To define alterations early in tumor formation, we studied nerve tumors in neurofibromatosis 1 (NF1), a tumor predisposition syndrome. Affected individuals develop neurofibromas, benign tumors driven by NF1 loss in Schwann cells (SCs). By comparing normal nerve cells to plexiform neurofibroma (PN) cells using single-cell and bulk RNA sequencing, we identified changes in 5 SC populations, including a de novo SC progenitor–like (SCP-like) population. Long after Nf1 loss, SC populations developed PN-specific expression of Dcn, Postn, and Cd74, with sustained expression of the injury response gene Postn and showed dramatic expansion of immune and stromal cell populations; in corresponding human PNs, the immune and stromal cells comprised 90% of cells. Comparisons between injury-related and tumor monocytes/macrophages support early monocyte recruitment and aberrant macrophage differentiation. Cross-species analysis verified each SC population and unique conserved patterns of predicted cell-cell communication in each SC population. This analysis identified PROS1-AXL, FGF-FGFR, and MIF-CD74 and its effector pathway NF-κB as deregulated in NF1 SC populations, including SCP-like cells predicted to influence other types of SCs, stromal cells, and/or immune cells in mouse and human. These findings highlight remarkable changes in multiple types of SCs and identify therapeutic targets for PN.
Leah J. Kershner, Kwangmin Choi, Jianqiang Wu, Xiyuan Zhang, Melissa Perrino, Nathan Salomonis, Jack F. Shern, Nancy Ratner
We previously reported that Smad Anchor for Receptor Activation (SARA) plays a critical role in maintaining epithelial cell phenotype. Here, we show that SARA suppresses myofibroblast precursor transdifferentiation in a mouse model of scleroderma. Mice overexpressing SARA specifically in PDGFRβ+ pericytes and pan-leukocytes (SARATg) developed significantly less skin fibrosis in response to bleomycin injection compared to wild-type littermates (SARAWT).Single cell RNASeq analysis of skin PDGFRβ+ cells implicated pericyte subsets assuming myofibroblast characteristics under fibrotic stimuli, and SARA overexpression blocked the transition. In addition, a cluster that expresses molecules associated with Th2 cells and macrophage activation was enriched in SARAWT, but not in SARATg mouse, after bleomycin treatment. Th2- specific Il-31 expression was increased in skin of the bleomycin-treated SARAWT mice, and scleroderma patients. Receptor-ligand analyses indicated that lymphocytes mediate pericyte transdifferentiation in SARAWT mice, while with SARA overexpression the myofibroblast activity of pericytes was suppressed. Together, these data suggest a novel crosstalk between myofibroblast precursors and immune cells in the pathogenesis of SSc, for which SARA plays a critical role.
Katia Corano-Scheri, Xiaoyan Liang, Vidhi Dalal, I. Caroline Le Poole, John Varga, Tomoko Hayashida
Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in non-vesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane-protected and disproportionately derived from non-tumour cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.
Ethan Z. Malkin, Steven De Michino, Meghan Lambie, Rita G. Gill, Zhen Zhao, Ariana Rostami, Andrea Arruda, Mark D. Minden, Scott V. Bratman
Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in DM1 patients resemble the appearance of an accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from DM1 patients and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which related to the accumulation of cellular senescence. The data from transcriptome analyses were corroborated in human myoblasts and blood samples as well as in mouse and Drosophila models of the disease. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts, whereas DM1 Drosophila model showed reduced longevity and impaired locomotor activity. Moreover, functional studies highlighted the impact of BMI1 and downstream p16INK4A/RB and ARF/p53/p21CIP pathways in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds, Quercetin, Dasatinib, or Navitoclax, reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in Drosophila in vivo. Our results identified the accumulation of senescence as part of DM1 pathophysiology and therefore, demonstrated the efficacy of senolytic compounds in the pre-clinical setting.
