Vascular calcification (VC) is regarded as an important pathological change lacking effective treatment and associated with high mortality. Sirtuin 6 (SIRT6) is a member of Sirtuin family, a class III histone deacetylase and a key epigenetic regulator. SIRT6 has a protective role in patients with chronic kidney disease (CKD), however the exact role and molecular mechanism of SIRT6 in VC in CKD patients remains unclear. Here, we demonstrated that SIRT6 was significantly downregulated in peripheral blood mononuclear cells (PBMCs) and in the radial artery tissue of CKD patients with VC. SIRT6-transgenic (SIRT6-Tg) mice showed alleviated VC, while vascular smooth muscle cells (VSMCs)-specific, SIRT6 knocked down mice showed severe VC, in CKD. SIRT6 suppressed the osteogenic transdifferentiation of VSMCs via regulation of runt-related transcription factor 2 (Runx2). Co-immunoprecipitation (co-IP) and immunoprecipitation (IP) assays confirmed that SIRT6 bound to Runx2. Moreover, Runx2 was deacetylated by SIRT6 and further promoted nuclear export via exportin 1(XPO1), which in turn caused degradation of Runx2 through the ubiquitin-proteasome system. These results demonstrated that SIRT6 prevented VC by suppressing the osteogenic transdifferentiation of VSMCs, and as such targeting SIRT6 may be an appealing therapeutic target for VC in CKD.
Wenxin Li, Weijing Feng, Xiaoyan Su, Dongling Luo, Zhibing Li, Yongqiao Zhou, Yongjun Zhu, Mengbi Zhang, Jie Chen, Baohua Liu, Hui Huang
Coding variants in apolipoprotein L1 (APOL1), termed G1 and G2, can explain most excess kidney disease risk in African Americans; however, the molecular pathways of APOL1-induced kidney dysfunction remain poorly understood. Here, we report that expression of G2 APOL1 in the podocytes of Nphs1rtTA/TRE-G2APOL1 (G2APOL1) mice leads to early activation of the cytosolic nucleotide sensor, stimulator of interferon genes (STING), and the NLR family pyrin domain–containing 3 (NLRP3) inflammasome. STING and NLRP3 expression was increased in podocytes from patients with high-risk APOL1 genotypes, and expression of APOL1 correlated with caspase-1 and gasdermin D (GSDMD) levels. To demonstrate the role of NLRP3 and STING in APOL1-associated kidney disease, we generated transgenic mice with the G2 APOL1 risk variant and genetic deletion of Nlrp3 (G2APOL1/Nlrp3 KO), Gsdmd (G2APOL1/Gsdmd KO), and STING (G2APOL1/STING KO). Knockout mice displayed marked reduction in albuminuria, azotemia, and kidney fibrosis compared with G2APOL1 mice. To evaluate the therapeutic potential of targeting NLRP3, GSDMD, and STING, we treated mice with MCC950, disulfiram, and C176, potent and selective inhibitors of NLRP3, GSDMD, and STING, respectively. G2APOL1 mice treated with MCC950, disulfiram, and C176 showed lower albuminuria and improved kidney function even when inhibitor treatment was initiated after the development of albuminuria.
Junnan Wu, Archana Raman, Nathan J. Coffey, Xin Sheng, Joseph Wahba, Matthew J. Seasock, Ziyuan Ma, Pazit Beckerman, Dorottya Laczkó, Matthew B. Palmer, Jeffrey B. Kopp, Jay J. Kuo, Steven S. Pullen, Carine M. Boustany-Kari, Andreas Linkermann, Katalin Susztak
Insulin resistance is a cornerstone of obesity related complications such as type 2 diabetes, metabolic syndrome, and non-alcoholic fatty liver disease. A high rate of lipolysis is known to be associated with insulin resistance, and inhibiting adipose tissue lipolysis improves obesity-related insulin resistance. Here, we demonstrate that inhibition of 5-HT signaling through serotonin receptor 2B (HTR2B) in adipose tissues ameliorates insulin resistance by reducing lipolysis in visceral adipocytes. Chronic high-fat diet (HFD) feeding increased Htr2b expression in eWAT, resulting in the increased HTR2B signaling in visceral white adipose tissue. Moreover, HTR2B expression in white adipose tissue was increased in obese humans and positively correlated with metabolic parameters. We further found that adipocyte-specific Htr2b-knockout mice are resistant to high-fat diet (HFD)-induced insulin resistance, visceral adipose tissue inflammation, and hepatic steatosis. Enhanced 5-HT signaling through HTR2B directly activated lipolysis through phosphorylation of hormone sensitive lipase in visceral adipocytes. Moreover, treatment with a selective HTR2B antagonist attenuated HFD-induced insulin resistance, visceral tissue inflammation, and hepatic steatosis. Thus, adipose HTR2B signaling could be a potential therapeutic target for treatment of obesity-related insulin resistance.
