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Published on: March 18, 2022
Hypoxia and Wnt signaling inversely regulate expression of chondroprotective molecule ANP32A in articular cartilage
J Quintiens1, A De Roover2, F M F Cornelis2
1Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Research Center, Department of Development and Regeneration, KU Leuven, Leuven, Belgium; Department of Rheumatology, University Hospitals Leuven, Leuven, Belgium.
Objectives:
ANP32A is a key protector of cartilage health, via preventing oxidative stress and Wnt hyper-activation. We aimed to unravel how ANP32A is regulated in cartilage.
Methods:
A bioinformatics pipeline was applied to identify regulators of ANP32A. Pathways of interest were targeted to study their impact on ANP32A in in vitro cultures of the human chondrocyte C28/I2 cell-line and primary human articular chondrocytes (hACs) from up to five different donors, using Wnt-activator CHIR99021, hypoxia-mimetic IOX2 and a hypoxia chamber. ANP32A was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In vivo, the effect of hypoxia was examined by immunohistochemistry in mice injected intra-articularly with IOX2 after destabilization of the medial meniscus. Effects of Wnt hyper-activation were investigated using Frzb-knockout mice and wild-type mice treated intra-articularly with CHIR99021. Wnt inhibition effects were assessed upon intra-articular injection of XAV939.
Results:
The hypoxia and Wnt signaling pathways were identified as networks controlling ANP32A expression. In vitro and in vivo experiments demonstrated increases in ANP32A upon hypoxic conditions (1.3-fold in hypoxia in C28/I2 cells with 95% confidence interval (CI) [1.11-1.54] and 1.90-fold in hACs [95% CI: 1.56-2] and 1.67-fold in ANP32A protein levels after DMM surgery with IOX2 injections [95% CI: 1.33-2.08]). Wnt hyper-activation decreased ANP32A in chondrocytes in vitro (1.23-fold decrease [95% CI: 1.02-1.49]) and in mice (1.45-fold decrease after CHIR99021 injection [95% CI: 1.22-1.72] and 1.41-fold decrease in Frzb-knockout mice [95% CI: 1.00-1.96]). Hypoxia and Wnt modulated ataxia-telangiectasia mutated serine/threonine kinase (ATM), an ANP32A target gene, in hACs (1.89-fold increase [95% CI: 1.38-2.60] and 1.41-fold decrease [95% CI: 1.02-1.96]).
Conclusions:
Maintaining hypoxia and limiting Wnt activation sustain ANP32A and protect against osteoarthritis.
Insights
Maintaining hypoxia and limiting Wnt activation are key to sustaining ANP32A, a crucial protein for cartilage health, thereby protecting against osteoarthritis. This study reveals how these pathways regulate ANP32A expression.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Acid ceramidase (ASAH1) is crucial for cellular lipid metabolism and homeostasis.
- Dysregulation of ASAH1 is implicated in various diseases, including cancer and neurological disorders.
- Understanding ASAH1 regulation is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the regulatory mechanisms of ANP32A in cartilage.
- To determine the role of hypoxia and Wnt signaling pathways in controlling ANP32A expression.
- To explore the therapeutic potential of modulating these pathways for osteoarthritis prevention.
Main Methods:
- Bioinformatic analysis to identify ANP32A regulators.
- In vitro studies using human chondrocyte cell lines (C28/I2) and primary human articular chondrocytes (hACs).
- In vivo studies in mice using hypoxia-mimetic agents, Wnt activators/inhibitors, and surgical induction of osteoarthritis models.
Main Results:
- Hypoxia and Wnt signaling pathways were identified as key regulators of ANP32A.
- Hypoxic conditions increased ANP32A expression in vitro and in vivo.
- Wnt hyper-activation decreased ANP32A expression in chondrocytes and in mice.
- ATM, an ANP32A target gene, was modulated by hypoxia and Wnt signaling.
Conclusions:
- Maintaining a hypoxic environment and limiting Wnt activation are essential for sustaining ANP32A levels.
- These regulatory mechanisms offer potential therapeutic strategies for protecting cartilage and preventing osteoarthritis.
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