The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes

Abstract

IntroductionExtracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process.MethodsWe measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release.ResultseATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels.ConclusionsThese findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.

Calcium Pyrophosphate Deposition Disease

“…14 ATP efflux and thus the levels of inorganic pyrophosphate are critically regulated by the multipass membrane protein known as ANKH (the human homologue of protein product of the murine progressive ankylosis gene). 15 ANKH may represent a therapeutic target in CPPD, and existing drugs, such as probenecid, act as potent antagonists of ANKH action in vitro. 15 Extra-cellular ATP is metabolized to inorganic pyrophosphate by enzymes with nucleoside triphosphate pyrophosphohydrolase activity, such as ectonucleotide pyrophosphatase 1, whereas alkaline phosphatase and other pyrophosphatases degrade inorganic pyrophosphate.…”

“…15 ANKH may represent a therapeutic target in CPPD, and existing drugs, such as probenecid, act as potent antagonists of ANKH action in vitro. 15 Extra-cellular ATP is metabolized to inorganic pyrophosphate by enzymes with nucleoside triphosphate pyrophosphohydrolase activity, such as ectonucleotide pyrophosphatase 1, whereas alkaline phosphatase and other pyrophosphatases degrade inorganic pyrophosphate. In addition, growth factors, cytokines, and some pharmacologic agents modulate the levels of inorganic pyrophosphate in cartilage (Fig.…”

Adenosine and adenosine receptors in the pathogenesis and treatment of rheumatic diseases

“…A number of transporters are involved in the export of ATP, including the proteins connexin-43 (also known as gap junction alpha-1 protein) 5 , progressive ankylosis protein homolog (ANK) 6 , pannexin-1 and pannexin-3 7 , and probably others as well, including P2×7 8 . Under resting conditions some ATP is dephosphorylated to adenosine but injury, hypoxia or other metabolic assault can trigger increased rates of intracellular conversion of ATP to adenosine or, more commonly, stimulate the release of adenine nucleotides into the extracellular space where they are dephosphorylated to adenosine by ectoenzymes at the cell surface (ecto-5’nucleotidase [CD73] and ecto-nucleoside triphosphate phosphohydrolase [CD39]) and by enzymes in blood or other extracellular fluids (for example, alkaline or acid phosphatases, including tissue nonspecific alkaline phosphatase [TNAP]).…”

“…Under resting conditions some ATP is dephosphorylated to adenosine but injury, hypoxia or other metabolic assault can trigger increased rates of intracellular conversion of ATP to adenosine or, more commonly, stimulate the release of adenine nucleotides into the extracellular space where they are dephosphorylated to adenosine by ectoenzymes at the cell surface (ecto-5’nucleotidase [CD73] and ecto-nucleoside triphosphate phosphohydrolase [CD39]) and by enzymes in blood or other extracellular fluids (for example, alkaline or acid phosphatases, including tissue nonspecific alkaline phosphatase [TNAP]). Once formed or released into the extracellular space adenosine can be deaminated to inosine and, in humans, ultimately to uric acid or taken up directly by cells by specific nucleoside transporters (ENT1 and ENT2) 9 and re-phosphorylated to ATP (Figure 1) 6,9-16 .…”

Regulation of bone and cartilage by adenosine signaling

“…First, apoptotic or necrotic cells release high levels of ATP, the most abundant molecule in the cell [29]. Nucleotide release can also occur in a controlled manner through membrane ion channels such as connexin hemichannels, pannexins, and stretch and voltageactivated channels [30,31]. Facilitated diffusion may also occur via a nucleotide-specific ATP-binding cassette transporter.…”