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A2229 - A Proteomic Analysis of Extracellular Matrix from Idiopathic Pulmonary Fibrosis Patient-Derived Fibroblasts in Response to Transforming Growth Factor β1
Author Block: L. Knüppel1, J. Merl-Pham2, E. Hennen1, R. Hatz3, J. Behr4, O. Eickelberg5, S. Hauck2, C. A. Staab-Weijnitz1; 1Comprehensive Pneumology Center, Helmholtz Zentrum Muenchen, Munich, Germany, 2Research Unit Protein Science, Helmholtz Zentrum Muenchen, Munich, Germany, 3Kunikum Grosshadern Univ of Munich, Munich 81377, Germany, 4Internal Medicine V, University of Munich, Comprehensive Pneumology Ctr., Munchen, Germany, 5Pulmonary Sciences and Critical Care Medicine, Univ of Colorado Denver, Denver, CO, United States.
Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible interstitial lung disease characterized by excessive deposition of extracellular matrix (ECM) in the interstitium leading to progressive dyspnea. Transforming growth factor β1 (TGF-β1) is a central profibrotic cytokine known to induce expression of many ECM components, but these effects have, to our knowledge, not been studied in a proteomic approach.
Methods: Primary human IPF fibroblasts were cultured with and without 2 ng/ml TGF-β1 for 48 h in presence of 0.1 mM 2-phospho-L-ascorbic acid. For enrichment of ECM components, the insoluble material after conventional protein extraction was pelleted and solubilized using 6 M guanidinium chloride-containing buffer. After reduction and alkylation of cysteine residues, ECM components were digested with LysC and trypsin and the resulting peptides analyzed by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) in a quadrupole orbitrap mass spectrometer. Identification and label-free quantification was performed using Progenesis IQ and Mascot. Regulation of gene expression of selected targets was validated by qPCR and Western Blot analysis.
Results: Counting proteins quantified with at least two unique peptides, we identified 1626 proteins in the ECM-enriched fraction. While only 7 % (115) of these proteins were known ECM components as defined by “Matrisome Annotator” (http://matrisome.org/), these accounted for 32 % of total protein abundance. TGF-β1 significantly induced levels of 34 ECM proteins including known targets like fibronectin 1 and the collagen I α1 and α2 chain, but also previously unrecognized targets like SERPINE2 and SEMA7A. At the same time, TGF-β1 significantly reduced levels of 29 ECM proteins including the collagen VI α1, α2, and α3 chains, and laminins α4 and β2. As collagen chain stoichiometry has been shown to strongly influence cleavage efficiency by collagenases, in particular for collagen I, we also assessed abundance ratios for collagen I and collagen VI chains, but found no significant changes.
Conclusions: We provide the first comprehensive proteomic analysis of TGF-β1-induced changes in the ECM. TGF-β1 induced, but, notably, also downregulated expression of a multitude of ECM proteins and regulators in IPF fibroblasts. Collagen I and VI chain stoichiometries were not affected.