Won Gun Choi, Wonsuk Choi, Tae Jung Oh, Hye-Na Cha, Inseon Hwang, Yun Kyung Lee, Seung Yeon Lee, Hyemi Shin, Ajin Lim, Dongryeol Ryu, Jae Myoung Suh, So-Young Park, Sung Hee Choi, Hail Kim
Ferroptosis, an iron-dependent non-apoptotic cell death, is a highly regulated tumor suppressing process. However, functions and mechanisms of RNA binding proteins in regulation of evasion of ferroptosis during lung cancer progression are still largely unknown. Here we reported that the RNA binding protein RBMS1 participated in lung cancer development through mediating ferroptosis evasion. Through an shRNA-mediated systematic screen, we discovered that RBMS1 was a key ferroptosis regulator. Clinically, RBMS1 was elevated in lung cancer and its high expression was associated with reduced patient survival. Conversely, depletion of RBMS1 inhibited lung cancer progression both in vivo and in vitro. Mechanistically, RBMS1 interacted with the translation initiation factor eIF3d directly to bridge the 3'- and 5'-UTRs of SLC7A11. RBMS1 ablation inhibited the translation of SLC7A11, reduced SLC7A11-mediated cystine uptake and promotes ferroptosis. In a drug screen that targeted RBMS1, we further uncovered that nortriptyline hydrochloride decreased the level of RBMS1, thereby promoting ferroptosis. Importantly, RBMS1 depletion or inhibition by nortriptyline hydrochloride sensitized radioresistant lung cancer cells to radiotherapy. Our findings established RBMS1 as a translational regulator of ferroptosis and a prognostic factor with therapeutic potentials and clinical values.
Wenjing Zhang, Yu Sun, Lu Bai, Lili Zhi, Yun Yang, Qingzhi Zhao, Chaoqun Chen, Yangfan Qi, Wenting Gao, Wenxia He, Luning Wang, Dan Chen, Shujun Fan, Huan Chen, Hai-Long Piao, Qinglong Qiao, Zhaochao Xu, Jinrui Zhang, Jinyao Zhao, Sirui Zhang, Yue Yin, Chao Peng, Xiaoling Li, Quentin Liu, Han Liu, Yang Wang
Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3′-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell–specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.
Eva M. Berghausen, Wiebke Janssen, Marius Vantler, Leoni L. Gnatzy-Feik, Max Krause, Arnica Behringer, Christine Joseph, Mario Zierden, Henrik ten Freyhaus, Anna Klinke, Stephan Baldus, Miguel A. Alcazar, Rajkumar Savai, Soni Savai Pullamsetti, Dickson W.L. Wong, Peter Boor, Jean J. Zhao, Ralph T. Schermuly, Stephan Rosenkranz
Epoxyeicosatrienoic acids (EETs) have potent anti-inflammatory properties. Hydrolysis of EETs by soluble epoxide hydrolase (sEH/EPHX2) to less active diols attenuates their anti-inflammatory effects. Macrophage activation is critical to many inflammatory responses; however, the role of EETs and sEH in regulating macrophage function remains unknown. Lung bacterial clearance of S. pneumoniae was impaired in Ephx2-deficient (Ephx2-/-) mice and in mice treated with an sEH inhibitor. The EET receptor antagonist, EEZE, restored lung clearance of S. pneumoniae in Ephx2-/- mice. Ephx2-/- mice had normal lung Il-1β, Il-6 and Tnfα expression and macrophage recruitment to lungs during S. pneumoniae infection; however, Ephx2 disruption attenuated proinflammatory cytokine induction, Tlr2 and Pgylrp1 receptor upregulation and Rac1/2 and Cdc42 activation in PGN-stimulated macrophages. Consistent with these observations, Ephx2-/-macrophages displayed reduced phagocytosis of S. pneumoniae in vivo and in vitro. Heterologous overexpression of TLR2 and PGLYRP1 in Ephx2-/- macrophages restored macrophage activation and phagocytosis. Human macrophage function was similarly regulated by EETs. Together, these results demonstrate that EETs reduce macrophage activation and phagocytosis of S. pneumoniae through down-regulation of TLR2 and PGLYRP1 expression. Defining the role of EETs and sEH in macrophage function may lead to development of new therapeutic approaches for bacterial diseases.
