Article
Calcium pyrophosphate dihydrate (CPPD) crystals but not basic calcium phosphate (BCP) crystals induce Syndecan-4 expression in cartilage
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Authors
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Sonia Nasi
- University Hospital of Münster, Institute of Musculoskeletal Medicine, Münster
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Jessica Bertrand
- Department of Orthopaedic Surgery, Otto-von-Guericke University, Magdeburg
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Miriam Bollmann
- Otto-von-Guericke-Universität Magdeburg, Magdeburg
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Richard Stange
- University Hospital of Münster, Münster
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Thomas Pap
- Institut für Muskuloskelettale Medizin, Universitätsklinikum Münster, Abteilung Molekulare Medizin, Münster
| Published: | September 9, 2020 |
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Outline
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Introduction: Chondrocalcinosis is a rheumatic condition caused by the deposition of calcium pyrophosphate dihydrate crystals (CPPD) in joint tissues. It is known that CPPD crystals cause inflammation and degenerative changes in joint, but the underlying mechanisms remain poorly understood. In particular, nothing is known about how these crystals and inflammation regulate transmembrane heparan sulphate proteoglycans (HSPGs). We focused on a family of HSPGs called syndecans, as they play important roles as adhesion molecules and as modulators of intracellular signaling triggered by cytokines and growth factors. The aim was to evaluate how CPPD crystals modulate syndecans expression in cartilage, and if this is linked to inflammation and downstream cartilage damage during chondrocylcinosis.
Methods: Murine chondrocytes were stimulated with 0,1 ng/ml CPPD crystals or with 0,1 ng/ml BCP (basic-calcium phosphate) crystals, a family of calcium-containing crystals typical of other rheumatic conditions such as osteoarthritis (OA). Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release in the supernatant at 30 minutes, and 3, 6, 24 hours after stimulation. At the same time-points, mRNA expression levels of syndecans (Synd-1, -2, -3, -4), matrix-degrading enzymes (Mmp-3, -9, -13; Adamts-4, -5), and of the pro-inflammatory cytokine Il-6 was analyzed by qRT-PCR. IL-6 secretion was measured by ELISA in supernatant of BCP and CPPD stimulated cells. Finally, Syndecan-4 protein expression was studied by immunohistochemistry (IHC) in cartilage samples of patients with chondrocalcinosis and in samples of patients with severe OA without chondrocalcinosis as control.
Results: LDH assay revealed no increased cytotoxicity by CPPD or BCP at any time-point. qRT-PCR indicated that CPPD crystals but not BCP crystals induced Synd-2 and -3 upregulation at 30 minutes after stimulation and Synd-4 upregulation at 3 hours, while no modulation of syndecans was seen at later time-points. CPPD also induced Adamts-4 expression at 3 hours after stimulation, and Mmp-9 expression at 3 and 6 hours. The expression of the other matrix-degrading enzymes was not affected. Il-6 mRNA was upregulated by both BCP and CPPD crystals at 30 minutes and 3 hours, and IL-6 secretion was significantly induced by both crystals at 6 and 24 hours. Human chondrocalcinosis cartilage exhibited enhanced Synd-4 expression compared to severe OA cartilage containing BCP calcification. Interestingly, Synd-4 expression was observed in the extracellular matrix but not on cell membrane, suggesting Synd-4 shedding (Figure 1 [Fig. 1]).
Conclusion: BCP and CPPD crystals differentially modulate syndecans and metalloproteases in chondrocytes. CPPD crystals induce Synd-4, Adamts-4 and Mmp-9 which are not induced by BCP crystals. It remains to be clarified whether the two events are interlinked and at which stage IL-6 plays a role. Also, further studies are required to investigate if Adamts-4 and Mmp-9 are involved in Synd-4 shedding or if viceversa Synd-4 regulates Adamts-4 and Mmp-9 activation and downstream cartilage breakdown in chondrocalcinosis.
Disclosures: nothing to disclose
