Reductive Stress Selectively Disrupts Collagen Homeostasis and Modifies Growth Factor-independent Signaling Through the MAPK/Akt Pathway in Human Dermal Fibroblasts
2019; Elsevier BV; Volume: 18; Issue: 6 Linguagem: Inglês
10.1074/mcp.ra118.001140
ISSN1535-9484
AutoresNaomi A. Carne, Steven Bell, Adrian P. Brown, Arto Määttå, Michael J. Flagler, Adam M. Benham,
Tópico(s)melanin and skin pigmentation
ResumoRedox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition. Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition. Dermal fibroblasts are a heterogeneous group of cells that can be categorized into three distinct subpopulations according to their location: papillary (upper lineage), where they are more densely packed; reticular (lower lineage) where there are fewer cells among a more organized extracellular matrix (ECM) 1The abbreviations used are: ECMextracellular matrixATFactivating transcription factorCOLcollagenDIAdata independent acquisitionDTTdithiothreitolERendoplasmic reticulumERKextracellular signal-regulated kinaseFCSfetal calf serumGAMPOgoat anti-mouse peroxidaseMAPKmitogen activated protein kinasePDGFRplatelet derived growth factorPDIprotein disulfide isomerasePERKPKR-like endoplasmic reticulum kinaseRAGPOrabbit anti-goat peroxidaseRIPAradio-immunoprecipitation assaySARPOswine anti-rabbit peroxidaseSFMserum free mediaSTATsignal transducer and activator of transcriptionSWATHsequential windowed acquisition of all theoretical fragment ion mass spectra. 1The abbreviations used are: ECMextracellular matrixATFactivating transcription factorCOLcollagenDIAdata independent acquisitionDTTdithiothreitolERendoplasmic reticulumERKextracellular signal-regulated kinaseFCSfetal calf serumGAMPOgoat anti-mouse peroxidaseMAPKmitogen activated protein kinasePDGFRplatelet derived growth factorPDIprotein disulfide isomerasePERKPKR-like endoplasmic reticulum kinaseRAGPOrabbit anti-goat peroxidaseRIPAradio-immunoprecipitation assaySARPOswine anti-rabbit peroxidaseSFMserum free mediaSTATsignal transducer and activator of transcriptionSWATHsequential windowed acquisition of all theoretical fragment ion mass spectra.; and fibroblasts associated with the hair follicle (upper lineage) (1Sorrell J.M. Caplan A.I. Fibroblast heterogeneity: more than skin deep.J. Cell Sci. 2004; 117: 667-675Crossref PubMed Scopus (360) Google Scholar). Skin fibroblasts play an important role in wound healing and repair and are responsible for the secretion of ECM components such as collagen and glycosaminoglycans. The secretion of collagens by fibroblasts requires a complex quality control process that begins in the endoplasmic reticulum (ER). Collagens are synthesized as procollagen α-peptides, three of which come together to form the distinctive triple-helix. The triple helix is stabilized by hydroxylation of proline and lysine residues by prolyl-4-hydroxylase (P4HA) and lysyl hydroxylase (LOX), permitting the formation of interchain hydrogen bonds. After secretion, these triple helical units assemble into ordered polymeric collagen fibrils and bundles. The fibrillar collagens (type I, II, III, V and XI) are organized into matrices in the dermis by a combination of fibrillar-associated collagens (type VI) and the contractile action of fibroblasts attached to the dispersed fibrils (2Malhotra V. Erlmann P. The pathway of collagen secretion.Annu. Rev. Cell Dev. Biol. 2015; 31: 109-124Crossref PubMed Scopus (104) Google Scholar, 3Shoulders M.D. Raines R.T. Collagen Structure and Stability.Annu. Rev. Biochem. 2009; 78: 929-958Crossref PubMed Scopus (2170) Google Scholar). extracellular matrix activating transcription factor collagen data independent acquisition dithiothreitol endoplasmic reticulum extracellular signal-regulated kinase fetal calf serum goat anti-mouse peroxidase mitogen activated protein kinase platelet derived growth factor protein disulfide isomerase PKR-like endoplasmic reticulum kinase rabbit anti-goat peroxidase radio-immunoprecipitation assay swine anti-rabbit peroxidase serum free media signal transducer and activator of transcription sequential windowed acquisition of all theoretical fragment ion mass spectra. extracellular matrix activating