Hypoxia and Hypoxia-Inducible Factor-1α Regulate Endoplasmic Reticulum Stress in Nucleus Pulposus Cells
2020; Elsevier BV; Volume: 191; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2020.11.012
ISSN1525-2191
AutoresEmanuel J. Novais, Hyowon Choi, Vedavathi Madhu, Kaori Suyama, Sandra I. Anjo, Bruno Manadas, Irving M. Shapiro, António J. Salgado, Makarand V. Risbud,
Tópico(s)Calpain Protease Function and Regulation
ResumoEndoplasmic reticulum (ER) stress is shown to promote nucleus pulposus (NP) cell apoptosis and intervertebral disc degeneration. However, little is known about ER stress regulation by the hypoxic disc microenvironment and its contribution to extracellular matrix homeostasis. NP cells were cultured under hypoxia (1% partial pressure of oxygen) to assess ER stress status, and gain-of-function and loss-of-function approaches were used to assess the role of hypoxia-inducible factor (HIF)-1α in this pathway. In addition, the contribution of ER stress induction on the NP cell secretome was assessed by a nontargeted quantitative proteomic analysis by sequential windowed data independent acquisition of the total high-resolution mass spectra–mass spectrometry. NP cells exhibited a lower ER stress burden under hypoxia. Knockdown of HIF-1α increased C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) levels, whereas HIF-1α stabilization decreased the expression of ER stress markers Ddit3, Hsp5a, Atf6, and Eif2a. Interestingly, ER stress inducers tunicamycin and thapsigargin induced HIF-1α activity under hypoxia while promoting the unfolded protein response. NP cell secretome analysis demonstrated an impact of ER stress induction on extracellular matrix secretion, with decreases in collagens and cell adhesion–related proteins. Moreover, analysis of transcriptomic data of NP tissues from aged mice and degenerated human discs showed higher levels of unfolded protein response markers and decreased levels of matrix components. Our study shows, for the first time, that hypoxia and HIF-1α attenuate ER stress responses in NP cells, and ER stress promotes inefficient extracellular matrix secretion under hypoxia. Endoplasmic reticulum (ER) stress is shown to promote nucleus pulposus (NP) cell apoptosis and intervertebral disc degeneration. However, little is known about ER stress regulation by the hypoxic disc microenvironment and its contribution to extracellular matrix homeostasis. NP cells were cultured under hypoxia (1% partial pressure of oxygen) to assess ER stress status, and gain-of-function and loss-of-function approaches were used to assess the role of hypoxia-inducible factor (HIF)-1α in this pathway. In addition, the contribution of ER stress induction on the NP cell secretome was assessed by a nontargeted quantitative proteomic analysis by sequential windowed data independent acquisition of the total high-resolution mass spectra–mass spectrometry. NP cells exhibited a lower ER stress burden under hypoxia. Knockdown of HIF-1α increased C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) levels, whereas HIF-1α stabilization decreased the expression of ER stress markers Ddit3, Hsp5a, Atf6, and Eif2a. Interestingly, ER stress inducers tunicamycin and thapsigargin induced HIF-1α activity under hypoxia while promoting the unfolded protein response. NP cell secretome analysis demonstrated an impact of ER stress induction on extracellular matrix secretion, with decreases in collagens and cell adhesion–related proteins. Moreover, analysis of transcriptomic data of NP tissues from aged mice and degenerated human discs showed higher levels of unfolded protein response markers and decreased levels of matrix components. Our study shows, for the first time, that hypoxia and HIF-1α attenuate ER stress responses in NP cells, and ER stress promotes inefficient extracellular matrix secretion under hypoxia. Low back pain is one of the leading causes of disability. Although the pathophysiology is not wholly understood, disc degeneration is considered one of the major factors contributing to the disease process.1Risbud M.V. Shapiro I.M. Role of cytokines in intervertebral disc degeneration: pain and disc content.Nat Rev Rheumatol. 