Phosphoproteomics of cellular mechanosensing reveals NFATC4 as a regulator of myofibroblast activity

Feedback connections between tissue stiffness and cellular contractile forces can instruct cell identity and activity via a process referred to as mechanosensing. Specific phosphoproteome changes during mechanosensing are poorly characterized. In this work, we chart the global phosphoproteome dynamics of primary human lung fibroblasts sensing the stiffness of injury relevant fibronectin coated Poly(dimethylsiloxane) substrates. We discovered a key signaling threshold at a Young’s modulus of eight kPa stiffness, above which cells activated a large number of pathways including RhoA, CK2A1, PKA, AMPK, AKT1, and Hippo-YAP1/TAZ mediated signaling. Time-resolved phosphoproteomics of cell spreading on stiff substrates revealed the temporal dynamics of these stiffness-sensitive signaling pathways. ECM substrate stiffness above eight kPA induced fibroblast contractility, cytoskeletal rearrangements, ECM secretion, and a fibroblast to myofibroblast transition. Our data indicate that phosphorylation of the transcriptional regulator NFATC4 at S213/S217 enhances myofibroblast activity, which is the key hallmark of fibrotic diseases. NFATC4 knock down cells display reduced stiffness induced collagen secretion, cell contractility, nuclear deformation and invasion, suggesting NFATC4 as a novel target for antifibrotic therapy. Synopsis How tissue stiffness regulates identity and activity of tissue fibroblasts is unclear. Mass spectrometry based analysis of tissue stiffness dependent phosphoproteome changes reveals how primary lung fibroblasts sense the mechanical properties of their environment and identifies NFATC4 as a novel regulator of the stiffness dependent transition of fibroblasts to ECM secreting myofibroblasts. Mass spectrometry analysis reveals the signaling landscape of fibroblast mechanosensing Time-resolved phosphoproteomic analysis of cell spreading on fibronectin NFATC4 regulates myofibroblast collagen secretion, cell contractility and invasion

[1]  C. Samakovlis,et al.  Autocrine Sfrp1 inhibits lung fibroblast invasion during transition to injury induced myofibroblasts , 2022, bioRxiv.

[2]  S. Dupont,et al.  Mechanical regulation of chromatin and transcription , 2022, Nature Reviews Genetics.

[3]  J. Lammerding,et al.  Mechanics and functional consequences of nuclear deformations , 2022, Nature Reviews Molecular Cell Biology.

[4]  M. Lindner,et al.  Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics , 2021, Science advances.

[5]  C. Albigès-Rizo,et al.  Calcium signaling mediates a biphasic mechanoadaptive response of endothelial cells to cyclic mechanical stretch , 2021, Molecular biology of the cell.

[6]  Brian A. Aguado,et al.  Nuclear mechanosensing drives chromatin remodelling in persistently activated fibroblasts , 2021, Nature Biomedical Engineering.

[7]  Fabian J. Theis,et al.  Alveolar regeneration through a Krt8+ transitional stem cell state that persists in human lung fibrosis , 2020, Nature Communications.

[8]  N. Kaminski,et al.  Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and relevance to fibrosis , 2020, Nature Communications.

[9]  G. Shivashankar,et al.  Regulation of nuclear architecture, mechanics, and nucleocytoplasmic shuttling of epigenetic factors by cell geometric constraints , 2019, Proceedings of the National Academy of Sciences.

[10]  Taotao Ma,et al.  Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling , 2019, Theranostics.

[11]  Alireza Hadj Khodabakhshi,et al.  Metascape provides a biologist-oriented resource for the analysis of systems-level datasets , 2019, Nature Communications.

[12]  David E. James,et al.  Illuminating the dark phosphoproteome , 2019, Science Signaling.

[13]  Martin Eisenacher,et al.  The PRIDE database and related tools and resources in 2019: improving support for quantification data , 2018, Nucleic Acids Res..

[14]  Sean J. Humphrey,et al.  High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform , 2018, Nature Protocols.

[15]  R. DePinho,et al.  FoxO3 an important player in fibrogenesis and therapeutic target for idiopathic pulmonary fibrosis , 2017, EMBO molecular medicine.

[16]  Fabian J Theis,et al.  SCANPY: large-scale single-cell gene expression data analysis , 2018, Genome Biology.

[17]  D. Navajas,et al.  Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores , 2017, Cell.

[18]  H. Schiller,et al.  Functional proteomics of cellular mechanosensing mechanisms. , 2017, Seminars in cell & developmental biology.

[19]  Haitao Mao,et al.  Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner , 2017, Scientific Reports.

[20]  Jorge Oliver-De La Cruz,et al.  YAP regulates cell mechanics by controlling focal adhesion assembly , 2017, Nature Communications.

[21]  Kevin W. Eliceiri,et al.  ImageJ2: ImageJ for the next generation of scientific image data , 2017, BMC Bioinformatics.

[22]  Marco Y. Hein,et al.  The Perseus computational platform for comprehensive analysis of (prote)omics data , 2016, Nature Methods.

[23]  Philipp Niethammer,et al.  The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation , 2016, Cell.

[24]  V. Hytönen,et al.  Mechanosensing in cell-matrix adhesions - Converting tension into chemical signals. , 2016, Experimental cell research.

[25]  E. Danen,et al.  A guide to mechanobiology: Where biology and physics meet. , 2015, Biochimica et biophysica acta.

[26]  Adam Byron,et al.  Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly , 2015, Nature Cell Biology.

