Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans

Abstract

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1 -/-mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (mCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1 -/-mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1 -/-mice than in wild-type, consistent with loss of ENT1-a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1 -/-mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1 -/-mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1 -/-mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders. ß

Lack of the nucleoside transporter ENT1 results in the Augustine-null blood type and ectopic mineralization

“…by guest www.bloodjournal.org From support this hypothesis. 13,14 The precise role of ENT1 in bone homeostasis remains to be established in mice and humans, but it is most likely linked to its ability to transport adenosine, which has emerged as a key regulator of bone metabolism. 15 In fact, other genes of the adenosine pathway have been found to be responsible for calcification disorders.…”

Juxta-articular joint-capsule mineralization in CD73 deficient mice: Similarities to patients withNT5Emutations

“…30 This mouse was developed as a model for diffuse idiopathic skeletal hyperostosis (DISH), a non-inflammatory spondyloarthropathy characterized by ectopic mineralization of spinal tissues. This mouse model was generated through targeted deletion of exons 2-4 of the gene encoding ENT1.…”

“…However, similar to our findings on Nt5e ¡/¡ mice, the Ent1 -/-mice showed no evidence of mineralization in blood vessels, indicating specificity of the mineralization process in these mice for the axial skeleton. 30 Collectively, the mouse models of ectopic mineralization, including those with CD73 and ENT1 deficiency, attest to the presence of a complex pro-mineralization/anti-mineralization network that under physiologic homeostatic conditions prevents ectopic mineralization of soft connective tissues. 12 Alterations in the components of this network, such as altered ratio of P i /PP i and the adenosine concentration, can result in mineralization pathways with phenotypic tissue specificity.…”

Equilibrative nucleoside transporters—A review

“…Choi et al (72) developed the first reported ENT1-null mouse and demonstrated that it maintained normal reproductive behavior, had no gross anatomical abnormalities, and survival rates were similar to wild-type mice, although, bodyweight was significantly less than wild-type littermates (72–74). Several studies have utilized ENT1 null mice (20, 72–77) and found that these mice possess elevated circulating adenosine and thymidine levels in the plasma, reduced cellular uptake of adenosine (20, 73, 75), aberrant bone density (73, 74), dysregulation of the calcification of soft tissues associated with the enthesis regions of the vertebral column and sternum (73), increased resistance to oxidative stress (76), deficit in locomotor activity and motor coordination (74, 77), increased voluntary ethanol self-seeking behaviors associated with increased resistance to acute ethanol intoxication and reduced aversive effects of ethanol (72, 77), and that the absence of ENT1 is associated with reduced anxiety-like behavior in mice (78). …”