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Transforming growth factorβ (TGFβ) signaling regulates diverse aspects of vertebrate skeletal muscle tissue including differentiation, homeostasis, regeneration and pathogenic degeneration. Ubiquitination of SMAD2, an intracellular transducer of TGFβ signaling, is a well-studied negative feedback regulation of the signaling pathway in the field of cell biology, but it’s relevance in skeletal muscle tissue has been elusive. In this study, to elucidate the in vivo role of SMAD2 ubiquitination, we generated Smad2dPY mutant mice in which a 15 bp sequence encoding the PY motif of SMAD2 protein is deleted from Smad2 gene. By removing this motif, the SMAD2 protein escapes from protein-protein interaction with NEDD4 family E3 ligases and thus is devoid of ubiquitination-dependent negative regulation. Smad2dPY mice showed no obvious abnormality in development, growth or fertility, indicating that SMAD2 ubiquitination through PY motif is dispensable for these processes. The skeletal muscle of Smad2dPY mice demonstrated reduced weight and myofiber size reduction at 12 months old. SMAD2 protein level was increased in the skeletal muscle of Smad2dPY mice while SMAD2 ubiquitination was reduced. Primary myoblasts of Smad2dPY mice displayed higher TGFβ responsiveness and suppressed terminal differentiation, which may explain the reduced muscle mass. The TGFβ responsiveness of the interstitial fibroblast population was also increased. Fibrotic tissue remodeling triggered by cardiotoxin injection was exacerbated in Smad2dPY mice. Altogether, our study identified SMAD2 ubiquitination through PY motif as an important regulatory mechanism operating in skeletal muscle tissue to maintain the TGFβ signaling pathway at the desired level in homeostasis and tissue remodeling.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2 , specifically by the WWP2-N terminal isoform. Importantly, the WWP2 -regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2. Pathological cardiac fibrosis is a hallmark of diseases leading to heart failure. Here, the authors used systems genetics to identify a pro-fibrotic gene network regulated by WWP2, a E3 ubiquitin ligase, which orchestrates the nucleocytoplasmic shuttling and transcriptional activity of SMAD2 in the diseased heart.

The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

AbstractMesenchymal stem cells (MSCs) and their derived extracellular vesicles have been reported as promising tools for the management of heart disease. The aim of this study was to explore the function of adipose-derived MSCs (adMSCs)-derived exosomes (Exo) in the progression of myocardial infarction (MI) and the molecules involved. Mouse cardiomyocytes were treated with oxygen-glucose deprivation (OGD) to mimic an MI condition in vitro. The adMSCs-derived Exo were identified and administrated into the OGD-treated cardiomyocytes, and then the viability and apoptosis of cells, and the secretion of fibrosis- and inflammation-related cytokines in cells were determined. Differentially expressed microRNAs (miRNAs) in cells after Exo treatment were screened using a microarray analysis. The downstream molecules regulated by miR-671 were explored through bioinformatic analysis. Involvements of miR-671 and transforming growth factor beta receptor 2 (TGFBR2) in the exosome-mediated events were confirmed by rescue experiments. A murine model with MI was induced and treated with Exo for functional experiments in vivo. Compared to phosphate-buffered saline treatment, the Exo treatment significantly enhanced viability while reduced apoptosis of cardiomyocytes, and in reduced myocardial fibrosis and inflammation both in vitro and in vivo. miR-671 was significantly upregulated in cells after Exo treatment. Downregulation of miR-671 blocked the protective functions of Exo. miR-671 targeted TGFBR2 and suppressed phosphorylation of Smad2. Artificial downregulation of TGFBR2 enhanced viability of the OGD-treated cardiomyocytes. This study suggested that adMSC-derived exosomal miR-671 directly targets TGFBR2 and reduces Smad2 phosphorylation to alleviate MI-like symptoms both in vivo and in vitro.

Following the publication of the above paper, it was drawn to the Editor's attention by a concerned reader that certain of the flow cytometric data shown in Fig. 5A on p. 5208 were strikingly similar to data that had appeared previously in other papers written by different authors at different research institutes. In view of the fact that the abovementioned data had already apparently been published prior to its submission to , the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 19: 5203-5210, 2019; DOI: 10.3892/mmr.2019.10186].

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.

P450 aromatase, encoded by the Cyp19 gene, catalyzes the synthesis of estrogen, which is crucial for mammalian germ cell differentiation. We have previously shown that transforming growth factor beta 1 (TGF-β1) attenuated the accumulation of steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) and eventually reduced the transcription of Cyp19 in rat Leydig cells (LCs). Here, we report that TGF-β1 treatment-induced phosphorylation of Smad2 and decreased the expression levels of SF-1 and LRH-1 by elevating the expression levels of microRNA-21-3p and microRNA-339-5p in vivo and in vitro. Furthermore, both TGF-β1 treatment and over-expression of Smad2 inhibited the SF-1 or LRH-1-regulated promoter activity of the Cyp19 gene, and p-Smad2 physically interacted with SF-1 and LRH-1. Our findings collectively suggest that TGF-β1 may inhibit the expression of CYP19 in LCs mainly through two ways. On the one hand, TGF-β1 acts through Smad2 to repress the accumulation of SF-1 and LRH-1 at post-transcriptional level by upregulating specific microRNAs. On the other hand, TGF-β1 inhibits the transcriptional activity of Cyp19 through the interaction of p-Smad2 with SF-1/LRH-1. Summary Sentence TGF-β1 inhibits Cyp19 transcription mediated by Smad2 in two ways: upregulation of miRNAs targeting SF-1/LRH-1 and inactivation of Cyp19 promoter. Graphical Abstract