Mikel García-Puga, Ander Saenz-Antoñanzas, Gorka Gerenu, Alex Arrieta, Roberto Fernandez-Torron, Miren Zulaica, Amets Saenz, Joseba Elizazu, Gisela Nogales-Gadea, Shahinaz M. Gadalla, Marcos J. Araúzo-Bravo, Adolfo Lopez de Munain, Ander Matheu
Cellular stress contributes to the capacity of melanoma cells to undergo phenotype switching into highly migratory and drug tolerant dedifferentiated states. Such dedifferentiated melanoma cell states are marked by loss of melanocyte specific gene expression and increase of mesenchymal markers. Two crucial transcription factors, MITF and SOX10, important in melanoma development and progression have been implicated in this process. In this study we describe that loss of MITF is associated with a distinct transcriptional program, MITF promoter hypermethylation and poor patient survival in metastatic melanoma. From a comprehensive collection of melanoma cell lines, we observed that MITF methylated cultures were subdivided in two distinct subtypes. Examining mRNA levels of neural crest associated genes we found that one subtype had lost the expression of several lineage genes including SOX10. Intriguingly, SOX10 loss was associated with SOX10 gene promoter hypermethylation and distinct phenotypic and metastatic properties. Depletion of SOX10 in MITF methylated melanoma cells using CRISPR/Cas9 confirmed these findings. In conclusion, this study describes the significance of melanoma state and the underlying functional properties explaining the aggressiveness of such states.
Adriana Sanna, Bengt Phung, Shamik Mitra, Martin Lauss, Jiyeon Choi, Tongwu Zhang, Ching-Ni Jenny Njauw, Eugenia Cordero, Katja Harbst, Frida Rosengren, Rita Cabrita, Iva Johansson, Karolin Isaksson, Christian Ingvar, Ana Carneiro, Kevin Brown, Hensin Tsao, My Andersson, Kristian Pietras, Göran Jönsson
Idiopathic pulmonary fibrosis (IPF) is a chronic disease of unmet medical need. It is characterized by formation of scar tissue leading to a progressive and irreversible decline in lung function. IPF is associated with repeated injury, which may alter the composition of the extracellular matrix (ECM). Here, we demonstrate that IPF patient–derived pulmonary ECM drives profibrotic response in normal human lung fibroblasts (NHLF) in a 3D spheroid assay. Next, we reveal distinct alterations in composition of the diseased ECM, identifying potentially novel associations with IPF. Growth differentiation factor 15 (GDF15) was identified among the most significantly upregulated proteins in the IPF lung–derived ECM. In vivo, GDF15 neutralization in a bleomycin-induced lung fibrosis model led to significantly less fibrosis. In vitro, recombinant GDF15 (rGDF15) stimulated α smooth muscle actin (αSMA) expression in NHLF, and this was mediated by the activin receptor-like kinase 5 (ALK5) receptor. Furthermore, in the presence of rGDF15, the migration of NHLF in collagen gel was reduced. In addition, we observed a cell type–dependent effect of GDF15 on the expression of cell senescence markers. Our data suggest that GDF15 mediates lung fibrosis through fibroblast activation and differentiation, implicating a potential direct role of this matrix-associated cytokine in promoting aberrant cell responses in disease.
Agata Radwanska, Christopher Travis Cottage, Antonio Piras, Catherine Overed-Sayer, Carina Sihlbom, Ramachandramouli Budida, Catherine Wrench, Jane Connor, Susan Monkley, Petra Hazon, Holger Schluter, Matthew J. Thomas, Cory M. Hogaboam, Lynne A. Murray
Junctional adhesion molecule-A (JAM-A) is expressed in several cell types including epithelial and endothelial cells as well as some leukocytes. In intestinal epithelial cells (IEC), JAM-A localizes to cell junctions and plays a role in regulating barrier function. In vitro studies with model cell lines have shown that JAM-A contributes to IEC migration, however in vivo studies investigating the role of JAM-A in cell migration-dependent processes such as mucosal wound repair have not been performed. In this study, we developed an inducible intestinal epithelial-specific JAM-A knockdown mouse model (Jam-aER-ΔIEC). While acute induction of IEC-specific loss of JAM-A did not result in spontaneous colitis, such mice had significantly impaired mucosal healing after chemically induced colitis and after biopsy colonic wounding. In vitro primary cultures of JAM-A deficient IEC demonstrated impaired migration in wound healing assays. Mechanistic studies revealed that JAM-A stabilizes formation of protein signaling complexes containing Rap1A/Talin/β1-integrin at focal adhesions of migrating IECs. Loss of JAM-A in primary IEC led to decreased Rap1A activity and protein levels of Talin and β1-integrin and a reduction in focal adhesion structures. These findings suggest that epithelial JAM-A plays a critical role in controlling mucosal repair in vivo through dynamic regulation of focal adhesions
Shuling Fan, Kevin Boerner, Chithra K. Muraleedharan, Asma Nusrat, Miguel Quiros, Charles A. Parkos
The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impairs SERCA-dependent SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracts the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from Duchenne myopathy patients display lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorates SR calcium homeostasis, and improves muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic interest for mitigating myopathy.
