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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.

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 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.

The discovery that endoplasmic reticulum (ER) luminal chaperones such as GRP78/BiP can escape to the cell surface upon ER stress where they regulate cell signaling, proliferation, apoptosis, and immunity represents a paradigm shift. Toward deciphering the mechanisms, we report here that, upon ER stress, IRE1α binds to and triggers tyrosine kinase SRC activation, leading to ASAP1 phosphorylation and Golgi accumulation of ASAP1 and Arf1-GTP, resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. At the cell surface, GRP78 binds to and acts in concert with a glycosylphosphatidylinositol-anchored protein, CD109, in blocking TGF-β signaling by promoting the routing of the TGF-β receptor to the caveolae, thereby disrupting its binding to and activation of Smad2. Collectively, we uncover a SRC-mediated signaling cascade that leads to the relocalization of ER chaperones to the cell surface and a mechanism whereby GRP78 counteracts the tumor-suppressor effect of TGF-β.

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-β family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-β signaling.

The aortic root is the predominant site for development of aneurysm caused by heterozygous loss-of-function mutations in positive effectors of the transforming growth factor-β (TGF-β) pathway. Using a mouse model of Loeys-Dietz syndrome (LDS) that carries a heterozygous kinase-inactivating mutation in TGF-β receptor I, we found that the effects of this mutation depend on the lineage of origin of vascular smooth muscle cells (VSMCs). Secondary heart field-derived (SHF-derived), but not neighboring cardiac neural crest-derived (CNC-derived), VSMCs showed impaired Smad2/3 activation in response to TGF-β, increased expression of angiotensin II (AngII) type 1 receptor (Agtr1a), enhanced responsiveness to AngII, and higher expression of TGF-β ligands. The preserved TGF-β signaling potential in CNC-derived VSMCs associated, in vivo, with increased Smad2/3 phosphorylation. CNC-, but not SHF-specific, deletion of Smad2 preserved aortic wall architecture and reduced aortic dilation in this mouse model of LDS. Taken together, these data suggest that aortic root aneurysm predisposition in this LDS mouse model depends both on defective Smad signaling in SHF-derived VSMCs and excessive Smad signaling in CNC-derived VSMCs. This work highlights the importance of considering the regional microenvironment and specifically lineage-dependent variation in the vulnerability to mutations in the development and testing of pathogenic models for aortic aneurysm.

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.

The formation of transcription regulatory complexes by the association of Smad4 with Smad2 and Smad3 (Smad2/3) is crucial in the canonical TGFβ pathway. Although the central requirement of Smad4 as a common mediator is emphasized in regulating TGFβ signaling, it is not obligatory for all responses. The role of Smad2/3 independently of Smad4 remains understudied. Here, we introduce a stepwise paradigm in which Smad2/3 regulate the lineage priming and differentiation of mouse embryonic stem cells (mESCs) by collaboration with different effectors. During the naïve-to-primed transition, Smad2/3 upregulate DNA methyltransferase 3b (Dnmt3b), which establishes the proper DNA methylation patterns and, in turn, enables Smad2/3 binding to the hypomethylated centers of promoters and enhancers of epiblast marker genes. Consequently, in the absence of Smad2/3, Smad4 alone cannot initiate epiblast-specific gene transcription. When primed epiblast cells begin to differentiate, Dnmt3b becomes less actively engaged in global genome methylation, and Smad4 takes over the baton in this relay race, forming a complex with Smad2/3 to support mesendoderm induction. Thus, mESCs lacking Smad4 can undergo the priming process but struggle with the downstream differentiation. This work sheds light on the intricate mechanisms underlying TGFβ signaling and its role in cellular processes. The role of Smad2/3 in the TGFβ pathway, independent of Smad4, is understudied. Authors reveal distinct roles for Smad2/3 and Smad4 during mESC priming to differentiation, and uncover a collaboration of Smad2/3 with Dnmt3b in epiblast formation.

An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here, we report the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genomewide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.

Non-small cell lung cancer (NSCLC) has the highest mortality rate among all malignancies worldwide. The role of long noncoding RNAs (lncRNAs) in the progression of cancers is a contemporary research hotspot. Based on an integrative analysis of The Cancer Genome Atlas database, we identified lncRNA-RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) as one of the most highly upregulated lncRNAs that are associated with poor survival in NSCLC. Furthermore, N(6)-methyladenosine (m6A) was highly enriched within RMRP and enhanced its RNA stability. In vitro and in vivo experiments showed that RMRP promoted NSCLC cell proliferation, invasion, and migration. In terms of mechanism, RMRP recruited YBX1 to the TGFBR1 promotor region, leading to upregulation of the transcription of TGFBR1. The TGFBR1/SMAD2/SMAD3 pathway was also regulated by RMRP. In addition, RMRP promoted the cancer stem cells properties and epithelial mesenchymal transition, which promote the resistance to radiation therapy and cisplatin. Clinical data further confirmed a positive correlation between RMRP and TGFBR1. In short, our work reveals that m6A RNA methylation-mediated RMRP stability renders proliferation and progression of NSCLC through regulating TGFBR1/SMAD2/SMAD3 pathway.