Cell biology
Abstract
Over the last years, a number of drugs have been approved for the treatment of cystic fibrosis (CF). Among them, the new Trikafta, a combination of three drugs, holds great promises to radically improve the quality of life for a large part of CF patients carrying one copy of the most frequent CFTR mutation: F508del. Currently available, disease-modifying, CF drugs work by rescuing the function of mutated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) anion channel. Recent research work shows that membrane lipids, and the cell lipidome in general, play a significant role in the mechanism of CFTR defective trafficking and, on the other hand, in its rescue. In this paper, by using untargeted lipidomics on CFBE41o– cells, we identified distinctive changes in bronchial epithelial cell lipidome associated with treatment with the triple combination VX-661/VX-445/VX-770 (drug name: Trikafta) and other CF drugs. Particularly interesting is the reduction of ceramide levels, known molecular players in the induction of apoptosis, that appears to be associated with a decrease in cell susceptibility to undergo apoptosis. This evidence could account for additional beneficial role of the triple combination on CF phenotypes.
Authors
Nara Liessi, Emanuela Pesce, Clarissa Braccia, Sine Mandrup Bertozzi, Alessandro Giraudo, Tiziano Bandiera, Nicoletta Pedemonte, Andrea Armirotti
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a relative paucity of cancer cells that are surrounded by an abundance of non-tumor cells and extracellular matrix, known as stroma. The interaction between stroma and cancer cells contributes to poor outcome, but how proteins from these individual compartments drive aggressive tumor behavior is not known. Here, we report the proteomic analysis of laser-capture microdissected (LCM) PDAC samples. We isolated stroma, tumor, and bulk samples from a cohort with long- and short-term survivors. Compartment-specific proteins were measured by mass spectrometry, yielding the largest PDAC proteome landscape to date. These analyses revealed that in bulk analysis, tumor-derived proteins were typically masked and that LCM was required to reveal biology and novel prognostic markers. We validated tumor CALB2 and stromal COL11A1 expression as compartment-specific prognostic markers. We identified and functionally addressed the contributions of the tumor cell receptor EPHA2 to tumor cell viability and motility, underscoring the value of compartment-specific protein analysis in PDAC.
Authors
Tessa Y.S. Le Large, G. Mantini, Laura L. Meijer, T.V. Pham, N. Funel, Nicole C.T. van Grieken, B. Kok, Jaco C. Knol, H.W.M. van Laarhoven, S.R. Piersma, C.R. Jimenez, G. Kazemier, E. Giovannetti, M.F. Bijlsma
Abstract
Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient’s risk of severe COPD. We determined 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in IGSF3 (immunoglobulin superfamily, member 3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs of which, rs1414272 and rs12066192 were directly- and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide- and sphingosine-1 phosphate-, but low glycosphingolipids- and gangliosides levels; were less apoptotic and more adherent; with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and a weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.
Authors
Kelly S. Schweitzer, Natini Jinawath, Raluca Yonescu, Kevin Ni, Natalia Rush, Varodom Charoensawan, Irina Bronova, Evgeny Berdyshev, Sonia M. Leach, Lucas A. Gillenwater, Russell P. Bowler, David B. Pearse, Constance A. Griffin, Irina Petrache
Abstract
Free light chains (FLCs) induce inflammatory pathways in proximal tubule cells (PTCs). The role of toll-like receptors (TLR) in these responses is unknown. Here we present findings on the role of TLRs in FLC-induced PTC injury. We exposed human kidney PTC cultures to κ and λ FLCs, and used cell supernatants and pellets for ELISA and gene expression studies. We also analyzed tissues from Stat1–/– and littermate control mice treated with daily intraperitoneal injections of a κ-FLC for 10 days. FLCs increased the expression of TLRs 2, 4, 6 via HMGB1, a damage-associated molecular pattern. Countering TLRs 2, 4, and 6 through GIT-27 or specific TLR-siRNAs reduced downstream cytokine responses. Blocking HMGB1 through siRNA or pharmacologic inhibition, or via STAT1 inhibition reduced FLC-induced TLRs 2, 4, and 6 expression. Blocking endocytosis of FLCs through silencing of megalin/cubilin, with bafilomycin-A1, or hypertonic sucrose attenuated FLC-induced cytokine responses in PTCs. Immunohistochemistry showed decreased TLR 4 and 6 expression in kidney sections from Stat1–/– mice compared to their littermate controls. PTCs exposed to FLCs released HMGB1, which induced TLRs 2, 4, 6 expression and downstream inflammation. Blocking FLCs’ endocytosis, Stat1 knock-down, HMGB1 inhibition, and TLR knock-down each rescued PTCs from FLC-induced injury.
Authors
Rohit Upadhyay, Wei-Zhong Ying, Zannatul Nasrin, Hana Safah, Edgar A. Jaimes, Wenguang Feng, Paul W. Sanders, Vecihi Batuman
Abstract
Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus non-active tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secrete factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of pro-angiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGFBR2, thereby sensitizing tumor cells to TGF-β ligands. Together these findings suggest that Wnt/β-catenin active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.
