Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-β signaling.

Stassen, RHMJ, van den Akker, GGH, Surtel, DAM, Housmans, BAC ORCID: 0000-0003-4428-7122, Cremers, A, Caron, MMJ ORCID: 0000-0001-5316-9211, Smagul, A, Peffers, MJ ORCID: 0000-0001-6979-0440, van Rhijn, LW and Welting, TJM (2023) Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-β signaling. Osteoarthritis and cartilage, 31 (8). pp. 1035-1046.

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Abstract

<h4>Objective</h4>Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods.<h4>Method</h4>Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-β) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors.<h4>Results</h4>Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signaling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signaling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression.<h4>Conclusion</h4>BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment.

Item Type:	Article
Uncontrolled Keywords:	Chondrocytes, Humans, Osteoarthritis, Calcium Phosphates, Transforming Growth Factor beta, Interleukin-6, Culture Media, Conditioned, Chromatography, Liquid, Proteomics, Signal Transduction, Tandem Mass Spectrometry, Secretome
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences
Depositing User:	Symplectic Admin
Date Deposited:	28 Apr 2023 09:57
Last Modified:	04 Aug 2023 05:21
DOI:	10.1016/j.joca.2023.02.079
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3170043