transcription factor collagen data independent acquisition dithiothreitol endoplasmic reticulum extracellular signal-regulated kinase fetal calf serum goat anti-mouse peroxidase mitogen activated protein kinase platelet derived growth factor protein disulfide isomerase PKR-like endoplasmic reticulum kinase rabbit anti-goat peroxidase radio-immunoprecipitation assay swine anti-rabbit peroxidase serum free media signal transducer and activator of transcription sequential windowed acquisition of all theoretical fragment ion mass spectra. The secretory demand to produce collagens and other ER clients can trigger an unfolded protein/ER stress response (4Marutani T. Yamamoto A. Nagai N. Kubota H. Nagata K. Accumulation of type IV collagen in dilated ER leads to apoptosis in Hsp47-knockout mouse embryos via induction of CHOP.J. Cell Sci. 2004; 117: 5913-5922Crossref PubMed Scopus (55) Google Scholar). A productive ER stress response results in the activation of the ER transmembrane sensors Ire1α and ATF6 to drive the production of compensatory ER chaperones and lipids, facilitating ER expansion. Meanwhile, PERK attenuates general translation while allowing the synthesis of a restricted set of target genes through the phosphorylation of eIF2α (5Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Transcriptional and translational control in the mammalian unfolded protein response.Annu. Rev. Cell Dev. Biol. 2002; 18: 575-599Crossref PubMed Scopus (808) Google Scholar). 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In addition to inducing an ER stress response, disruption of oxidative protein folding disturbs the redox balance of the cell. Reductants such as DTT can trigger an ER stress response by disrupting disulfide bonds, leading to an accumulation of newly synthesized proteins in the ER (8Jamsa E. Simonen M. Makarow M. Selective retention of secretory proteins in the yeast endoplasmic reticulum by treatment of cells with a reducing agent.Yeast Chichester Engl. 1994; 10: 355-370Crossref PubMed Scopus (92) Google Scholar). However, little is known about the quantitative response of cells to reductants and how the global cell proteome is affected by the combined effect of redox and ER stress, particularly during metabolically demanding conditions such as proteostasis in response to growth factors. To investigate this question, we have studied the effect of reductive ER stress in human dermal skin fibroblasts subject to stimulation by PDGF. The PDGF pathway in fibroblasts is well understood and is an important contributor to wound healing in the skin. PDGF stimulates the dimerization and autophosphorylation of PDGFR family molecules, followed by recruitment of the signal transduction machinery (e.g. GRB2, Src, GAP, PI3 kinase, PLCγ, and NCK), culminating in the activation of STAT transcription factors. Various signaling pathways are initiated, leading to the control of cell growth, proliferation and differentiation (by src, MAPK and PKC pathways); and actin reorganization and cell migration (by the PKC and Akt/PKB pathways) (9Deuel T.F. Silverman N.J. Kawahara R.S. Platelet-derived growth factor: a multifunctional regulator of normal and abnormal cell growth.BioFactors. 1988; 1: 213-217PubMed Google Scholar). Data-independent acquisition (DIA) is a robust and reproducible mass spectrometry method (10Collins B.C. Hunter C.L. Liu Y. Schilling B. Rosenberger G. Bader S.L. Chan D.W. Gibson B.W. Gingras A.-C. Held J.M. Hirayama-Kurogi M. Hou G. Krisp C. Larsen B. Lin L. Liu S. Molloy M.P. Moritz R.L. Ohtsuki S. Schlapbach R. Selevsek N. Thomas S.N. Tzeng S.-C. Zhang H. Aebersold R. Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry.Nat. Commun. 2017; 8: 291Crossref PubMed Scopus (269) Google Scholar) for label-free relative quantification of all detectable analytes within a defined range based on their fragment ion spectra. Purvine et al. first reported a shotgun strategy to identify peptides after collision-induced dissociation in parallel on a TOF-MS machine (11Purvine S. Eppel J.-T. Yi E.C. Goodlett D.R. Shotgun collision-induced dissociation of peptides using a time of flight mass analyzer.Proteomics. 