2014; 10: 44-56Crossref PubMed Scopus (732) Google Scholar The intervertebral disc is composed of three compartments: nucleus pulposus (NP), the central, aneural, and avascular tissue, rich in proteoglycans; annulus fibrosus, circumferential, collagen-rich tissue; and superior and inferior cartilaginous endplates. More importantly, because of the physiologically hypoxic environment of the NP, resident cells rely on hypoxia-inducible factor (HIF)-1α signaling to function.2Choi H. Merceron C. Mangiavini L. Seifert E.L. Schipani E. Shapiro I.M. Risbud M.V. Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling.Autophagy. 2016; 12: 1631-1646Crossref PubMed Scopus (62) Google Scholar In contrast to other cell types, regulation of HIF-1α is unique in these cells; it is robustly expressed under normoxia and shows only a minimal increase in its stability under hypoxia.3Schoepflin Z.R. Silagi E.S. Shapiro I.M. Risbud M.V. PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis.FASEB J. 2017; 31: 3831-3847Crossref PubMed Scopus (17) Google Scholar Importantly, previous studies of FoxA2Cre;HIF-1αf/f mice with targeted deletion of HIF-1α in the notochord have unequivocally shown that the absence of this key transcription factor compromises NP compartment development and cell survival in vivo.4Merceron C. Mangiavini L. Robling A. Wilson T.L. Giaccia A.J. Shapiro I.M. Schipani E. Risbud M.V. Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus.PLoS One. 2014; 9: e110768Crossref PubMed Scopus (57) Google Scholar The endoplasmic reticulum (ER) is a highly conserved organelle responsible for folding and maturing newly synthesized secretory and transmembrane proteins. ER homeostasis can be compromised by different stimuli, such as high protein demand, inflammatory processes, reactive oxygen species, or mutant proteins, resulting in the accumulation of misfolded and unfolded proteins in the ER lumen. This condition is termed ER stress and leads to an adaptive response, termed the unfolded protein response (UPR), known to be an essential mechanism for cell survival.5Oslowski C.M. Urano F. Measuring ER stress and the unfolded protein response using mammalian tissue culture system.Methods Enzymol. 2011; 490: 71-92Crossref PubMed Scopus (488) Google Scholar The chaperone glucose-regulated protein 78/immunoglobulin heavy-chain–binding (GRP78/BiP), encoded by the HSPA5 gene, is a master regulator of ER homeostasis. It directly promotes protein folding, contributes to proteasomal degradation of misfolded proteins through retrograde transport across ER membrane, and interacts with UPR transducers: inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6).6Lee A.S. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress.Methods. 2005; 35: 373-381Crossref PubMed Scopus (709) Google Scholar Accumulation of unfolded proteins in the ER results in dissociation of BiP from these transducers, initiating their respective UPR pathways that involve molecular chaperones, protein processing enzymes, and autophagy to prevent or promote the clearance of unwanted proteins. Briefly, IRE1 senses ER stress and activates IRE1, which then splices X-box binding protein 1 mRNA, up-regulating UPR target genes responsible for ER-associated protein degradation and folding proteins such as protein disulfide isomerase.7Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA.Nature. 2002; 415: 92-96Crossref PubMed Scopus (2002) Google Scholar In the presence of high levels and unresolved ER stress, IRE1 can also activate tumor necrosis factor receptor–associated factor 2 and apoptosis-signaling kinase 1, leading to C-Jun N-terminal kinase (JNK) activation and apoptosis.8Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. ASK1 is essential for JNK/SAPK activation by TRAF2.Mol Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar Likewise, PERK is also an ER transmembrane kinase whose activation prevents the formation of ribosomal complexes, decreasing global mRNA translation and attenuating ER work loading.9Harding H.P. Zhang Y. Bertolotti A. Zeng H. Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response.Mol Cell. 2000; 5: 897-904Abstract Full Text Full Text PDF PubMed Scopus (1471) Google Scholar In parallel, PERK can also activate ATF4, which up-regulates other UPR target genes involved in apoptosis, such as C/EBP homologous protein (CHOP).10Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2276) Google Scholar In addition, UPR activates ATF6, driving ATF6 to the Golgi, where it is cleaved and then translocated to the nucleus to promote the transcription of UPR genes involved in protein folding, processing, and degradation.11Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response.Mol Cell Biol. 2000; 20: 6755-6767Crossref PubMed Scopus (754) Google Scholar Several studies have shown the importance of the UPR in various disease contexts, including intervertebral disc degeneration.12Fujii T. Fujita N. Suzuki S. Tsuji T. Takaki T. Umezawa K. Watanabe K. Miyamoto T. Horiuchi K. Matsumoto M. Nakamura M. The unfolded protein response mediated by PERK is casually related to the pathogenesis of intervertebral disc degeneration.J Orthop Res. 2018; 36: 1334-1345Crossref PubMed Scopus (15) Google Scholar Recent studies show that modulation of ER stress protects NP cells from apoptosis and ameliorates intervertebral disc degeneration.13Liao Z. Luo R. Li G. Song Y. Zhan S. Zhao K. Hua W. Zhang Y. Wu X. Yang C. Exosomes from mesenchymal stem cells modulate endoplasmic reticulum stress to protect against nucleus pulposus cell death and ameliorate intervertebral disc degeneration in vivo.Theranostics. 2019; 9: 4084-4100Crossref PubMed Scopus (98) Google Scholar Interestingly, a cross talk between hypoxia, HIF-1α, and UPR activation has been reported in other cell types.14Delbrel E. Soumare A. Naguez A. Label R. Bernard O. Bruhat A. Fafournoux P. Tremblais G. Marchant D. Gille T. Bernaudin J.F. Callard P. Kambouchner M. Martinod E. Valeyre D. Uzunhan Y. Planès C. Boncoeur E. HIF-1α triggers ER stress and CHOP-mediated apoptosis in alveolar epithelial cells, a key event in pulmonary fibrosis.Sci Rep. 2018; 8: 17939Crossref PubMed Scopus (44) Google Scholar,15Burman A. Kropski J.A. Calvi C.L. Serezani A.P. Pascoalino B.D. Han W. Sherrill T. Gleaves L. Lawson W.E. Young L.R. Blackwell T.S. Tanjore H. Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein.JCI Insight. 2018; 3: e99543Crossref PubMed Scopus (34) Google Scholar However, little is known regarding how ER stress is governed and regulated in the hypoxic niche of the NP compartment. Therefore, we asked the questions: Is ER stress regulated by hypoxia in NP cells, and does HIF-1α play a role in this regulation? Our study shows, for the first time, that in NP cells, hypoxia reduces the ER stress burden, and HIF-1α activity modulates the UPR. We also demonstrate that induction of ER stress results in an inefficient secretion of extracellular matrix, characterized by decreased secretion of a wide spectrum of collagens and cell adhesion–related proteins in vitro and in vivo. NP cells were isolated from adult male Sprague Dawley rats (350 g) in accordance with the protocol (number 00959) approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University (Philadelphia, PA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Corning, Corning, NY; 10-013-CV) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO; F6178) supplemented with antibiotics in T25 flasks until confluent [passage 0 (P0)]. Cells were passaged into T75 flasks for expansion (P1) and used for experiments until P2/3. Cells were maintained in a Hypoxia Workstation (InvivO2 300; Baker Ruskinn, Sanford, ME) with a mixture of 1% O2, 5% CO2, and 94% or 90% N2 from 8 hours up to 5 days; cells were also treated with either tunicamycin (1 to 10 μg/mL; Sigma, St. Louis, MO; T7765) or thapsigargin (20 to 80 nmol/L; Sigma; T9033) for 24 hours. Concentrations of these ER stress–inducing drugs were chosen on the basis of published literature and the absence of reduced cell viability during the treatment period.16Peters L.R. Raghavan M. Endoplasmic reticulum calcium depletion impacts chaperone secretion, innate immunity, and phagocytic uptake of cells.J Immunol. 