Purpose: Ageing, which is a complex physiological process, consists in a gradual decline in certain functions, coordination, loss of homeostasis and physiological integrity, is one of the main risk factors for OA. Like aging, osteoarthritis is commonly described as the result of a disruption of cartilage tissue homeostasis where catabolic products like damage-associated molecular patterns (Damps), accumulate in the joint and induce inflammation. Articular chondrocytes must rely on the autophagic process as the principal mechanism for the maintenance of normal cell function and survival. However, during aging, autophagy gradually decreases in chondrocytes thereby inducing senescence that ultimately leads to increase the severity of OA. Given that aging is one of the main risk factor for OA, antiaging factors have been contemplated with interest for the treatment of OA. The antiaging factor Klotho has been shown to regulate autophagy in a variety of cell types, and Klotho polymorphisms have been associated with higher risks of OA. The aim of this study was thus to investigate the role of Klotho in OA.
Methods: The expression of Klotho and autophagy markers (LC3b and Beclin-1) and OA onset were evaluated in C57BL/6 mice from 1 to 24 months of age as an age-related spontaneous model of OA. Klotho-deficient mice knee joints were harvested and prepared for Safranin-O staining, for immunohistochemistry of autophagy (LC3b, Beclin-1) and apoptosis (cleaved caspase 3 and TUNEL) markers. In parallel, to investigate the relationship between Klotho and autophagy, immature murine articular chondrocytes (iMACs) were stimulated with increasing doses of soluble recombinant Klotho. The effect of Klotho on autophagy was also evaluated in a pathological context, i.e. following IL1-β stimulation (10 ng/ml) for hours. Bax/Bcl-2 ratio, a marker of the intrinsic apoptotic pathway, was also evaluated in IL1-β-treated chondrocytes.
Results: Our results revealed a gradual decrease in Klotho and autophagy markers expression, which correlated with OA progression appearance in the knee joints of mice from escalating ages. In the knee joint of Klotho-deficient mice, an increase in the OA histological score, associated with increased autophagy (LC3b) and chondrocyte death (caspase 3 and TUNEL) in articular cartilage were observed, suggesting a protective anti-apoptotic role of Klotho in cartilage. In vitro, IL1-β treatment in murine primary chondrocytes resulted in autophagy induction as well as an increase in Bax/Bcl-2 ratio, which were both counteracted by the addition of recombinant Klotho protein.
Conclusions: In summary, we described an early articular cartilage degradation in the absence of Klotho, suggesting a potential protective role of Klotho in OA development. The increase in autophagic process in Klotho mutant mice associated with the decrease in autophagy and Klotho expression with age clearly indicate intimate relationships between these two players. In vitro experiments suggested that Klotho may not have a direct effect on autophagy but rather through reducing apoptosis induced by pro-inflammatory cytokines such as IL1-β. This hypothesis is further reinforced by the increased expression of apoptotic and autophagic markers in the articular cartilage of Klotho-deficient mice, and the increasing number of studies demonstrating that excessive induction of autophagy in OA-chondrocytes led to cell death by apoptosis. Further work is needed to decipher the link between autophagy and apoptosis in the pathogenesis of OA in order to develop new treatment to prevent chondrocyte death.
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