Abstract
Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.
Highlights
The formation of calcium crystals [both basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD)] within adult human cartilage is pathological and is a contributory factor in the development and progression of osteoarthritis (OA) (Fuerst et al, 2009; McCarthy and Dunne, 2018; Yan et al, 2020)
We found that chondrocyte IL-6 secretion was inhibited by both febuxostat and allopurinol in a dose-dependent manner (Figure 2B)
A large body of evidence supports the idea that calciumcontaining crystals are active players in the initiation and progression of OA (Conway and McCarthy, 2018; FIGURE 3 | Xanthine oxidase form of xanthine oxidoreductase (XOR) is preferentially involved in chondrocyte mineralization, reactive oxygen species (ROS) production and IL-6 secretion. (A) xanthine oxidase (XO) activity measurement by AmplexRed Kit in cell homogenates from xanthine dehydrogenase (XDH) ki and XO-locked (W338A/F339L) mutant protein (XO ki) chondrocytes. ∗∗p < 0.01. (B) Mitochondrial ROS production in XDH ki and XO ki chondrocytes stimulated or not with 500 μg/ml HA crystals for 2 h. ∗p < 0.05. (C) Representative Alizarin red staining of XDH ki and XO ki chondrocytes cultured with calciprotein particles (CPP) for 24 h
Summary
The formation of calcium crystals [both basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD)] within adult human cartilage is pathological and is a contributory factor in the development and progression of osteoarthritis (OA) (Fuerst et al, 2009; McCarthy and Dunne, 2018; Yan et al, 2020). A molybdopterin-containing enzyme, is transcribed from the xdh gene and exists in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH) Both forms oxidize hypoxanthine/xanthine to uric acid. The conversion of XDH to XO is possible via either irreversible proteolysis or by reversible oxidation of thiol groups at positions 535 and 992 (Nishino and Okamoto, 2015) Both ROS and uric acid generation are inhibited by pharmacological xanthine oxidase inhibitors such as allopurinol (used in the treatment of gout) (Day et al, 2016). We have investigated the role of XOR, a potential source of ROS, in chondrocyte calcification by pharmacological and genetic approaches and studied its link to calcification in OA cartilage samples obtained from patients undergoing knee replacement surgery. Immature chondrocytes were isolated from 5 to 7 days old mice as described (Nasi et al, 2016b) and amplified for 7 days in DMEM + 10%FBS to reach chondrocyte differentiation (Gosset et al, 2008)
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