factors and cytokines that are expressed in the injured tissues, such as substance P (SP), stromal cell derived factor-1 (SDF-1), and transforming growth factor-beta (TGF-β). Extracellular bioactive lipid metabolites such as ceramide-1-phosphate and sphingosine-1-phosphate also modulate BM-MSC migration as SP, SDF-1, and TGF-β. However, the roles of intrinsic lipid kinases of BM-MSCs in the stem cell migration are unclear. Here, we demonstrated that ceramide kinase mediates the chemotactic migration of BM-MSCs in response to SP, SDF-1, or TGF-β. Furthermore, a specific inhibitor of ceramide kinase inhibited TGF-β-induced migration of BM-MSCs and N-cadherin that is necessary for BM-MSCs migration in response to TGF-β. Therefore, these results suggest that the intracellular ceramide kinase is required for the BM-MSCs migration and the roles of the intrinsic ceramide kinase in the migration are associated with N-cadherin regulation. Highlights • TGF-β increases the migration and N-cadherin expression of BM-MSC. • Ceramide kinase mediates BM-MSC migration mediated with TGF-β. • Ceramide kinase regulates N-cadherin expression. [ABSTRACT FROM AUTHOR]

Abstract Endotoxin tolerance is an important state for the prevention of lethal infection and inflammatory response, which is closely associated with the participation of innate immune cells. Moreover, mesenteric lymph nodes (MLNs)-resident immune cells, such as CD4+Foxp3+ regulatory T (Treg) cells and dendritic cells, play important roles in the maintenance of peripheral immune tolerance. However, the potential roles of these cells in MLNs in the development of endotoxin tolerance remain largely unknown. Recent research work showed that CD4+Foxp3+ Treg cells contributed to the development of endotoxin tolerance. Here, we further analyzed the possible change on CD4+Foxp3+Tregs population in MLNs in murine LPS-induced endotoxin tolerance model. Our data showed that the proportion and absolute number of CD4+Foxp3+Tregs, expressing altered levels of CTLA4 and GITR, significantly increased in MLNs of murine LPS-induced tolerance model. Moreover, the expression level of TGF-β in MLNs also increased obviously. Furthermore, TGF-β blockade could obviously reduce the proportion and absolute number of CD4+Foxp3+Tregs in MLNs and subsequently impair the protection effect against LPS rechallenge. Of note, we found that tolerogenic dendritic cell (Tol-DC), expressing lower levels of MHC-II and CD86 molecules, dominantly secreted TGF-β in MLNs in murine LPS-induced tolerance model. In all, our data provided an unknown phenomenon that the total cell number of CD4+Foxp3+Tregs significantly increased in MLNs in endotoxin tolerance, which was related to MLN-resident TGF-β secreting CD11c+DCs, providing a new fundamental basis for the understanding on the potential roles of MLN-resident immune cells in the development of endotoxin tolerance. Highlights • The number of CD4+Foxp3+Tregs increased in MLNs in endotoxin tolerance. • The expression level of TGF-β in MLNs increased obviously. • TGF-β was critical for the increased cell number of CD4+Foxp3+ Tregs. • CD11c+DCs, which dominantly secreted TGF-β, exhibited a tolerance phenotype in MLNs. [ABSTRACT FROM AUTHOR]

