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Genetic ablation of adenosine receptor A3 results in articular cartilage degeneration

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Abstract

Osteoarthritis (OA), the most common form of arthritis, is characterized by inflammation of joints and cartilage degradation leading to disability, discomfort, severe pain, inflammation, and stiffness of the joint. It has been shown that adenosine, a purine nucleoside composed of adenine attached to ribofuranose, is enzymatically produced by the human synovium. However, the functional significance of adenosine signaling in homeostasis and pathology of synovial joints remains unclear. Adenosine acts through four cell surface receptors, i.e., A1, A2A, A2B, and A3, and here, we have systematically analyzed mice with a deficiency for A3 receptor as well as pharmacological modulations of this receptor with specific analogs. The data show that adenosine receptor signaling plays an essential role in downregulating catabolic mechanisms resulting in prevention of cartilage degeneration. Ablation of A3 resulted in development of OA in aged mice. Mechanistically, A3 signaling inhibited cellular catabolic processes in chondrocytes including downregulation of Ca2+/calmodulin-dependent protein kinase (CaMKII), an enzyme that promotes matrix degradation and inflammation, as well as Runt-related transcription factor 2 (RUNX2). Additionally, selective A3 agonists protected chondrocytes from cell apoptosis caused by pro-inflammatory cytokines or hypo-osmotic stress. These novel data illuminate the protective role of A3, which is mediated via inhibition of intracellular CaMKII kinase and RUNX2 transcription factor, the two major pro-catabolic regulators in articular cartilage.

Key messages

  • Adenosine receptor A3 (A3) knockout results in progressive loss of articular cartilage in vivo.

  • Ablation of A3 results in activation of matrix degradation and cartilage hypertrophy.

  • A3 agonists downregulate RUNX2 and CaMKII expression in osteoarthritic human articular chondrocytes.

  • A3 prevents articular cartilage matrix degradation induced by inflammation and osmotic fluctuations.

  • A3 agonist inhibits proteolytic activity of cartilage-degrading enzymes.

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Funding

This work is supported by NIH grant R01AR071734, DOD grant W81XWH-13-1-0465, and CIRM grant TRAN1-09288, all to DE. This work was also supported by grants from the Swedish Heart and Lung Foundation (Dnr: 20170124 & 20140448, M.C.), the Swedish Research Council (Dnr: 2016-01381 to M.C. and Dnr. 2016-02835 to ASC), and by KID-funding from the Karolinska Institutet (Dnr 2415/2012-225 and Dnr 2-3707/2013, M.C.).

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Authors and Affiliations

  1. Department of Orthopaedic Surgery, University of Southern California (USC), Los Angeles, CA, USA

    Ruzanna Shkhyan, Siyoung Lee, Francesca Gullo, Nicholas W. Banks, Sean Limfat, Nancy Q. Liu & Denis Evseenko

  2. Department of Orthopaedic Surgery, David Geffen School of Medicine (DGSOM), UCLA, Los Angeles, CA, USA

    Ruzanna Shkhyan, Siyoung Lee, Francesca Gullo & Denis Evseenko

  3. Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

    Lei Li, Maria Peleli, Mattias Carlstrom & Andrei S. Chagin

  4. Institute for Regenerative Medicine, Sechenov Moscow Medical University, Moscow, Russia

    Andrei S. Chagin

  5. Department of Stem Cell Research and Regenerative Medicine, University of Southern California (USC), Los Angeles, CA, USA

    Denis Evseenko

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  1. Ruzanna Shkhyan
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  2. Siyoung Lee
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  3. Francesca Gullo
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  4. Lei Li
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  10. Nancy Q. Liu
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Contributions

RS, SL, FG, KLL, NWB, SL, NQL, MC, ASC and MP conducted experiments. MC provided the A3 KO mice and corresponding WT. RS and DE designed experiments. RS and DE analyzed data. RS, MC, ASC, and DE provided conceptual insight. RS and DE wrote the manuscript. All authors reviewed and revised the manuscript.

Corresponding author

Correspondence to Denis Evseenko.

Ethics declarations

The experiments were approved by the institutional review board (the Stockholm Ethical Committee for Animal Experiments; Protocols: N139/15 & N314/12) and were conducted in accordance with the National Institutes of Health guidelines for the conduct of experiments in animals.

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The authors declare that they have no conflict of interest.

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Shkhyan, R., Lee, S., Gullo, F. et al. Genetic ablation of adenosine receptor A3 results in articular cartilage degeneration. J Mol Med 96, 1049–1060 (2018). https://doi.org/10.1007/s00109-018-1680-3

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  • DOI: https://doi.org/10.1007/s00109-018-1680-3

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