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pmid:32586320
Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines.
Bernadette Jeremiasse · Csaba Matta · Christopher R Fellows · David J Boocock · Julia R Smith · Susan Liddell · Floris Lafeber · Willem E van Spil · Ali Mobasheri
BMC molecular and cell biology · 2020 doi:10.1186/s12860-020-00288-9
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Experiment
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Analysis
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Complete file
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In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the 'surfaceome'. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting.\nRESULTS: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1-2 and annexin A1 were considered to be of special interest and were analysed further by western blotting.\nCONCLUSIONS: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.", "claims": [{"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [88] partner repository with the dataset identifier PXD014773.1", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:32586320", "locator": "sections[25] \"Availability of data and materials\" paragraph 2", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [88] partner repository with the dataset identifier PXD014773.1"}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": null, "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:32586320"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": [], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "complete", "blocked_on": null, "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": [], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": ["step:obtain_source_data blocked on data:accession_absent"]}, "data": {"state": "partial", "assets": [{"id": "source:pmid:32586320", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1186/s12860-020-00288-9", "sha256": "sha256:3a4cbba5d6b80624d6c113631ac6f64e780cad7a7288693ec22fe37f88beacf7", "bytes": 83081, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1186/s12860-020-00288-9", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC7318434", "source": "europe_pmc_fulltext", "coverage": "whole_body", "text_hash_scope": "rendered_section_body"}}, {"id": "asset:table:pmid:32586320:1", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:81c3a469c673f142bb271cfaf54819d31c5da84834e0af75819415c5278cc2cf", "bytes": 3146, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[7]/table-wrap[1]#Tab2", "version": null, "note": "Results of an overrepresentation analysis on the identified proteins in unstimulated equine articular chondrocytes carried out using the Reactome resource. 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The analysis is based on a statistical (hypergeometric distribution) test that determines whether certain Reactome pathways are over-represented (enriched) in the submitted data. This test produces a probability score, which is corrected for false discovery rate using the Benjamani-Hochberg method. The 25 most relevant pathways sorted by p-value are shown. Table extracted from Reactome Pathway Analysis Report", "structure": {"pmcid": "PMC7318434", "section_index": 7, "section_title": "Cytokine treatment results in the over-representation of different pathways and protein interactions", "row_count": 27, "column_count": 7}, "table": {"label": "Table 3", "caption": "Results of an overrepresentation analysis on the identified proteins following cytokine stimulation in equine articular chondrocytes carried out using the Reactome resource. 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The experimental workflow is presented in F", "10 ng/mL) for 1 h at 37 °C. The samples for western blotting were paired: for each pair, controls and cyt"], "order_basis": "paper_text_order:1247", "id": "assay:2"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.77, "evidence": ["e primary equine articular chondrocytes were cultured in T175 flasks for 72 h with or without IL-1β a"], "order_basis": "paper_text_order:2776", "id": "assay:3"}, {"role": "data_generation", "definition_id": "apoptosis_assay", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.69, "evidence": ["e upon cytokine exposure. Figure S2. 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