Abstract Cilostazol (Ciloz) a potent Type III phosphodiesterase inhibitor is effective against inflammation, insulin resistance and cardiomyopathy. However, the effect of Ciloz on obesity-associated left ventricular diastolic dysfunction has not been explored yet. Hence, we examined the effect of Ciloz on cardiac remodelling and dysfunction in non-obese and obese-insulin resistant mice infused with AngiotensinII (AngII). Male C57BL/6 J mice were initially subjected to 19 weeks of chow or high fat diet (HFD) regimen and thereafter animals were randomised for AngII (1500 ng/kg/min, s.c) infusion or saline and Ciloz (50 mg/kg, p.o) for another 1 week. Obese and non-obese mice infused with AngII exhibited significant diastolic dysfunction and features of heart failure with preserved ejection fraction (HFpEF) since a decrease in fractional shortening and no change in ejection fraction were observed when compared with respective controls. Administration of AngII and Ciloz in HFD fed mice significantly improved the left ventricular function compared with AngII infused HFD mice as evinced from the echocardiographic data. Further, Ciloz treatment significantly reduced cardiomyocyte area, interstitial and perivascular fibrosis; and collagen deposition. Moreover, Ciloz reduced the inflammatory milieu in the heart as evinced by decreased F4/80+ and CD68+ cells; IL-1β and IL-6 gene transcripts. Quantitative assessment of the expression levels revealed substantial upregulation of MMP-9 (pro- and mature-forms) and α-SMA in the left ventricle of AngII infused HFD-fed mice, which was considerably suppressed by Ciloz regimen. The beneficial effect of Ciloz was associated with the normalization in gene expression of hypertrophic and fibrotic markers. Likewise, Ciloz administration markedly reduced the AngII and HFD induced TGF-β1/SMAD3 and Akt/mTOR signalling. Additionally, AngII administered and HFD-fed mice showed increased glycolytic flux, which was considerably diminished by Ciloz treatment as indicated from suppressed PKM2, HK-2, PDK-2, HIF-1α mRNA and GLUT-1 protein expression. Taken together, Ciloz might be therapeutically exploited against AngII and obesity-associated diastolic dysfunction thereby preventing overt heart failure. Highlights • AngII induced diastolic dysfunction and HFpEF in obese and non-obese mice. • Cilostazol ameliorates AngII induced diastolic dysfunction and HFpEF in obese and non-obese mice. • Cilostazol reduced obesity linked LV geometric anomalies, inflammation and fibrosis. • Cilostazol Suppressed obesity-induced myocardial metabolic defects. • Cilostazol might be useful in the subset of obese patients with high renin and AngII levels. [ABSTRACT FROM AUTHOR]

Epilepsy is a chronic disorder in which immune dysregulation is shown to be involved. Imbalances in the cytokine levels both in serum and brain tissue have been demonstrated in epileptic patients. In the present study, we assessed mRNA expression of TNF-α, TGF-β, IFN-γ, CXCL8, IL-1β, IL-2, 1L-4, IL-6, IL-17 and CXCL8 in blood samples of 40 epileptic patients compared with age- and sex-matched healthy controls by means of quantitative real time PCR. The relative levels of CXCL8 transcripts were significantly higher in total epileptic patients compared with healthy subjects (P = .023). Relative mRNA expression of IFN-γ was significantly higher in female patients compared with female healthy subject (P = .048). In addition, significant correlations have been found between the mRNA levels of mentioned cytokines. Such imbalance between expression of pro-inflammatory and anti-inflammatory cytokines might be implicated in the pathogenesis of epilepsy. [ABSTRACT FROM AUTHOR]

Fibrosis is a pathophysiological state characterized by the excessive formation/deposition of fibrous extracellular matrix. Transforming growth factor-beta (TGF-β) is a central profibrotic mediator, and targeting TGF-β is a promising strategy in the development of drugs for the treatment of fibrosis. Therefore, the effect of LY2109761, a small molecule inhibitor against TGF-β with targets beyond TGF-β signaling, on fibrogenesis was elucidated in vitro (HepG2 cells and LX-2 cells) and ex vivo (human and rat precision-cut liver slices). Our results displayed an anti-fibrotic effect of LY2109761, as it markedly down-regulated gene and protein expression of collagen type 1, as well as gene expression of the inhibitor of metalloproteinases 1. This effect on fibrosis markers was partially mediated by targeting TGF-β signaling, seeing that LY2109761 inhibited TGF-β1 gene expression and SMAD2 protein phosphorylation. Interestingly, particularly at a high concentration, LY2109761 decreased SMAD1 protein phosphorylation and gene expression of the inhibitor of DNA binding 1, which appeared to be TGF-β-independent effects. In conclusion, LY2109761 exhibited preclinical anti-fibrotic effects via both TGF-β-dependent and -independent pathways. These results illustrate that small molecule inhibitors directed against TGF-β could possibly influence numerous signaling pathways and thereby mitigate fibrogenesis. [ABSTRACT FROM AUTHOR]

