Roderick H.M.J Stassen, Guus G.H. Van Den Akker, Don A.M Surtel, Andy Cremers, Mandy J. Peffers, Marjolein M.C. Caron, Lodewijk W. Van Rhijn, Tim J.M. Welting
Affiliation(s):
Maastricht Univeristy
Objectives: Determine the changes in the chondrocyte protein secretome in response to BCP crystal stimulation, using an unbiased combinatory method approach.
Status: The role of calcium-containing crystals in the progression of osteoarthritis (OA) have become more evident over the past decades. Various cell types present in the OA-affected joint were shown to be responsive to BCP crystals by increasing secretion of pro-inflammatory factors and matrix metalloproteinases (MMPs). Although progress has been made, a broader overview of the BCP crystal response of human OA articular chondrocytes remains to be elucidated.
Methods: BCP crystals were produced by alkaline hydrolysis of brushite and chemical composition was determined by FTIR. Stimulations of human knee OA articular chondrocytes (OA HACs) were performed with 50 µg/ml BCP crystals. Gene expression levels were determined by RT-qPCR and normalized to PPIA. Secretion of IL-6 was determined by ELISA. Label-free LC-MS/MS quantitative proteomics and cytokine array were performed on 48 hour-conditioned medium from control and BCP-stimulated HACs of 14 individual donors or a pools thereof (Figure 1A).
Findings: To verify biological activity of our synthetically produced BCP crystals, we investigated IL-6 gene expression and protein secretion upon exposure. After 24 and 48 hours of BCP exposure, a significant increase in both IL-6 gene expression and protein was observed. To determine the effect of BCP crystals on extracellular matrix alterations, we examined gene expression of COL1A1, COL2A1 and ACAN. We observed a significant decrease of the expression of these genes, which was further accompanied by increased expression of MMP-1. To broaden the scope beyond the classical OA-related molecules and get a better understanding of the underlying processes that take place in chondrocytes upon BCP exposure, we utilized label-free LC-MS/MS and a cytokine array on medium samples of chondrocytes that were exposed to BCP crystals for 48 hours. LC-MS/MS revealed proteins either induced or decreased by BCP, and involved in a variety of processes such as matrix reorganization and fibrinolysis. Additional analysis of the pooled conditioned media with the cytokine array revealed, amongst others, upregulation of TGF-β signaling-related molecules, such as Activin A and TGF-β2 in response to BCP crystals. Further investigation of TGF-β target genes and SBE luciferase reporters confirmed increased TGF-β signaling upon BCP crystal stimulation. To investigate downstream consequences of this increased TGF-β signaling, and how this relates to BCP-driven IL-6, we inhibited BCP-driven TGF-β signaling with the use of inhibitors. ALK5 or TAK-1 inhibition resulted in decreased IL-6 gene expression and protein levels after 48 hours (Figure 1B).
Significance: Due to the high prevalence of BCP crystals in OA cartilage, there is an increased need for a better understanding of chondrocyte-specific BCP crystal responses. We aimed to map the BCP-induced changes in the chondrocyte’s secretome with the use of two unbiased methods. Our results show that human articular chondrocytes respond to BCP crystals in a complex and diverse manner, including stimulation of matrix reorganization, secretion of pro-inflammatory cytokines and secretion of TGF-β superfamily members. Overall, we revealed new mechanisms in the chondrocyte-specific BCP crystal response potentially aiding the identification of new OA therapeutic targets.