It is all about calcification

It is well known that ectopic (soft-)tissue mineralization can be due to aging and environmental factors such as smoking. In addition, Mendelian disorders with soft tissue calcification provide clues on genes that involved in this complex pathological condition. It is generally accepted that ectopic mineralization is linked to the metabolic pathway controlling the plasma phosphate (Pi) pyrophosphate (PPi) ratio, since Pi facilitates, and PPi inhibits calcium hydroxyapatite crystal formation. Research has shown that the pathway has metabolic components but may also be affected by the local environment of the calcifying tissue. The paper published by Li et al in the present issue¹ characterizes the Nt5e knock out mouse deficient in the gene coding for one the key components of the pathway.

The first metabolite of this cascade is ATP released from liver cells, facilitated by the plasma membrane transporter ABCC6². Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), a late onset multiorgan calcification disorder. ATP is immediately hydrolyzed in the liver vasculature to AMP and PPi by an ectonuclease, ENPP1. Mutations in the ENPP1 gene result in generalized arterial calcification in infancy (GACI), an early onset inborn disorder with massive calcification of the vascular system. Since approx. 60% of the plasma PPi is provided by the liver, PPi levels are low in both PXE and GACI^2,3. The third step is the hydrolysis of AMP to Pi and adenosine, catalyzed by a membrane-bound ecto-5’-nucleotidase (NT5E/CD73) at the peripheral site of soft tissue calcification. Adenosine down-regulates the expression of tissue non-specific alkaline phosphatase (TNAP), which cleaves PPi to 2Pi molecules. Thus, the unregulated, high activity of NT5E (in the absence of adenosine) results in an elevated Pi/PPi ratio, and ultimately in increased calcification. The human condition associated with mutations in the NT5E/CD73 gene is calcification of joints and arteries (CALJA) also called arterial calcification due to deficiency of CD73 (ACDC).⁴

Interestingly, Nt5e knock out mouse models were generated a decade ago, but the link between this protein and ectopic calcification was not known until the identification of NT5E as the disease gene of ACDC in 2011, and no systematic investigation of calcifying tissues or organs of these mouse strains were performed.

The paper published by Li et al. in the 13(16) issue of Cell Cycle makes an important contribution by filling up this gap. This study is part of a larger project conducted by the Uitto group at Thomas Jefferson University and The Jacksons Laboratory. The ultimate aim is unravel genetic and metabolic components of pathological ectopic calcification by a systematic characterization of mouse strains deleted for key components controlling ectopic calcification (Abcc6, Enpp1 and Nt5e).

Li et al showed that the Nt5e knock out mice have an elevated Pi/PPi ratio with higher Pi and lower PPi plasma levels, this observation is in harmony with the regulatory function of NT5E on TNAP as described above. They demonstrated calcification of costochondral junctions, in juxta-articular spaces of the legs and in various ligaments and capsules adjacent to bones. These symptoms are similar to those found in the corresponding human disease. They also performed careful studies to find mineralization in the vascular system, typical symptoms of the human disease, but – in spite of the shifted Pi/PPi ratio in plasma – no sign of calcification was found. This important finding urges for research for modifying genes to explain the difference between the mouse model and the human disease. Nevertheless, the Nt5e knock out mouse strain is a valuable preclinical model for pharmacological intervention in ACDC. Indeed, a phase 1 clinical trial in ACDC using etidronate (1-hydroxyethan-1,1-diyl)bis(phosphonic acid), a bisphosphonate has been recently approved.⁵ It would be very interesting to see the effect of this particular drug in the mouse Li et al characterized in this paper.