POS0228 HYPOXIA AND INHIBITION OF WNT SIGNALING PROMOTE EXPRESSION OF THE PROTECTIVE MOLECULE ANP32A IN CARTILAGE

Abstract

BackgroundOsteoarthritis (OA) is the most common chronic joint disease and is characterized by damage to the articular cartilage. To date, no cure exists (1). Previously, an association between polymorphisms in the acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) gene and OA was described (2). Our group showed that ANP32A plays a key protective role in OA by preventing oxidative stress (3). In OA cartilage, ANP32A is downregulated as compared to non-OA cartilage (3).ObjectivesMaintaining levels of ANP32A seems crucial to preserve joint health, yet the factors that regulate ANP32A in the joint remain unknown. In this study, we aim to investigate factors that regulate ANP32A expression since such knowledge may lead to the identification of specific novel targets for OA therapy.MethodsA bioinformatic analysis pipeline was applied to identify potential regulatory factors of ANP32A expression. We then investigated two selected pathways in vitro, using pharmacological compounds and incubation in a hypoxia chamber in the human articular chondrocyte cell line C28/I2 and in primary human articular chondrocytes (hACs). The effects on ANP32A expression were evaluated using RT-qPCR and Western blot analysis. For in vivo validation, we performed immunohistochemistry to determine ANP32A protein levels in several mouse models.ResultsThe bioinformatic analysis pipeline identified the Wnt signaling pathway and the hypoxia pathway as likely regulatory pathways of ANP32A expression. Wnt hyper-activation is known to contribute to OA (4). We found that Wnt hyper-activation with CHIR99021 decreased ANP32A expression at the mRNA level and at the protein level in hACs. In vivo, ANP32A protein expression was decreased in Frzb knock-out mice and wild-type mice that were treated with CHIR99021 intra-articularly (two Wnt gain-of-function mouse models). ANP32A protein levels increased in wild-type mice upon intra-articular injection of the Wnt inhibitor XAV939. In healthy conditions, cartilage is in hypoxic status but in OA, hypoxia is lost (5). Treatment with the hypoxia mimetic IOX2 and incubation in a hypoxia chamber increased ANP32A expression in vitro in both the C28/I2 cell line and in hACs. In vivo, ANP32A expression was increased in wild-type mice treated with the hypoxia mimetic IOX2 intra-articularly after induction of OA by destabilization of the medial meniscus (DMM).ConclusionLimiting activation of Wnt signaling and maintaining the homeostatic hypoxic environment within the joint are strategies to sustain ANP32A levels and its protective effect against the development of osteoarthritis.References[1]OARSI White Paper - Osteoarthritis as a serious disease 2016 [Available from: https://www.oarsi.org/sites/default/files/docs/2016/oarsi_white_paper_oa-serious-disease.pdf].[2]Valdes AM, Lories RJ, van Meurs JB, Kerkhof H, Doherty S, Hofman A, et al. Variation at the ANP32A gene is associated with risk of hip osteoarthritis in women. Arthritis Rheum. 2009;60(7):2046-54.[3]Cornelis FMF, Monteagudo S, Guns LKA, den Hollander W, Nelissen R, Storms L, et al. ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone. Sci Transl Med. 2018;10(458).[4]Lories RJ, Monteagudo S. Review Article: Is Wnt Signaling an Attractive Target for the Treatment of Osteoarthritis? Rheumatol Ther. 2020;7(2):259-70.[5]Bouaziz W, Sigaux J, Modrowski D, Devignes CS, Funck-Brentano T, Richette P, et al. Interaction of HIF1alpha and beta-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice. Proc Natl Acad Sci U S A. 2016;113(19):5453-8.AcknowledgementsFellowship FWO (The Research Foundation – Flanders)Disclosure of InterestsJolien Quintiens: None declared, Frederique M.F. Cornelis: None declared, Astrid De Roover: None declared, Ana Escribano Núñez: None declared, Silvia Monteagudo: None declared, Rik Lories Speakers bureau: consultancy & speaker fees from BioSplice (formerly Samumed), Consultant of: consultancy & speaker fees from BioSplice (formerly Samumed)