The host defence peptide cathelicidin (LL-37 in humans, mCRAMP in mice) is released from neutrophils by de-granulation, NETosis and necrotic death; it has potent anti-pathogen activity as well as being a broad immunomodulator. Here we report that cathelicidin is a powerful Th17 potentiator which enhances aryl hydrocarbon receptor (AHR) and RORγt expression, in a TGF-β1-dependent manner. In the presence of TGF-β1, cathelicidin enhanced SMAD2/3 and STAT3 phosphorylation, and profoundly suppressed IL-2 and T-bet, directing T cells away from Th1 and into a Th17 phenotype. Strikingly, Th17, but not Th1, cells were protected from apoptosis by cathelicidin. We show that cathelicidin is released by neutrophils in mouse lymph nodes and that cathelicidin-deficient mice display suppressed Th17 responses during inflammation, but not at steady state. We propose that the neutrophil cathelicidin is required for maximal Th17 differentiation, and that this is one method by which early neutrophilia directs subsequent adaptive immune responses. Neutrophils secrete numerous immune effector molecules including cathelicidin which is associated with antimicrobial properties. Here the authors implicate neutrophil derived cathelicidin in modulation of CD4 T cell homoeostasis and the promotion of Th17 CD4 T cells.

Circular RNAs (circRNAs) are key regulatory factors in the development of multiple cancers. This study is targeted at exploring the effect of circ_0002623 on bladder cancer (BCa) progression and its mechanism. Circ_0002623 was screened out by analyzing the expression profile of circRNAs in BCa tissues. Circ_0002623, miR‐1276, and SMAD2 mRNA expression levels in clinical sample tissues and cell lines were detected through quantitative real‐time polymerase chain reaction (qRT‐PCR). After circ_0002623 had been overexpressed or silenced in BCa cells, the cell proliferation, migration, and cell cycle were evaluated by CCK‐8, BrdU, Transwell assay, and flow cytometry. Tumor xenograft model was used to validate the biological function of circ_0002623 in vivo. Bioinformatics analysis and dual‐luciferase reporter gene assay were conducted for analyzing and confirming, respectively, the targeted relationship between circ_0002623 and miR‐1276, as well as between miR‐1276 and SMAD2. The regulatory effects of circ_0002623 and miR‐1276 on the expression levels of TGF‐β, WNT1, and SMAD2 in BCa cells were detected by Western blot. We reported that, in BCa tissues and cell lines, circ_0002623 was upregulated, whereas miR‐1276 was downregulated. Circ_0002623 positively regulated BCa cell proliferation, migration, and cell cycle progression. Additionally, circ_0002623 could competitively bind with miR‐1276 to increase the expression of SMAD2, the target gene of miR‐1276. Furthermore, circ_0002623 could regulate the expression of TGF‐β and WNT1 via modulating miR‐1276 and SMAD2. This study helps to better understand the molecular mechanism underlying BCa progression. Mechanism research indicated that circ_0002623 increased SMAD2 expression via sponging miR‐1276, thereby promoting the malignant phenotype of bladder cancer (BCa) cells.

Missense variants in , encoding a key transcriptional regulator of transforming growth factor beta signalling, were recently reported to cause arterial aneurysmal disease. The aims of the study were to identify the genetic disease cause in families with aortic/arterial aneurysmal disease and to further define genotype-phenotype correlations. Using gene panel sequencing, we identified a nonsense variant and four missense variants, all affecting highly conserved amino acids in the MH2 domain. The premature stop codon (c.612dup; p.(Asn205*)) was identified in a marfanoid patient with aortic root dilatation and in his affected father. A p.(Asn318Lys) missense variant was found in a Marfan syndrome (MFS)-like case who presented with aortic root aneurysm and in her affected daughter with marfanoid features and mild aortic dilatation. In a man clinically diagnosed with Loeys-Dietz syndrome (LDS) that presents with aortic root dilatation and marked tortuosity of the neck vessels, another missense variant, p.(Ser397Tyr), was identified. This variant was also found in his affected daughter with hypertelorism and arterial tortuosity, as well as his affected mother. The third missense variant, p.(Asn361Thr), was discovered in a man presenting with coronary artery dissection. Variant genotyping in three unaffected family members confirmed its absence. The last missense variant, p.(Ser467Leu), was identified in a man with significant cardiovascular and connective tissue involvement. Taken together, our data suggest that heterozygous loss-of-function variants can cause a wide spectrum of autosomal dominant aortic and arterial aneurysmal disease, combined with connective tissue findings reminiscent of MFS and LDS.