Alexis Boulinguiez, Christian Duhem, Alicia Mayeuf-Louchart, Benoit Pourcet, Yasmine Sebti, Kateryna Kondratska, Valérie Montel, Stéphane Delhaye, Quentin Thorel, Justine Beauchamp, Aurore Hebras, Marion Gimenez, Marie Couvelaere, Mathilde Zecchin, Lise Ferri, Natalia Prevarskaya, Anne Forand, Christel Gentil, Jessica Ohana, France Piétri-Rouxel, Bruno Bastide, Bart Staels, Helene Duez, Steve Lancel
Acute lung injury (ALI) can cause acute respiratory distress syndrome (ARDS), a lethal condition with limited treatment options and currently a common global cause of death due to COVID-19. ARDS secondary to transfusion-related ALI (TRALI) has been recapitulated preclinically by anti–MHC-I antibody administration to LPS-primed mice. In this model, we demonstrate that inhibitors of PTP1B, a protein tyrosine phosphatase that regulates signaling pathways of fundamental importance to homeostasis and inflammation, prevented lung injury and increased survival. Treatment with PTP1B inhibitors attenuated the aberrant neutrophil function that drives ALI and was associated with release of myeloperoxidase, suppression of neutrophil extracellular trap (NET) formation, and inhibition of neutrophil migration. Mechanistically, reduced signaling through the CXCR4 chemokine receptor, particularly to the activation of PI3Kγ/AKT/mTOR, was essential for these effects, linking PTP1B inhibition to promoting an aged-neutrophil phenotype. Considering that dysregulated activation of neutrophils has been implicated in sepsis and causes collateral tissue damage, we demonstrate that PTP1B inhibitors improved survival and ameliorated lung injury in an LPS-induced sepsis model and improved survival in the cecal ligation and puncture–induced (CLP-induced) sepsis model. Our data highlight the potential for PTP1B inhibition to prevent ALI and ARDS from multiple etiologies.
Dongyan Song, Jose M. Adrover, Christy Felice, Lisa N. Christensen, Xue-Yan He, Joseph R. Merrill, John E. Wilkinson, Tobias Janowitz, Scott K. Lyons, Mikala Egeblad, Nicholas K. Tonks
Aristolochic acid nephropathy (AAN) is characterized by acute proximal tubule necrosis and immune cell infiltration, contributing to the global burden of chronic kidney disease and urothelial cancer. Although the proximal tubule has been defined as the primary target of aristolochic acids I (AAI), the mechanistic underpinning of gross renal deterioration caused by AAI has not been explicitly explained, prohibiting effective therapeutic intervention. To this point, we employed integrated single-cell RNA-sequencing, bulk RNA-sequencing, and mass spectrometry-based proteomics to analyze mouse kidney post-acute AAI exposure. Our results revealed a dramatic reduction of proximal tubule epithelial cells, associated with apoptotic and inflammatory pathways, indicating permanent damage beyond repair. We found the enriched development pathways in other nephron segments, suggesting activation of reparative programs triggered by AAI. The divergent response may be attributed to the segment-specific distribution of organic anions channels along the nephron, including OAT1 and OAT3. Moreover, we observed dramatic activation and recruitment of cytotoxic T and macrophage M1 cells, highlighting inflammation as a principal contributor to permanent renal injury. Ligand-receptor pairing revealed critical intercellular crosstalk underpins damage-induced activation of immune cells. These results provide novel insight into AAI-induced kidney injury and point out potential pathways for future therapeutic intervention.
Jiayun Chen, Piao Luo, Chen Wang, Chuanbin Yang, Yunmeng Bai, Xueling He, Qian Zhang, Junzhe Zhang, Jing Yang, Shuang Wang, Jigang Wang
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