P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity

Abstract

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E (PGE) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE release and was attenuated by inhibition of either phospholipase A or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.

P2X7 Receptor Induces Pyroptotic Inflammation and Cartilage Degradation in Osteoarthritis via NF‐κB/NLRP3 Crosstalk

“…The interference of anabolic and catabolic activities can cause negative cellular changes and even death [ 24 – 27 ]. Dying cells and damaged tissues release large amounts of ATP, which is a critical signal for P2X7 activation [ 28 ]. Extracellular ATP concentrations measured at the inflammation site are extremely high, reaching hundreds of micromolar, in contrast to the low nanomolar range found in healthy tissues.…”

“…For example, AZD9056 inhibited P2X7 to relieve MIA-induced joint swelling and inflammatory-factor release in Wistar rats [ 31 ]. Continuous A740003 blockade of P2X7 suppressed MIA-induced mechanical allodynia [ 29 ], while BzATP was shown to be a potent and selective P2X7 activator [ 28 ]. Interestingly, we found that A740003 alone did not affect chondrocyte activity but that BzATP and cell activity were inversely correlated.…”

Purinergic System Signaling in Metainflammation-Associated Osteoarthritis

“…Interestingly, the P2X7 receptor needs a high concentration of ATP for full activation, suggesting a specific role under pathological conditions ( 201 ). Accumulation of ATP released by osteoarthritic chondrocytes could act as a warning signal via P2X7, aggravating the inflammatory process and pain and causing cell death ( 168 ). Low concentrations of extracellular ATP produce a positive response of the P2X7 receptor activation toward cell proliferation.…”

“…Extracellular concentrations of ATP modulate the activation of these receptors, which act differently according to the tissue where they are expressed. The P2X7 receptor is related to the inflammatory processes during OA development, with high levels of extracellular ATP ( 168 ). However, in adipose tissue, this receptor shows an anti-adipogenic effect ( 171 ).…”

P2X7‐induced nociception in the temporomandibular joint of rats depends on inflammatory mechanisms and C‐fibres sensitization

“…Specifically, P2X7 receptors are predominantly expressed in cells of immunological origin, type B synoviocytes, and chondrocytes of synovial joints (Caporali et al, 2008;Collo et al, 1997;Kim et al, 2001;Surprenant & North, 2009;Tanigawa et al, 2018). Its activation can trigger membrane permeabilization, inflammasome and caspases activation, cell proliferation and pro-inflammatory cytokine release (Caporali et al, 2008;Franceschini et al, 2015;Kahlenberg & Dubyak, 2004;Verhoef et al, 2003), contributing to processes of pain and hyperalgesia in articular tissues (Broom et al, 2008;Hu et al, 2016;McIlwrath et al, 2017;Teixeira et al, 2017Teixeira et al, , 2018.…”