Authors
Allegra G. Hawkins, Elisabeth A. Pedersen, Sydney Treichel, Kelsey Temprine, Colin Sperring, Jay A. Read, Brian Magnuson, Rashmi Chugh, Elizabeth R. Lawlor
Abstract
Tissue regeneration capacity declines with aging in association with heightened oxidative stress. Expression of the oxidant-generating enzyme, NADPH oxidase 4 (Nox4) is elevated in aged mice with diminished capacity for fibrosis resolution. Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal (BET) family of proteins that function as epigenetic “readers” of acetylated lysine groups on histones. In this study, we explored the role of Brd4 and its interaction with the p300 acetyltransferase in the regulation of Nox4, and the in-vivo efficacy of a BET inhibitor to reverse established age-associated lung fibrosis. BET inhibition interferes with the association of Brd4, p300, and acetylated histone H4K16 with the Nox4 promoter in lung fibroblasts stimulated with the pro-fibrotic cytokine, transforming growth factor-β1 (TGF-β1). This Brd4-Nox4 epigenetic axis is constitutively upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. A number of BET inhibitors, including I-BET-762, JQ1, and OTX015, downregulate Nox4 gene expression and activity. Aged mice with established and persistent lung fibrosis recovered capacity for fibrosis resolution with OTX015 treatment. This study implicates epigenetic regulation of Nox4 by Brd4 and p300, and supports BET/Brd4 inhibition as an effective strategy for the treatment of age-related fibrotic lung disease.
Authors
Yan Y. Sanders, Xing Lyu, Q. Jennifer Zhou, Zheyi Xiang, Denise Stanford, Sandeep Bodduluri, Steven M. Rowe, Victor J. Thannickal
Abstract
Increased microvascular leakage is a cardinal feature of many critical diseases. Regular exercise is associated with improved endothelial function and reduced risk of cardiovascular disease. Irisin, secreted during exercise, contributes to many health benefits of exercise. However, the effects of irisin on endothelial function and microvascular leakage remain unknown. In this study, we found that irisin remarkably strengthened endothelial junctions and barrier function via binding to integrin αVβ5 receptor in LPS-treated endothelial cells. The beneficial effect of irisin was associated with suppression of the Src-MLCK-β-catenin pathway, activation of the AMPK-Cdc42/Rac1 pathway and improvement of mitochondrial function. In preclinical models of microvascular leakage, exogenous irisin improved pulmonary function, decreased lung edema and injury, suppressed inflammation, and increased survival. In ARDS patients, serum irisin levels were decreased and inversely correlated with disease severity and mortality. In conclusion, irisin enhances endothelial barrier function and mitigates microvascular leakage related diseases.
Authors
Jianbin Bi, Jia Zhang, Yifan Ren, Zhaoqing Du, Yuanyuan Zhang, Chang Liu, Yawen Wang, Lin Zhang, Zhihong Shi, Zheng Wu, Yi Lv, Rongqian Wu
Abstract
Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading, and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ), and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared to the wild type Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically-stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage, and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.
Authors
Jonathan P. Gumucio, Martin M. Schonk, Yalda A. Kharaz, Eithne Comerford, Christopher L. Mendias
Abstract
In pulmonary hypertension and certain forms of congenital heart disease, ventricular pressure overload manifests at birth and is an obligate hemodynamic abnormality that stimulates myocardial fibrosis which leads to ventricular dysfunction and poor clinical outcomes. Thus, an attractive strategy is to attenuate the myocardial fibrosis to help preserve ventricular function. Here, by analyzing RNA-sequencing databases and comparing the transcript and protein levels of fibrillar collagen in wild-type and global knockout mice, we found that SLIT3 was predominantly present in fibrillar collagen-producing cells and that SLIT3 deficiency attenuated collagen production in the heart and other non-neuronal tissues. We then performed transverse aortic constriction or pulmonary artery banding in wild-type and knockout mice to induce left and right ventricular pressure overload, respectively. We discovered that SLIT3 deficiency abrogates fibrotic and hypertrophic changes and promotes long-term ventricular function and overall survival in both left and right ventricular pressure overload. Furthermore, we found that SLIT3 stimulated fibroblast activity and fibrillar collagen production, which coincided with the transcription and nuclear localization of the mechanotransducer YAP1. These results indicate that SLIT3 is important for regulating fibroblast activity and fibrillar collagen synthesis in an autocrine manner, making it a potential therapeutic target for fibrotic diseases, especially myocardial fibrosis and adverse remodeling induced by persistent afterload elevation.
Authors
Lianghui Gong, Shuyun Wang, Li Shen, Catherine Liu, Mena Shenouda, Baolei Li, Xiaoxiao Liu, John Shaw, Alan Wineman, Yifeng Yang, Dingding Xiong, Anne Eichmann, Sylvia M. Evans, Stephen J. Weiss, Ming-Sing Si
Abstract
The Wnt/beta-catenin signaling pathway plays an important role in renal development and is re-expressed in the injured kidney and other organs. Beta-catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/beta-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular beta-catenin signaling in CKD remains unclear, we genetically stabilized (i.e. activated) beta-catenin specifically in murine proximal tubules. Mice with increased tubular beta-catenin signaling were protected in two different murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional TCF/LEF-dependent beta-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular beta-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine gamma-lyase (CSE) as a novel transcriptional target of beta-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturn the conventional dogma about beta-catenin signaling and CKD by showing a protective effect of proximal tubule beta-catenin in CKD and identified a new transcriptional target of beta-catenin/FoxO3 signaling that has therapeutic potential for CKD.
Authors
Stellor Nlandu-Khodo, Yosuke Osaki, Lauren Scarfe, Hai-chun Yang, Melanie Phillips-Mignemi, Jane Tonello, Kenyi Saito-Diaz, Surekha Neelisetty, Alla V. Ivanova, Tessa Huffstater, Robert S. McMahon, Makoto M. Taketo, Mark deCaestecker, Balakuntalam S. Kasinath, Raymond C. Harris, Ethan Lee, Leslie Gewin
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