2003; 3: 847-850Crossref PubMed Scopus (130) Google Scholar). The feasibility of automating the quantitative analysis of complex peptides was demonstrated by Venable et al. (12Venable J.D. Dong M.-Q. Wohlschlegel J. Dillin A. 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Cell. Proteomics. 2012; 11 (O111.016717)Abstract Full Text Full Text PDF PubMed Scopus (1777) Google Scholar) and commercialized by ABSCIEX, that we have used in this study to quantitate the response of human skin-derived (dermal) fibroblasts to reductive stress. In recent years, this methodology has been adopted to quantitate protein interactomes, to develop disease biomarkers, to reveal how organisms respond to stress and to map proteostasis, for example in fibroblasts from individuals with Down syndrome (15Gao Y. Wang X. Sang Z. Li Z. Liu F. Mao J. Yan D. Zhao Y. Wang H. Li P. Ying X. Zhang X. He K. Wang H. Quantitative proteomics by SWATH-MS reveals sophisticated metabolic reprogramming in hepatocellular carcinoma tissues.Sci. Rep. 2017; 7: 45913Crossref PubMed Scopus (45) Google Scholar, 16Martins-Marques T. Anjo S.I. Pereira P. Manadas B. Girao H. Interacting network of the gap junction (GJ) protein connexin43 (Cx43) is modulated by ischemia and reperfusion in the heart.Mol. Cell. Proteomics. 2015; 14: 3040-3055Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17Xiao W. Duan X. Lin Y. Cao Q. Li S. Guo Y. Gan Y. Qi X. Zhou Y. Guo L. Qin P. Wang Q. Shui W. Distinct proteome remodeling of industrial Saccharomyces cerevisiae in response to prolonged thermal stress or transient heat shock.J. Proteome Res. 2018; 17: 1812-1825Crossref PubMed Scopus (14) Google Scholar, 18Liu Y. Borel C. Li L. Muller T. Williams E.G. Germain P.-L. Buljan M. Sajic T. Boersema P.J. Shao W. Faini M. Testa G. Beyer A. Antonarakis S.E. Aebersold R. Systematic proteome and proteostasis profiling in human Trisomy 21 fibroblast cells.Nat. Commun. 2017; 8: 1212Crossref PubMed Scopus (63) Google Scholar). Here, we use the technology to discover new redox-responsive protein targets that provide insight into how the redox environment could be modulated for medical and cosmetic benefit. We find that in skin fibroblasts, reductants stimulate the chronic dephosphorylation of p42/44 MAPK (ERK1/2) while concomitantly inducing the phosphorylation of Akt in a growth factor-independent and redox-specific fashion. DIA proteomics revealed that, remarkably, only 1% of the total identified fibroblast proteome was significantly changed after chronic exposure to DTT. Of the proteins that were altered, all but one was diminished, revealing that ER stress induced by DTT does not result in the up-regulation of the pool of secretory pathway clients. Rather, reductive stress destabilizes a select set of proteins that includes collagens, ECM components and MAPK signaling pathway targets. Standard laboratory chemicals were purchased from Sigma Aldrich, UK unless otherwise stated. LiChrosolv LC-MS chromatography solvents were from VWR, UK. All primary antibodies for Western blotting were purchased from Cell Signaling, Danvers, MA unless stated otherwise. Antibodies used were: PathScan® PDGFR Activity Assay Multiplex Western Detection Mixture II (#5304, 1:2000); Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) XP® rabbit mAb (#4370, 1:2000); Phospho-Akt (Ser473) (#9271, 1:1000); Akt (pan) (40D4) mouse mAb (#2920, 1:2000); Phospho-eIF2á (Ser51) rabbit pAb (#9721, 1:1000); PDI mouse mAb RL90 (ab2792, 1:100 for IF, Abcam, Cambridge, UK); β-actin mouse mAb (ab8226, 1:15000, Abcam); collagen type I goat pAb (1310-01, 1:1000 Southern Biotech, Birmingham, AL; raised by immunization against collagen type I and cross-absorped to remove any reactivity against type II, III, IV, V and VI collagens); collagen type VI rabbit pAb (14853-1-AP, 1:500, Proteintech, Rosemont, IL; raised against the human COL6A2-GST fusion protein catalogue number Ag6635). Goat anti-mouse peroxidase (GAMPO), swine anti-rabbit peroxidase (SARPO) and rabbit anti-goat peroxidase (RAGPO)-coupled secondary antibodies for Western blotting were used at 1:3000 and purchased from DAKO (Agilent, Santa Clara, CA) (#P0447, #P0217 and #P0449 respectively). Goat anti-mouse Alexa-Fluor® 488-conjugated