2011; 187: 919-931Crossref PubMed Scopus (68) Google Scholar Primary rat NP cells were plated in 10-cm plates and were cultured in either normoxia or hypoxia for 24 hours. Cells were trypsinized, collected as a pellet, and fixed overnight at 4°C with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4. After subsequent buffer washes, the samples were post-fixed in 2.0% osmium tetroxide for 1 hour at room temperature, and then washed again in cacodylate buffer, followed by distilled water (dH2O). After dehydration through a graded ethanol series, the cell pellet was infiltrated and embedded in EMbed-812 (Electron Microscopy Sciences, Hatfield, PA; 14900). Thin sections were stained with lead citrate and examined with a JEOL 1010 electron microscope (Peabody, MA) fitted with a Hamamatsu digital camera (Bridgewater, NJ; C4742-95) and AMT Advantage image capture software (Woburn, MA). Assessment of ER morphology was performed by analyzing three randomly selected regions of interest at a magnification of ×26,000 from 13 to 14 NP cells from normoxia and hypoxia groups, respectively. LV-shHIF-1α (clone 232222) and control pLKO.1 lentiviral plasmids were purchased from Sigma-Aldrich. The following plasmids were obtained from the Addgene (Watertown, MA) repository: luciferase reporter vector containing a hypoxia response element (HRE-Luc; number 26731), developed by Navdeep Chandel; and psPAX2 (number 12260) and pMD2G (number 12259), developed by by Didier Trono.17Emerling B.M. Weinberg F. Liu J.L. Mak T.W. Chandel N.S. PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through forkhead transcription factor 3a (FOXO3a).Proc Natl Acad Sci U S A. 2008; 105: 2622-2627Crossref PubMed Scopus (151) Google Scholar HEK 293T cells (ATCC, Manassas, VA; CRL-3216) were plated in 10-cm plates (5 × 106 cells per plate) in complete DMEM 1 day before transfection. Cells were transfected with 9 μg of short hairpin RNA plasmid control (ShCtr) or HIF1A knockdown (ShHIF1A) plasmids along with 6 μg psPAX2 and 3 μg pMD2.G. After 16 hours, transfection medium was removed and replaced with complete DMEM. Lentiviral medium was harvested at 48 to 60 hours after transfection, mixed with 7% PEG 6000 (Sigma-Aldrich; 81253) solution, and incubated overnight at 4°C to precipitate virus particles. The PEG solution was removed from the virus medium before transduction by centrifugation at 1500 × g for 30 minutes to pellet the virus particles. NP cells were plated in complete DMEM 1 day before transduction. Cells in 10-cm plates were transduced with 8 mL of fresh DMEM with 10% heat-inactivated fetal bovine serum containing viral particles, along with 8 μg/mL polybrene. Twenty-four hours later, the medium was removed and replaced with complete DMEM. Cells were harvested for protein extraction 4 days after transduction to ensure maximum knockdown efficiency without affecting cell viability. Total RNA was extracted from NP cells using RNAeasy mini columns (Qiagen, Hilden, Germany). Before elution from the column, RNA was treated with RNase-free DNase I (Qiagen). Purified RNA was converted to cDNA using EcoDry Premix (Clontech, Mountain View, CA). Template cDNA and gene-specific primers were added to the SYBR Green master mixture (Applied Biosystems, Foster City, CA), and mRNA expression was quantified using the Step One Plus Real-Time PCR System (Applied Biosystems). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used to normalize gene expression.18Seol D. Choe H. Zheng H. Jang K. Ramakrishnan P.S. Lim T.H. Martin J.A. Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc.BMC Res Notes. 