[27]  Sean J Humphrey,et al.  High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics , 2015, Nature Biotechnology.

[28]  M. Irmler,et al.  Validated prediction of pro-invasive growth factors using a transcriptome-wide invasion signature derived from a complex 3D invasion assay , 2015, Scientific Reports.

[29]  Pengke Yan,et al.  Transcriptional Regulation of BACE1 by NFAT3 Leads to Enhanced Amyloidogenic Processing , 2015, Neurochemical Research.

[30]  R. Krishnan,et al.  Active mechanics and dynamics of cell spreading on elastic substrates. , 2014, Soft matter.

[31]  D. Worth,et al.  Drebrin contains a cryptic F-actin–bundling activity regulated by Cdk5 phosphorylation , 2013, The Journal of cell biology.

[32]  Ulrich S. Schwarz,et al.  Physics of adherent cells , 2013, 1309.2817.

[33]  R. Fässler,et al.  Mechanosensitivity and compositional dynamics of cell–matrix adhesions , 2013, EMBO reports.

[34]  M. Mann,et al.  β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments , 2013, Nature Cell Biology.

[35]  M. Lindner,et al.  Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models , 2013, PloS one.

[36]  Hui Liu,et al.  Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis. , 2013, The Journal of clinical investigation.

[37]  Tamar Geiger,et al.  Opening the floodgates: proteomics and the integrin adhesome. , 2012, Current opinion in cell biology.

[38]  David M Reynolds,et al.  Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation. , 2012, Cell stem cell.

[39]  Samuel A. Safran,et al.  Mechanical consequences of cellular force generation , 2011 .

[40]  Weiwei Yang,et al.  Ras-Induced and Extracellular Signal-Regulated Kinase 1 and 2 Phosphorylation-Dependent Isomerization of Protein Tyrosine Phosphatase (PTP)-PEST by PIN1 Promotes FAK Dephosphorylation by PTP-PEST , 2011, Molecular and Cellular Biology.

[41]  Brenton D. Hoffman,et al.  Dynamic molecular processes mediate cellular mechanotransduction , 2011, Nature.

[42]  John R. Yates,et al.  Analysis of the myosinII-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation , 2011, Nature Cell Biology.

[43]  H. Schiller,et al.  Quantitative proteomics of the integrin adhesome show a myosin II‐dependent recruitment of LIM domain proteins , 2011, EMBO reports.

[44]  A. Kho,et al.  Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression , 2010, The Journal of cell biology.

[45]  Benjamin Geiger,et al.  The switchable integrin adhesome , 2010, Journal of Cell Science.

[46]  Adam Byron,et al.  Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator of Rac1 and Arf6 , 2009, Science Signaling.

[47]  Marta García,et al.  NFAT isoforms control activity-dependent muscle fiber type specification , 2009, Proceedings of the National Academy of Sciences.

[48]  J. de Gunzburg,et al.  Dissecting Activation of the PAK1 Kinase at Protrusions in Living Cells* , 2009, The Journal of Biological Chemistry.

[49]  Huan Sun,et al.  Repression of NFAT3 transcriptional activity by estrogen receptors , 2008, Cellular and Molecular Life Sciences.

[50]  H. R. Bergen,et al.  Nuclear factor of activated T3 is a negative regulator of Ras-JNK1/2-AP-1 induced cell transformation. , 2007, Cancer research.

[51]  H. R. Bergen,et al.  RSK2 Mediates Muscle Cell Differentiation through Regulation of NFAT3* , 2007, Journal of Biological Chemistry.

[52]  E. Olson,et al.  Two Novel Members of the ABLIM Protein Family, ABLIM-2 and -3, Associate with STARS and Directly Bind F-actin* , 2007, Journal of Biological Chemistry.

[53]  G. Schlunck,et al.  Contractility as a prerequisite for TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts. , 2006, Investigative ophthalmology & visual science.

[54]  M. Sheetz,et al.  Local force and geometry sensing regulate cell functions , 2006, Nature Reviews Molecular Cell Biology.

[55]  Xinming Cai,et al.  Glycogen Synthase Kinase 3- and Extracellular Signal-Regulated Kinase-Dependent Phosphorylation of Paxillin Regulates Cytoskeletal Rearrangement , 2006, Molecular and Cellular Biology.

[56]  Sylvie Garneau-Tsodikova,et al.  Protein posttranslational modifications: the chemistry of proteome diversifications. , 2005, Angewandte Chemie.

[57]  Erkki Ruoslahti,et al.  Cell spreading controls endoplasmic and nuclear calcium: A physical gene regulation pathway from the cell surface to the nucleus , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[58]  L. Pinna,et al.  One‐thousand‐and‐one substrates of protein kinase CK2? , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[59]  Roger J. Davis,et al.  Phosphorylation of NFATc4 by p38 Mitogen-Activated Protein Kinases , 2002, Molecular and Cellular Biology.

[60]  Ronald V. Maier,et al.  Mitogen-activated protein kinases. , 2002, Critical care medicine.

[61]  D. Allen,et al.  Different Pathways Regulate Expression of the Skeletal Myosin Heavy Chain Genes* , 2001, The Journal of Biological Chemistry.

[62]  R. Davis,et al.  Requirement of Two NFATc4 Transactivation Domains for CBP Potentiation* , 2001, The Journal of Biological Chemistry.

[63]  T. Hoey,et al.  Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins. , 1995, Immunity.