Degenerative arthritis is the most common cause of disability in the US and affects more than 50% of the population over age 65. One form of degenerative arthritis, calcium pyrophosphate (CPP) deposition disease (CPPD) is strongly associated with common conditions in the Veteran population including advanced age, prior joint injury and osteoarthritis (OA). While CPP crystals are known to accelerate articular tissue damage though multiple mechanisms, the etiology of CPP crystal formation remains unclear. Overproduction of extracellular inorganic pyrophosphate (ePPi) by chondrocytes is an absolute requirement for CPP crystal formation and enzymatic hydrolysis of extracellular ATP (eATP) by the ecto-enzyme ENPP1 is the rate limiting step in ePPi production. As there is ample ENPP1 on articular chondrocytes to convert all available substrate, it follows that increased concentrations of eATP in cartilage cause CPPD. We recently reported that the multipass transmembrane protein, ANK, which was originally described as a PPi transporter, critically regulates eATP levels in adult articular chondrocyte cultures. ANK is a highly conserved protein which is widely distributed, uniquely structured, and lacks a clearly defined function. The purpose of the work proposed here is to prove that ANK functions as an eATP transporter, that ANK is the dominant ATP efflux pathway in OA chondrocytes, and that ANK suppression with the FDA-approved drug, probenecid will reduce synovial fluid ATP levels in CPPD. The following specific aims will be accomplished with Xenopus oocytes, human and murine chondrocytes, and patients with CPPD.
Specific Aim 1. To prove that ANK functions as an ATP transporter by A) demonstrating that ANK increases eATP efflux in a Xenopus oocyte expression system, and B) using site-directed mutagenesis to determine structure-function relationships in ANK null murine chondrocytes Specific Aim 2. To test the hypotheses that ANK over-expression A) causes CPPD in human OA cartilage by dysregulating normal eATP efflux mechanisms in an arachidonic acid-dependent manner, and B) will produce CPP crystals in murine cartilage in the presence of pyrophosphatase inhibitors Specific Aim 3. To demonstrate that probenecid, an ANK inhibitor and FDA approved drug, reduces synovial fluid [ATP] and [PPi] in patients with CPPD. In addition to providing a source of ePPi for CPP crystal formation, eATP produces cartilage damage by signaling through purinergic receptors to elicit catabolic effectors and to cause pain, and serves as a cofactor for innate immune signals leading to inflammation. ANK is a highly conserved protein which is widely distributed, uniquely structured, and remains poorly understood. Once this work is complete, we will understand ANK's role in eATP efflux in normal and OA chondrocytes, have established the feasibility of creating a mouse model of CPPD, and will have collected pilot data rationalizing the use of the ANK inhibitor, probenecid, to improve clinical outcomes in patients suffering from CPPD.

Public Health Relevance

This project involves an exploration of the role of the transmembrane protein known as ANK in a type of degenerative arthritis called calcium pyrophosphate disease (CPPD). Risk factors for developing CPPD include characteristics which are particularly common in the Veteran population such as advanced age, prior joint injury, and osteoarthritis. There are currently no available therapies for this disease and many Veterans suffer from this painful and debilitating arthritis. The work included in this proposal will demonstrate that ANK protein levels on cartilage cells critically regulate the production of extracellular ATP, a key factor in the development of CPPD. This proposal includes a clinical trial to treat CPPD patients with an FDA-approved gout drug, called probenecid, which inhibits ANK activity. After this work is complete, we will have a better understanding of the causes of this common disease and some novel management strategies.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Immunology A (IMMA)
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Clement J. Zablocki VA Medical Center
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