2011; 4: 162Crossref PubMed Scopus (32) Google Scholar Melting curves were analyzed to verify the specificity of the real-time RT-PCR and the absence of primer dimer formation. Each sample was analyzed in duplicate and included a template-free control. All primers used were synthesized by IDT, Inc. (Coralville, IA). Following treatment, cells were placed on ice and washed with ice-cold phosphate-buffered saline. All of the wash buffers and the final cell lysis/resuspension buffers included 1× Complete Mini Protease Inhibitor Cocktail (Roche, South San Francisco, CA; 11836153001), NaF (5 mmol/L; Sigma-Aldrich; 201154), and Na3VO4 (200 μmol/L; Sigma-Aldrich; S6508). Total cell proteins were resolved by electrophoresis on 8% to 12% SDS-polyacrylamide gels and electroblotted to polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA; IPVH00010). The membranes were blocked with 5% nonfat dry milk in 1% Tween 20 (Bio-Rad, Hercules, CA; 161–0781) in tris-buffered saline and then incubated overnight at 4°C in 5% nonfat dry milk in 1% Tween 20 in tris-buffered saline with antibodies against glyceraldehyde-3-phosphate dehydrogenase (1:1000; number 3583S), CHOP (1:1000; number 2895), ATF4 (1:1000; number 11815), BiP (1:1000; number 3183), and PERK (1:1000; number 5683p), from Cell Signaling Technology (Danvers, MA), and ATF6 (1:1000; number NBP1-40256), from Novus Biologicals (Littleton, CO). The membrane was also incubated with primary antibodies against HIF-1α (1:1000; BD Transduction Laboratories, San Jose, CA; 610958) or β-tubulin (1:10,000; Developmental Studies Hybridoma Bank, Iowa City, IA; E7). Immunolabeling was detected using the Amersham ECL Reagent (Thermo Fisher Scientific, Waltham, MA; 45-002-401). Tubulin and glyceraldehyde-3-phosphate dehydrogenase were used as loading controls and to normalize protein expression analysis for densitometry quantification in ImageJ software version 1.53e (NIH, Bethesda, MD; http://imagej.nih.gov/ij).19Madhu V. Boneski P.K. Silagi E. Qiu Y. Kurland I. Guntur A.R. Shapiro I.M. Risbud M.V. Hypoxic regulation of mitochondrial metabolism and mitophagy in nucleus pulposus cells is dependent on HIF-1α -BNIP3 axis.J Bone Miner Res. 2020; 35: 1504-1524Crossref PubMed Scopus (20) Google Scholar The secretome of NP cells (P2) was assessed following the protocol by Fraga et al.20Fraga J.S. Silva N.A. Lourenço A.S. Gonçalves V. Neves N.M. Reis R.L. Rodrigues A.J. Manadas B. Sousa N. Salgado A.J. Unveiling the effects of the secretome of mesenchymal progenitors from the umbilical cord in different neuronal cell populations.Biochimie. 2013; 95: 2297-2303Crossref PubMed Scopus (28) Google Scholar Briefly, cells were seeded at a density of 12,000 cells/cm2 and maintained for 2 days in culture under hypoxia. Cells were then washed three times with phosphate-buffered saline and incubated with Opti-MEM medium (31985070; Thermo Fisher), with or without tunicamycin (5 mg/mL) or thapsigargin (40 nmol/L) for 24 hours. Following the treatment, the medium was collected and centrifuged at 400 × g for 10 minutes to remove any cell debris, concentrated (100×) by centrifugation using a 5-kDa cutoff concentrator (Vivaspin; GE Healthcare, Chicago, IL), and frozen at −80°C until used for proteomic analysis. To characterize the secretome, a nontargeted quantitative proteomic analysis by sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH)–mass spectrometry (MS) was performed.21Anjo S.I. Santa C. Manadas B. Short GeLC-SWATH: a fast and reliable quantitative approach for proteomic screenings.Proteomics. 2015; 15: 757-762Crossref PubMed Scopus (45) Google Scholar,22Anjo S.I. Santa C. Manadas B. SWATH-MS as a tool for biomarker discovery: from basic research to clinical applications.Proteomics. 2017; 17: 3-4Crossref Scopus (92) Google Scholar Dried secretome samples were solubilized in 30 μL Laemmli buffer containing 1 μg of a recombinant protein [maltose-binding protein (MBP)–green fluorescent protein] to be used as internal standard (IS) and retention time calibrator.23Anjo S.I. Simões I. Castanheira P. Grãos M. Manadas B. Use of recombinant proteins as a simple and robust normalization method for untargeted proteomics screening: exhaustive performance assessment.Talanta. 2019; 205: 120163Crossref PubMed Scopus (5) Google Scholar Each sample (25 μL) was used for SWATH-MS analysis, and 5 μL of each replicate was combined to generate representative pools of each experimental group to be used for protein identification/library generation [information-dependent acquisition (IDA) experiments]. All samples were digested with trypsin by Short-GeLC approach,21Anjo S.I. Santa C. Manadas B. Short GeLC-SWATH: a fast and reliable quantitative approach for proteomic screenings.Proteomics. 