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.

Keywords: alkaline phosphatase; arteries; calcification; lower extremity.

Figure 1.

AMP induces aberrant signaling in arterial calcification due to deficiency of CD73 (ACDC) cells. A, Schematic of CD73 and downstream adenosine receptor signaling. B, Baseline measurement of adenosine receptor gene expression. Results representative of n=6 to 8 per group using 4 control and 3 ACDC patient cell lines. Statistical analysis performed using unpaired t test with Welch correction. C, Quantification of cAMP levels in control and ACDC cells in response to exogenous AMP. Cells were pretreated with 0.5 mmol/L IBMX for 30 min then treated with 30 µM AMP for 15 min. Results representative of n=7 per group using 3 control and 4 ACDC patient cell lines. **P=0.0013 using unpaired t test with Welch correction. D, Control and ACDC fibroblasts were given exogenous 100 µM AMP for 10 min and total cell lysates were analyzed by Western blotting for VASP and AKT phosphorylation. Results representative of n=3 per group using 3 control patient and 3 ACDC patient cell lines. For P-VASP, ***P=0.0003, P-AKT T308 *P=0.0155, P-AKT S473 ***P=0.0005. Two-way ANOVA with Tukey multiple comparisons test was used for statistical analysis. E, ACDC fibroblasts were treated with 100 µM exogenous AMP or pretreated with 10 µM forskolin and 10 µM IBMX for 30 min before treatment with 100 µM exogenous AMP, 10 µM forskolin, and 10 µM IBMX (AFI) for 10 min. Total cell lysates were analyzed by Western blotting for VASP and AKT phosphorylation. Results representative of n=3 to 4 per group using 2 ACDC patient cell lines. For P-VASP *P=0.0183, P-AKT T308 **P=0.0025, P-AKT S473 ***P=0.0002. One-way ANOVA with Tukey multiple comparisons test was used for statistical analysis.

Figure 2.

Inducing autophagy reduces calcification in CD73-deficient cells. A, ALPL gene expression. Results representative of n=4 to 7 per group using 3 control and 3 arterial calcification due to deficiency of CD73 (ACDC) patient cell lines. ***P=0.003 ****P<0.0001. B, Staining for TNAP activity in control and ACDC fibroblasts cultured in osteogenic media and supplemented with 200 nM rapamycin for 5 d. Results representative of n=4 to 7 per group using 3 control and 3 ACDC patient cell lines. C, Alizarin Red S stain was used to image and quantify calcification content in control and ACDC fibroblasts cultured under osteogenic conditions for 21 d and supplemented with 200 nM rapamycin. Results representative of n=6 per group using 2 control and 2 ACDC patient cell lines. *P=0.037. D, Alizarin Red S staining of control and ACDC cells treated with autophagy-inducing peptide Tat-Beclin 1 D11. Results representative of n=5 per group using 4 control and 3 ACDC patient cell lines. **P=0.0043 ****P<0.0001. Scale bar represents 200 µm. Two-way ANOVA with Tukey multiple comparisons test was used for all statistical analyses in this figure.

Figure 3.

AMP induces FOXO1 (forkhead box O1 protein) nuclear localization. A, The putative FOXO1-binding site in the human ALPL promoter shown with the percentage that the sequence located 445 upstream of the transcriptional start site (TSS) on the human ALPL promoter is found in experimentally proven FOXO1-binding sites according to the TRANSFAC database. B, Immunofluorescent staining was used to detect FOXO1 localization in response to exogenous AMP. Control and arterial calcification due to deficiency of CD73 (ACDC) fibroblasts were pretreated with vehicle, 0.5 mmol/L 8-Br-cAMP, or 10 µM each of CGS-21680 and BAY 60-6583 (A2a and A2b agonists, respectively) for 30 min, then 100 µM AMP was added to each group for 2 h. Cells were stained with anti-FOXO1 antibody and an IgG control, and pixel intensity was quantified and normalized to DAPI. **P=0.0054, ##P=0.0027, @@P=0.0027 using 2-way ANOVA with Tukey multiple comparisons test for statistical analysis. Results representative of 5 replicates of 3 control patient cell lines and 3 ACDC patient cell lines per group. Scale bar represents 50 µm.

Figure 4.

FOXO1 (forkhead box O1 protein) inhibitor prevents calcification via reducing ALPL promoter activation. A, RT-qPCR analysis of ALPL mRNA isolated from cells in osteogenic conditions receiving 1 µM AS-184856 for 5 d. Results representative of n=5 per group using 3 control and 3 arterial calcification due to deficiency of CD73 (ACDC) patient cell lines. **P=0.0077, ##P=0.0039. Two-way ANOVA with Tukey multiple comparison test was used for statistical analysis. B, TNAP activity staining in cells cultured for 5 d in osteogenic media supplemented daily with the FOXO1 inhibitor AS-1842856 (1 µM) or vehicle control. Results representative of n=5 per group using 4 control and 3 ACDC patient cell lines. Scale bar represents 200 µm. C, Alizarin Red S stain in cells cultured under osteogenic conditions for 21 d and supplemented 1 µM AS-1842856. Results representative of n=6 per group using 4 control and 3 ACDC patient cell lines. ****P>0.0001 by 2-way ANOVA with Tukey multiple comparison test. Scale bar represents 200 µm. D, Luciferase assay of plasmid DNA containing the Gaussia luciferase under control of the human ALPL promoter (hALPL). Cells were transfected with 1 µg of plasmid and cultured for 5 d under osteogenic stimulation. Results representative of n=3 per group using 3 control and 3 ACDC patient cell lines. ***P=0.0011 by 2-way ANOVA with Sidak multiple comparisons test. E, Luciferase assay of hALPL plasmid in cells treated with 100 µmol/L AMP with and without pretreatment of 200 nM rapamycin. Results representative of n=3 using 1 ACDC patient cell line. *P=0.0406. F, Luciferase assay of hALPL and Luciferase assay of hALPL with site-directed mutagenesis (SDM) of the putative FOXO1-binding site. Results representative of n=3 using 1 ACDC patient cell line. ***P=0.0008. Unpaired t test with Welch correction was used for E and F.

Figure 5.

CD73 levels reduced in nonarterial calcification due to deficiency of CD73 (ACDC) calcified femoropopliteal artery exhibiting higher levels of FOXO1 (forkhead box O1 protein). Sections of femoropopliteal arteries from 20 different patients were stained with Von Kossa stain to detect calcification. Of the 20 patients, no calcification was detected in 7 vessels and the remaining 13 show calcification. Serial sections were stained with CD73, FOXO1, and TNAP antibody, and an IgG control to match concentration of TNAP at 20 µg/mL. Pixel quantification was done using ImageJ and normalized to DAPI. **P=0.0023, @@P=0.0067, $P=0.0151, $$P=0.0043, #P=0.0133. M=tunica media. I=tunica intima. L=lumen. Von Kossa scale bar represents 100 µm, fluorescent images scale bar represents 50 µm.