secondary antibodies were used for immunofluorescence (Invitrogen ThermoFisher, CA). The phospho-antibodies against pPDGFR, pAkt and p44/42 were validated against a PDGF-BB stimulated fibroblast lysate (supplemental Fig. S1). Human BJ fibroblasts were bought from ATCC®(#CRL-2522™) with initial passage number of 3, and maintained at low passage number in minimum Eagle's medium supplemented with 10% fetal calf serum, 2 mm GlutaMAX, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Cells were passaged twice weekly, when ∼90% confluent, by washing twice in sterile PBS (Sigma, St. Louis, MO or Severn Biotech, Worcestershire, UK) and dispersing in sterile trypsin (ThermoFisher) before reseeding. For cell lysis, cells were seeded in 6 cm dishes or 25 cm2 flasks and lysates were generated by scraping cells in RIPA lysis buffer (1% v/v Triton X-100, 50 mm Tris HCl pH 8, 150 mm NaCl, 0.5% w/v Na-deoxycholate, 0.1% SDS) supplemented with 10 μg/ml of the protease inhibitors antipain, chymostatin, leupeptin and pepstatin A; and 1× phosphatase inhibitors against acid, alkaline, serine/threonine, tyrosine, and dual-specificity phosphatases (PhosSTOP, Roche, Basel, Switzerland). Lysates were cleared by centrifugation at 16,000 × g and post-nuclear supernatants used for subsequent analysis. Platelet Derived Growth Factor-BB isoform (PDGF-BB, Corning #47743-598) was reconstituted in 0.1% BSA, 10 mm acetic acid. Before stimulation with PDGF-BB, spent media was removed and the cells washed with PBS. The cells were replaced into serum-free minimum Eagle's medium supplemented only with 2 mm GlutaMAX, 100 units/ml penicillin, and 100 μg/ml streptomycin (ThermoFisher), with PDGF-BB added to a final concentration of 10 ng/ml. Protein concentration was calculated using an acidified Bradford assay or the commercially available Pierce™ BCA assay kit (ThermoFisher Scientific, 23225). For the Bradford assay, a standard curve was prepared using 10 μl 0, 1, 2, 5, 8, and 10 μg/ml bovine serum albumin (BSA) in RIPA buffer, and samples were prepared as 2 μl sample, 8 μl RIPA buffer. These were each added to 10 μl 0.1 m HCl, 80 μl water and 900 μl Bradford dye (BioRad, Hercules, CA), vortexed and incubated at room temperature for 10 min. Absorbance was read at 595 nm using an Eppendorf Biophotometer for the Bradford assay (Eppendorf AG, Hamburg, Germany). The BCA assay was performed according to manufacturer's instructions in a microplate. Postnuclear supernatants were taken up in 2× sample buffer (65.8 mm Tris, pH 6.8, 2.1% SDS, 26.3% glycerol, 50 mm DTT and 0.01% bromphenol blue), denatured at 95 °C for 5 min, and then subjected to 10% SDS-PAGE. Gels were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) for 2 h at 150 mA or 30 V overnight, and then blocked in 5% milk in TBS-Tween for 1 h at room temperature or 8 h at 4 °C. Membranes were incubated with primary antibodies in 5% BSA or 5% milk in TBS-Tween overnight at 4 °C before being washed five times with TBS-Tween. Membranes were then incubated with GAMPO, RAGPO or SARPO secondary antibodies for 1 h at room temperature and washed a further five times with TBS-Tween. Proteins were visualized with 500 μl enhanced chemiluminescence fluid (GE Healthcare, IL) per membrane and exposed to film (Kodak) before development in an X-ray developer machine (XOMAT). Staining for senescence-associated β-galactosidase activity was performed using the Senescence β-Galactosidase Staining Kit from Cell Signaling (9860) according to the manufacturer's instructions. Briefly, spent media from treated BJ fibroblast cells was removed and cells were washed once with PBS before fixing in a proprietary fixative solution for 10–15 min at room temperature. Cells were then washed a further two times before application of pH-adjusted β-galactosidase staining solution and incubated overnight in a dry incubator at 37 °C. Staining solution was then removed, and cells washed twice with PBS. Following staining, dishes of stained cells were either stored in glycerol at 4 °C or immediately DAPI stained and cover slips mounted on microscope slides for analysis. Images were taken using a CMEX WiFi 5 camera (Euromex, DC.5000-WIFI) attached to an inverted wide field fluorescence microscope for analysis of DAPI stained cells (Zeiss Apotome). The wound closure assay was performed according to the method of Liang et al. (19Liang C.