2015; 15: 757-762Crossref PubMed Scopus (45) Google Scholar, 22Anjo S.I. Santa C. Manadas B. SWATH-MS as a tool for biomarker discovery: from basic research to clinical applications.Proteomics. 2017; 17: 3-4Crossref Scopus (92) Google Scholar, 23Anjo S.I. Simões I. Castanheira P. Grãos M. Manadas B. Use of recombinant proteins as a simple and robust normalization method for untargeted proteomics screening: exhaustive performance assessment.Talanta. 2019; 205: 120163Crossref PubMed Scopus (5) Google Scholar, 24Anjo S.I. Santa C. Saraiva S.C. Freitas K. Barah F. Carreira B. Araújo I. Manadas B. Neuroproteomics using short GeLC-SWATH: from the evaluation of proteome changes to the clarification of protein function.Neuromethods. 2017; 127: 107-138Crossref Scopus (5) Google Scholar with acrylamide as the alkylating agent. Samples were analyzed on a Triple TOF 5600 System (ABSciex, Framingham, MA) in two phases: IDA of the pooled samples and SWATH acquisition of each individual sample for protein quantification. Differentially secreted proteins between control versus tunicamycin and control versus thapsigargin groups were identified using multiple t-test analyses (P < 0.1).25Diz A.P. Carvajal-Rodríguez A. Skibinski D.O.F. Multiple hypothesis testing in proteomics: a strategy for experimental work.Mol Cell Proteomics. 2011; 10: 10Abstract Full Text Full Text PDF Scopus (113) Google Scholar,26Pascovici D. Handler D.C.L. Wu J.X. Haynes P.A. Multiple testing corrections in quantitative proteomics: a useful but blunt tool.Proteomics. 2016; 16: 2448-2453Crossref PubMed Scopus (79) Google Scholar Similarly, differentially secreted proteins between the three groups were identified using the Kruskal-Wallis test (P < 0.1 cutoff).25Diz A.P. Carvajal-Rodríguez A. Skibinski D.O.F. Multiple hypothesis testing in proteomics: a strategy for experimental work.Mol Cell Proteomics. 2011; 10: 10Abstract Full Text Full Text PDF Scopus (113) Google Scholar,26Pascovici D. Handler D.C.L. Wu J.X. Haynes P.A. Multiple testing corrections in quantitative proteomics: a useful but blunt tool.Proteomics. 2016; 16: 2448-2453Crossref PubMed Scopus (79) Google Scholar Principal component analysis was performed using Z-score values calculated from the normalized protein signals of all identified secreted proteins. Differential secreted proteins were then used for hierarchical clustering analysis, using the Pearson correlation method. Graphical analysis was performed with the Multiple Experiment Viewer (MeV) software version 4.9.0 (Bioinformatics, Austin, TX). Dynamic profiles of the proteins among the experimental conditions were determined by heat map and unsupervised clustering analyses using the R package Mfuzz,27Kumar L. Futschik M.E. Mfuzz: A software package for soft clustering of microarray data..Bioinformation. 2007; 2: 5-7Crossref PubMed Google Scholar which performs fuzzy c-means clustering. Unsupervised clustering was performed for the standardized levels of the 355 altered proteins, with a P-value cutoff of 10% using a Kruskal-Wallis test. The number of clusters was set to 4, and the fuzzifier coefficient was set to 4.08.28Schwämmle V. Jensen O.N. A simple and fast method to determine the parameters for fuzzy c-means cluster analysis.Bioinformatics. 2010; 26: 2841-2848Crossref PubMed Scopus (124) Google Scholar The optimal number of clusters was determined by first plotting the minimum centroid distance across a range of cluster values and then observing the cluster value at which this distance plateaued. Membership value represents how well the protein profile fits the average cluster profile. All of the proteins with a membership value <0.5 were not associated with any cluster. The final list of proteins from clusters 1 and 3 was used for gene ontology (GO) process characterization of up-regulated and down-regulated common proteins. GO process characterization of protein lists was performed using Protein Analysis Through Evolutionary Relationships (PANTHER) overrepresentation analysis version PANTHER15.0.29Mi H. Muruganujan A. Huang X. Ebert D. Mills C. Guo X. Thomas P.D. Protocol update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0).Nat Protoc. 