Dysregulation of FOXO1 (Forkhead Box O1 Protein) Drives Calcification in Arterial Calcification due to Deficiency of CD73 and Is Present in Peripheral Artery Disease

Abstract

Objective: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL ), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. Conclusions: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.

The elegant complexity of mammalian ecto-5′-nucleotidase (CD73)

“…To overcome this limitation, ACDC patient fibroblasts and iPSC-derived mesenchymal stromal cells (MSCs) have been used to study signaling mechanisms altered in the absence of functional CD73 [56,57]. These studies reveal that ACDC patient fibroblasts have dysregulated transcription factor Forkhead box O1 protein (FOXO1) activity [57].…”

“…To overcome this limitation, ACDC patient fibroblasts and iPSC-derived mesenchymal stromal cells (MSCs) have been used to study signaling mechanisms altered in the absence of functional CD73 [56,57]. These studies reveal that ACDC patient fibroblasts have dysregulated transcription factor Forkhead box O1 protein (FOXO1) activity [57]. Furthermore, ACDC patient MSCs display increased activation of AKT kinase, mechanistic target of rapamycin (mTOR), and the 70 kDa ribosomal protein S6 kinase (p70S6K) in the presence of osteogenic stimuli [56].…”

AMPK: Potential Therapeutic Target for Vascular Calcification

“…Humans lacking ectonucleotide pyrophosphorylase1 (extracellular PPi synthesizer) develop severe VC at an early age ( 97 ). What is more, recent studies demonstrate that adenosine might serve as an endogenous inhibitor of VC through regulating the expression of tissue non-specific alkaline phosphatase (TNAP) ( 98 , 99 ).…”

Biomolecules Orchestrating Cardiovascular Calcification

“…Thus, to investigate the mechanism, Moorhead and colleagues utilized CD73-deficient iPSC-derived human mesenchymal cells. They found that CD73 inhibits calcification by activating the A2a/A2b adenosine receptor and downstream cAMP, which inhibits the Akt/FOXO1 induction of TNAP [120,121].…”