The etiology of Autism Spectrum Disorders (ASDs) as severe neurodevelopmental ailments is not known. However, several evidences point to dysregulation of immune system as an underlying cause of ASD. In the present study we evaluated the mRNA expression levels of TNF-α, TGF-β, IFN-γ, CXCL8, IL-1β, IL-2, 1L-4, IL-6, IL-17 in whole blood samples of 30 ASD patients and 41 age and sex-matched healthy subjects with means of real-time PCR. TNF-α, IL-6 and IL-17 have been shown to be significantly up-regulated in ASD patients compared with healthy subjects (P < 0.0001, P = 0.001 and P < 0.0001 respectively). IL-2 has been shown to be significantly down-regulated in total ASD patients (P < 0.0001). No significant difference has been found in expression levels of other cytokines between patients and healthy subjects. The present study provides further evidences for dysregulation of immune response in ASD patients. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC) is the most malignant subtype of breast cancer in which the cell surface lacks usual targets for drug to exhibit its effects. Epirubicin (Epi) is widely used for TNBC, but a substantial number of patients develop Epi resistance that is usually associated with poor prognosis. Transforming growth factor (TGF-β) is a multifunctional cytokine. In recent study, it appears that TGF-β influences the cancer stem cell population, thus, the drug resistance of cancer may also be affected. We used epirubicin to treat MDA-MB-231 (MB-231) cells and found that TGF-β and breast cancer stem cell markers CD44 + CD24 − were increased and were dose-dependent of epirubicin. We established drug-resistant cell line from parental MB-231 cells by chronic treatment with low-concentration epirubicin. The MB-231/Epi cell line showed relatively slow growth rate with varied morphology. Transwell assay and drug sensitivity assay revealed that the malignant cell behaviors in terms of migration, invasion and epirubicin-resistant properties were markedly increased in the MB-231/Epi cells. Western blot, immunofluorescence assay, and flow cytometry were used to analyze the expression levels of the breast cancer stem cell markers, CD44 and CD24. Mammospheres assay showed that the stemness of MB-231/Epi was increased compared to their parental cells. Interestingly, MB-231/Epi cells showed different expression levels of apoptosis-related markers: Bcl2, Bax; EMT-related markers E-cadherin, N-cadherin and cell cycle-related marker cyclinD1. These genes have all been shown to be regulated by the TGF-β pathway. Taken together, our findings suggest that TGF-β plays a vital role in TNBC epirubicin-resistance through regulating stemness, EMT and apoptosis. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S), an endogenous gas mediator with multifaced biological functions, has been shown to be effective in the prevention and treatment of renal sclerosis in several models of chronic renal diseases. The mechanisms involved are still unclear. Given that Ang II- and TGF-β-induced renal tubular epithelial-mesenchymal transition (EMT) is a pivotal cellular event leading to renal sclerosis, we examined whether and how H 2 S intervened the processes of EMT. Ang II stimulated EMT in renal tubular epithelial cells, as indicated by the increased level of α-smooth muscle actin and a decreased level of E-cadherin. This effect of Ang II was blocked by a TGF-β receptor kinase inhibitor, indicative of a mediating role of TGF-β. Consistently, Ang II stimulated TGF-β activation and addition of the exogenous TGF-β1 also induced EMT. In the presence of H 2 S donor NaHS, the EMT-promoting actions of Ang II and TGF-β1 were abolished, which was associated with a reduced TGF-β activity. Further analysis using a human recombinant active TGF-β1 revealed that H 2 S cleaved the disulfide bond in the dimeric active TGF-β1 and promoted the formation of inactive TGF-β1 monomer. Collectively, these results indicate that H 2 S counteracted Ang II- and TGF-β1-induced EMT through mechanisms involving direct inactivation of TGF-β1. Our study thus provides novel mechanistic insight into the anti-fibrotic actions of H 2 S and suggest that H 2 S could be used to treat renal sclerotic diseases. [ABSTRACT FROM AUTHOR]