2019; 14: 703-721Crossref PubMed Scopus (403) Google Scholar The MS proteomics data were deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via Proteomics Identifications database (PRIDE) (https://www.ebi.ac.uk/pride, last accessed December 3, 2020), with the data set identifier PXD019151.30Perez-Riverol Y. Csordas A. Bai J. Bernal-Llinares M. Hewapathirana S. Kundu D.J. Inuganti A. Griss J. Mayer G. Eisenacher M. Pérez E. Uszkoreit J. Pfeuffer J. Sachsenberg T. Yilmaz Ş. Tiwary S. Cox J. Audain E. Walzer M. Jarnuczak A.F. Ternent T. Brazma A. Vizcaíno J.A. The PRIDE database and related tools and resources in 2019: improving support for quantification data.Nucleic Acids Res. 2019; 47: D442-D450Crossref PubMed Scopus (3230) Google Scholar,31Deutsch E.W. Bandeira N. Sharma V. Perez-Riverol Y. Carver J.J. Kundu D.J. García-Seisdedos D. Jarnuczak A.F. Hewapathirana S. Pullman B.S. Wertz J. Sun Z. Kawano S. Okuda S. Watanabe Y. Hermjakob H. Maclean B. Maccoss M.J. Zhu Y. Ishihama Y. Vizcaíno J.A. The ProteomeXchange consortium in 2020: enabling "big data" approaches in proteomics.Nucleic Acids Res. 2020; 48: D1145-D1152PubMed Google Scholar Samples were analyzed on a Triple TOF 5600 System in two phases: IDA of the pooled samples and SWATH acquisition of each individual sample. Peptides were resolved by liquid chromatography (nanoLC Ultra 2D; Eksigent, Dublin, CA) on a MicroLC column ChromXP C18CL (300-μm internal diameter × 15-cm length; 3-μm particles; 120-Å pore size; Eksigent) at 5 μL/minute with the following multistep gradient: 0 to 2 minutes linear gradient from 2% to 5%, 2 to 45 minutes linear gradient from 5% to 30%, and 45 to 46 minutes at 35% of acetonitrile in 0.1% formamide (FA) and 5% dimethyl sulfoxide. Peptides were eluted into the mass spectrometer using an electrospray ionization source (DuoSpray Source; ABSciex) with a 50-μm internal diameter stainless steel emitter (NewObjective, Littleton, MA). IDA experiments were performed for three fractions of the condition-specific pooled samples: the mass spectrometer set to scanning full spectra (350 to 1250 m/z) for 250 milliseconds, followed by up to 100 MS/MS scans (100 to 1500 m/z from a dynamic accumulation time, minimum 30 milliseconds for precursor above the intensity threshold of 1000, to maintain a cycle time of 3.3 seconds). Candidate ions with a charge state between 2 and 5 and counts above a minimum threshold of 10 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 25 seconds (mass spectrometer operated by Analyst TF 1.7; ABSciex). Rolling collision was used with a collision energy spread of 5. For SWATH-MS–based experiments, the mass spectrometer was operated in a looped product ion mode,32Collins B.C. Gillet L.C. Rosenberger G. Röst H.L. Vichalkovski A. Gstaiger M. Aebersold R. Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system.Nat Methods. 2013; 10: 1246-1253Crossref PubMed Scopus (229) Google Scholar and the same chromatographic conditions were used as in the IDA run described above. A set of 60 windows (Supplemental Table S1) of variables (width containing 1 m/z for the window overlap) was constructed, covering the precursor mass range of 350 to 1250 m/z. A 250-millisecond survey scan (350 to 1500 m/z) was acquired at the beginning of each cycle for instrument calibration, and SWATH MS/MS spectra were collected from 100 to 1500 m/z for 50 milliseconds, resulting in a cycle time of 3.3 seconds from the precursors ranging from 350 to 1250 m/z. The collision energy for each window was determined according to the calculation for a charge 2 ion centered on the window with variable collision energy spread in accordance with the window. A specific library of precursor masses and fragment ions was generated by combining all files from the IDA experiments and used for subsequent SWATH processing. Libraries were obtain
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