-C. Park A.Y. Guan J.-L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.Nat. Protoc. 2007; 2: 329Crossref PubMed Scopus (3101) Google Scholar). BJ fibroblasts were seeded onto 6-well plates (Greiner) which had been pre-treated with 10 μg/ml fibronectin (Sigma) and grown to confluency. Cells were serum starved for 24 h and the bottom of each well was scratched with a pipette tip (0.2 ml) to create a linear wound area free of cells. After washing twice with PBS (ThermoFisher) the cells were incubated in 10 ng/ml PDGF supplemented culture media ± 5 mm DTT (Sigma). After 10 min, the cells were washed and fresh SFM containing 10 ng/ml PDGF was added. Cell migration was recorded with an automated Zeiss Cell Observer for 12 h with images taken every 30 min. The cells were kept at 37 °C and 5% CO2 during image capture. Cell viability was confirmed by monitoring cell movement. Migration into the wound site was quantified by counting how many cells passed the scratch point after 10 h; and by calculating the percentage of cells in the field of view that had passed the scratch point after 10 h. A 1-way ANOVA with a Dunnett's multiple comparison test was conducted in Prism 8 to assess statistical significance (mean ± S.D., n = 3). Cells for analysis by immunofluorescence were seeded onto coverslips in 6 cm dishes before treatment. Treatments were performed once cells had reached ∼80% confluence, after which the cells were washed twice with PBS containing 1 mm CaCl2 and 0.5 mm MgCl2 (PBS++). Cells were fixed for 10 min in 4% paraformaldehyde, washed in PBS++, permeabilized using 0.1% Triton X-100 for 10 min, washed once for 5 min with PBS++ and blocked with 2% BSA for 1 h at room temperature. Coverslips were incubated with the anti-PDI antibody RL90 at 1:100 dilution in PBS++/0.2% BSA overnight at 4 °C. The cells were washed in PBS++ and then incubated with Alexa-Fluor® 488-conjugated secondary antibody (Invitrogen ThermoFisher) for 1 h at room temperature. The cells were washed three times in PBS++ and stained with DAPI to visualize nuclei (Sigma). The immunofluorescence images were captured with a brightfield fluorescence microscope (Zeiss Axio Imager M1). Cells (∼106 in a 25 cm2 flask) at ∼90% confluence were subjected to a 1 h or 6 h stress with 5 mm DTT in the presence or absence of 10 ng/ml PDGF stimulation for 24 h. Cells were washed twice in PBS, lysed in 300 μl TRI reagent and RNA extracted with 50 μl chloroform. RNA content was measured using an ND-1000 spectrophotometer (Nanodrop® Technologies Inc) and 50 ng RNA was subjected to RT-PCR, using the Access-Quick RT-PCR kit (Promega). Primers for Actin were: CCACACCTTCTACAATGAGC and ACTCCTGCTTGCTGATCCAC and for XBP1 were: GAAACTGAAAAACAGAGTAGCAGC and GCTTCCAGCTTGGCTGATG. PCR was carried out using a PTC-200 DNA engine (MJ Research) with 30 cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min. Actin cDNA was analyzed on a 1% agarose gel whereas XBP1 cDNA was subjected to Pst1 digestion for 2 h at 37 °C and DNA purified using a PCR purification kit (Qiagen, Hilden, Germany) to distinguish between the IRE1-spliced and non-spliced forms of XBP1. The resulting digested cDNA was then analyzed on a 2% agarose gel and both 1 and 2% gels were visualized by UV light with an INGenius bioimager (Syngene). White on black images were inverted using ImageJ software. Peptide samples were prepared using a commercial FASP Protein Digestion Kit (Expedeon #44250) and sequencing grade-modified trypsin (Promega, #V5111). Spin-filter eluates were freeze-dried and resuspended in 3% acetonitrile, 0.1% TFA and de-salted using C18 ZipTips (Millipore). Sample fractions containing 5 μg peptides were analyzed using an ekspert™ nanoLC 425 with low micro gradient flow module (Eksigent) coupled to a quadrupole Time-Of-Flight (QTOF) mass spectrometer (TripleTOF 6600, SCIEX, MA) with a DuoSpray source (SCIEX) and a 50-micron ESI electrode (Eksigent). Samples were loaded and washed on a TriArt C18 Capillary guard column 1/32″, 5 μm, 5 × 0.5 