Role of the anti-aging protein Klotho in the autophagy and senescence-associated development of osteoarthritis

Purpose: Human aging is defined as a progressive deterioration of physiological function with increasing age. The incidence of osteoarthritis (OA), one of the most common and debilitating joint diseases, is increasing during aging. Pathological mechanisms underlying OA are not fully characterized but include cell death, production of inflammatory mediators, abnormal differentiation of remaining chondrocytes and degradation of extracellular matrix. During OA, oxygen radical production is increased and oxidant defense mechanisms are compromised. Indeed, there is a progressive accumulation of damaged molecules and organelles due to an impairment in the autophagic process in chondrocytes. Concomitantly, the excessive oxidative stress observed during OA development induces a “stress-induced senescent state” leading to a progressive loss of metabolic activity of chondrocytes. Aging being one of the most important risk factor of OA, we focused our attention on the anti-aging protein Klotho which has been associated with osteoarthritic polymorphisms in humans, and has been shown to up-regulate autophagy and down-regulate senescence in a variety of cell types. As a first step in understanding the role of Klotho in OA, the aim of this study was to assess the expression pattern of Klotho in articular cartilage during aging and OA onset, and correlate its expression with autophagy and senescence markers in a mouse model of the disease, and in human cartilage.

Methods: To perform the longitudinal analysis of Klotho expression in articular cartilage, we used C57BL/6 male mice from 1 to 18 months of age as a spontaneous model of OA. Mice’s hind limbs were harvested and fixed in 4% formaldehyde for 48h. They were then decalcified with Ethylene Diamine Tetraacetic Acid (EDTA) 0.5M at 4°C for 7 to 14 days, embedded in paraffin, and cut in 4 μm sections. Human articular cartilage samples were also obtained from osteoarthritic patients who have undergone total knee replacement. Immunohistochemistry (IHC) and Safranin-O staining were performed using standard procedures. The expression of Klotho, autophagy (LC3b, Beclin-1 and ATG5) and senescence (p16INK4A) markers was determined in these samples.

Results: Analysis of the IHC experiments revealed the expression of Klotho in chondrocytes of articular cartilage, both in mice and in human. In mouse knee joints, the expression of Klotho gradually decreased with age, from 1 month to 18 months. Similarly, LC3b, Beclin-1 and ATG5 markers followed the same expression profile, confirming that the autophagy process in OA cartilage is decreasing with age. Surprisingly, the expression of Klotho in human cartilage was stronger in the affected regions of the articular cartilage than in the intact cartilage regions of the same knee. The expression of both autophagic markers (LC3b, Beclin-1 and ATG5) and the p16INK4A senescence marker was also increased in the affected regions of the human cartilage. Importantly, the expression of these markers in humans was detected in the numerous chondrocyte clusters that were present in the affected cartilage regions. These clusters were absent from mice OA cartilage, underlying different mechanisms at work in the two species.

Conclusions: We have shown for the first time the expression of Klotho in articular cartilage, both in mice and humans. In both species, a similar expression pattern between the anti-aging protein Klotho, and autophagy and senescence markers was observed. However, the presence of cell clusters in human OA cartilage but not in mice reveals fundamental differences in the underlying mechanisms of osteoarthritis development.