Hong Li, J. Alyce Bradbury, Matthew L. Edin, Joan P. Graves, Artiom Gruzdev, Jennifer Cheng, Samantha L. Hoopes, Laura Miller-Degraff, Michael B. Fessler, Stavros Garantziotis, Shepherd H. Schurman, Darryl Craig Zeldin
In this study, we demonstrate that Forkhead Box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, is retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as a key player in lung allograft remodeling and fibrosis. Utilizing Foxf1_tdTomato BAC (Foxf1_tdTomato) and Foxf1_tdTomato;Col1a1_GFP mice, we show that Lin-Foxf1+ cells encompass the Sca1+CD34+ subset of collagen I-expressing mesenchymal cells (MCs) with capacity to generate colony forming units and lung epithelial organoids. Histologically, Foxf1-expressing MCs formed a three-dimensional network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA sequencing confirmed distinct transcriptional signatures of Foxf1pos/neg MCs, with Foxf1-expressing cells delineated by their high Gli1 and low Integrin α8 expression, from other collagen-expressing MCs. Foxf1+Gli1+ MCs demonstrated proximity to Sonic hedgehog (Shh) expressing bronchial epithelium, and mesenchymal Foxf1/Gli1 expression was found to be dependent on the paracrine Shh signaling in epithelial organoids. Utilizing a murine lung transplant model, we show dysregulation of the epithelial mesenchymal Shh/Gli1/Foxf1 crosstalk and expansion of this specific peri-bronchial MC population in chronically rejecting fibrotic lung allografts.
Russell R. Braeuer, Natalie M. Walker, Keizo Misumi, Serina Mazzoni-Putman, Yoshiro Aoki, Ruohan Liao, Ragini Vittal, Gabriel G. Kleer, David S. Wheeler, Jonathan Z. Sexton, Carol F. Farver, Joshua D. Welch, Vibha N. Lama
Formation of nitric oxide (NO) by the endothelial NO-synthase (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and result in the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as Ca2+ and PDK1, plays a pivotal role in this process. Active PKN2 promoted phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation while active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone and blood pressure.
Young-June Jin, Ramesh Chennupati, Rui Li, Guozheng Liang, ShengPeng Wang, András Iring, Johannes Graumann, Nina Wettschureck, Stefan Offermanns
The transcription factor NFATC2 induces β-cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified ~2,200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle, and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse are less responsive to NFATC2-induced β-cell proliferation, suggesting the FOXP family works to regulate β-cell proliferation in concert with NFATC2. NFATC2 induced β-cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified ~250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle-associated transcripts, and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce β-cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of non-coding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate β-cell proliferation.
Shane P. Simonett, Sunyoung Shin, Jacob A. Herring, Rhonda Bacher, Linsin A. Smith, Chenyang Dong, Mary E. Rabaglia, Donnie S. Stapleton, Kathryn L. Schueler, Jeea Choi, Matthew N. Bernstein, Daniel R. Turkewitz, Carlos Perez-Cervantes, Jason Spaeth, Roland Stein, Jeffery S. Tessem, Christina Kendziorski, Sunduz Keles, Ivan P. Moskowitz, Mark P. Keller, Alan D. Attie
Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, CD146 was found to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the anti-tumor response of T cells. Together, our data reveal a previously unrecognized LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.
Hongxia Duan, Lin Jing, Xiaoqing Jiang, Yanbin Ma, Daji Wang, Jianquan Xiang, Xuehui Chen, Zhenzhen Wu, Huiwen Yan, Junying Jia, Zheng Liu, Jing Feng, Mingzhao Zhu, Xiyun Yan
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