mm trap column (YMC) and online chromatographic separation performed over 57 min on a Triart C18 Capillary column 1/32″, 12 nm, S-3 μm, 150 × 0.3 mm (YMC) at a flow rate of 5 μl/min with a linear gradient of 3–32% acetonitrile, 0.1% formic acid over 43 min, then to 80% acetonitrile, 0.1% formic acid over 2 min, held for 3 min before returning to 3% acetonitrile, 0.1% formic acid and re-equilibrated. SWATH acquisition was for 55 min with a 3.2 s cycle time. Each cycle consisted of MS-spectrum acquisition at 400 to 1,250 m/z for 250 msec followed by MS/MS (100 to 1500 m/z) using 100 variable SWATH windows (parameters downloaded from http://sciex.com/community/entitty/1217), 25 msec accumulation for each in high sensitivity mode with rolling CE and 2+ ions selected. Analyst software version 1.7.1 (SCIEX) was used to acquire all MS and MS/MS data. Samples were spiked with iRT peptides (Biognosys) at a ratio of 1 μg protein to 0.1 μl 10 × RT peptide mix. Three biological and three technical replicates were obtained for each treatment condition, widely accepted as appropriate to permit the use of statistical tests in analysis. Protein identifications were obtained by searching spectra against the 10316 entries in the panhuman 10000 protein 2014 spectral library PDX000954 (20Rosenberger G. Koh C.C. Guo T. Röst H.L. Kouvonen P. Collins B.C. Heusel M. Liu Y. Caron E. Vichalkovski A. Faini M. Schubert O.T. Faridi P. Ebhardt H.A. Matondo M. Lam H. Bader S.L. Campbell D.S. Deutsch E.W. Moritz R.L. Tate S. Aebersold R. A repository of assays to quantify 10,000 human proteins by SWATH-MS.Sci. Data. 2014; 1: 140031Crossref PubMed Scopus (266) Google Scholar) in PeakView version 2.2 with the MS/MSALL with SWATH™ acquisition microapp version 2.0. Chromatographic retention time calibration was performed using iRT peptides, and SWATH data processing carried out with the default settings as advised by SCIEX (300 peptides per protein, 5 transitions per peptide, 95% peptide confidence threshold, 1% peptide false discovery rate threshold, 3.0 XIC extraction window and XIC width 75 ppm). Following processing, data was exported to MarkerView version 1.2.1 and normalized by total area sums before analysis by t test. FDR correction of t test associated p values was performed using the p.adjust function in R. Co-efficient of variance calculations were performed using peak area values for each protein manually in excel. R was used to produce graphics. Statistical testing for overrepresentation or enrichment of GO terms was performed using the Panther tools available at pantherdb.org including Bonferroni correction for multiple testing (21Mi H. Huang X. Muruganujan A. Tang H. Mills C. Kang D. Thomas P.D. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements.Nucleic Acids Res. 2017; 45: D183-D189Crossref PubMed Scopus (1455) Google Scholar). The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE (20Rosenberger G. Koh C.C. Guo T. Röst H.L. Kouvonen P. Collins B.C. Heusel M. Liu Y. Caron E. Vichalkovski A. Faini M. Schubert O.T. Faridi P. Ebhardt H.A. Matondo M. Lam H. Bader S.L. Campbell D.S. Deutsch E.W. Moritz R.L. Tate S. Aebersold R. A repository of assays to quantify 10,000 human proteins by SWATH-MS.Sci. Data. 2014; 1: 140031Crossref PubMed Scopus (266) Google Scholar) partner repository with the data set identifier PXD010747. PDGF proteins occur as a family of disulfide-bonded, dimeric isoforms (PDGF-AA, AB, BB, CC and DD). For our experiments, we stimulated cells with PDGF-BB, because this isoform binds to PDGFRαβ, αα and αβ receptor combinations (22Bergsten E. Uutela M. Li X. Pietras K. Ostman A. Heldin C.H. Alitalo K. Eriksson U. PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor.Nat. Cell Biol. 2001; 3: 512-516Crossref PubMed Scopus (462) Google Scholar). Further, PDGF-BB is known from the literature to stimulate the fibroblasts used in this study, and exogenous PDGF-BB is effective in promoting wound healing in vitro and in ameliorating wound healing disorders in vivo (23Beer H.D. Longaker M.T. Werner S. Reduced expression of PDGF and PDGF receptors during impaired wound healing.J. Invest. 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