Research ArticleGENETIC DISORDERS

Ectopic calcification in pseudoxanthoma elasticum responds to inhibition of tissue-nonspecific alkaline phosphatase

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Science Translational Medicine  07 Jun 2017:
Vol. 9, Issue 393, eaal1669
DOI: 10.1126/scitranslmed.aal1669
  • Fig. 1. Crossing Abcc6 to Enpp1 or Nt5e mutant mice reveals genetic interaction.

    Abcc6 mutant mice were crossed to Enpp1 or Nt5e mutant mice to generate all possible genetic allele combinations. (A and C) Micro-CT scans of the muzzle to evaluate the extent of vibrissae fibrous capsule calcification were obtained at 15 weeks of age. Representative coronal z-stacked images of the mouse muzzle with the nasal bones and sinuses midline (indicated by white asterisk) and the pathological calcification seen as radiodense lesions (indicated by the yellow arrow) in the surrounding soft tissue. (B and D) Quantification of ectopic calcification from micro-CT images. A two-way ANOVA with Tukey’s honest significance difference post hoc analysis was performed. Two-way ANOVA: (B) Abcc6 effect, P = 2.2 × 10−16; Enpp1 effect, P = 2.2 × 10−16; interaction effect, P = 2.2 × 10−16; (D) Abcc6 effect, P = 2.2 × 10−16; Nt5e effect, P = 1.2 × 10−4; interaction effect, P = 1.1 × 10−3. P values of post hoc comparisons are indicated in the figure.

  • Fig. 2. Evidence for a provoked cell-autonomous defect and alterations in enzymes integral to the extracellular catabolism of ATP in ABCC6 mutant cells.

    (A) Primary dermal fibroblasts derived from patients with biallelic mutations in ABCC6 (ABCC6Mut/Mut) calcify in vitro when stimulated with osteogenic media, as indicated by alizarin red staining. Representative images demonstrating the spectrum of calcification are shown. (B) Quantification of the alizarin red staining was determined by colorimetric analysis. One-tailed Student’s t test was performed (P = 0.045). (C to F) Quantification of enzyme activity and gene expression for ENPP1 (ENPP1) and CD73 (NT5E) in primary dermal fibroblasts derived from patients with biallelic mutations in ABCC6, ENPP1, or NT5E. A one-way ANOVA with Tukey’s honest significance difference post hoc analysis was performed. One-way ANOVA: (C) P = 0.001; (D) P = 0.016; (E) P = 3.51 × 10−5; (F) P = 0.038. P values of post hoc comparisons are indicated in the figure.

  • Fig. 3. Liver-specific deletion of Abcc6 does not phenocopy constitutive ablation of Abcc6 in mice.

    Micro-CT scans of the muzzle to evaluate the extent of vibrissae fibrous capsule calcification were obtained at 20 weeks (A) and 1 year (D) of age. (B and E) Quantification of ectopic calcification from micro-CT images. A one-way ANOVA with Tukey’s honest significance difference post hoc analysis was performed. One-way ANOVA: (B) P = 2.2 × 10−16; (E) P = 4.68 × 10−11. P values of post hoc comparisons are indicated in the figure. (C) Visualization and confirmation of Cre-targeted tissues and cell types using the RosamTmG reporter mouse line. All cells that are successfully recombined transition from expression of membrane Tomato (mT; red fluorescence) to green fluorescent protein (mG; green fluorescence). Representative images are shown.

  • Fig. 4. Evidence that both local and systemic defects in ATP metabolism are needed to promote PXE-associated ectopic calcification in mice.

    (A) Micro-CT scans of the muzzle demonstrating ectopic calcification at 20 weeks of age upon deletion of Abcc6 in both the liver and local Wnt1+ cells in the fibrous capsule, albeit with reduced penetrance compared to constitutive targeting (Fig. 3, A and B). (B) Quantification of ectopic calcification from micro-CT images. (C) Visualization and confirmation of Cre-targeted tissues and cell types using the RosamTmG reporter mouse line. Representative images are shown.

  • Fig. 5. Circulating PPi concentration does not correlate with severity of calcification phenotype in mice.

    (A) Micro-CT scans of the muzzle to evaluate the extent of vibrissae fibrous capsule calcification were obtained at 1 year of age. (B) Quantification of ectopic calcification from micro-CT images. (C) Quantification of plasma PPi concentration. (B and C) A one-way ANOVA with Tukey’s honest significance difference post hoc analysis was performed. One-way ANOVA: (B) P = 1.09 × 10−5; (C) P = 0.0087. P values of post hoc comparisons are indicated in the figure.

  • Fig. 6. TNAP inhibition attenuates calcification in a PXE mouse model.

    (A and B) Micro-CT scans revealed significant attenuation of the calcification phenotype in both SBI-425 and etidronate-treated Abcc6−/− mice. Control mice did not calcify. A two-way ANOVA with Tukey’s honest significance difference post hoc analysis was performed. Two-way ANOVA: genotype effect, P = 1.2 × 10−12; treatment effect, P = 3.7 × 10−5; interaction effect, P = 2.1 × 10−5. P values of post hoc comparisons are indicated in the figure. (C) Micro-CT results were validated by dissolving the muzzle tissue and quantifying the calcium phosphate precipitate. OD575, optical density at 575 nm. (D) Quantification of TNAP activity from plasma from mice treated with SBI-425 or etidronate. (C and D) A one-way ANOVA with Tukey’s honest significance difference post hoc analysis was performed. One-way ANOVA: (C) P = 8.4 × 10−4; (D) P = 1.5 × 10−13. P values of post hoc comparisons are indicated in the figure.

  • Fig. 7. Proposed involvement of ABCC6 in extracellular ATP metabolism and the suppression of ectopic calcification.

    ENPP1 metabolizes ATP into AMP and PPi, whereas CD73 further degrades AMP into adenosine and inorganic phosphate (Pi). Adenosine can bind to its cell surface receptor to repress ALPL, the gene encoding TNAP. TNAP degrades PPi into Pi and is a primary distal regulator of PPi, a major negative inhibitor of calcification. Our data suggest that ABCC6 is integral to the extracellular ATP metabolism pathway and likely works downstream of ENPP1 and in tandem with CD73 to maintain low TNAP amount and prevent pathological calcification.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/393/eaal1669/DC1

    Materials and Methods

    Fig. S1. Location of calcification in the fibrous capsule surrounding the vibrissae.

    Fig. S2. Demonstration of genetic interaction between Abcc6 and Nt5e mice when aged to 1 year.

    Fig. S3. Demonstration of efficient liver-specific deletion of Abcc6 in mice.

    Fig. S4. Primary dermal fibroblasts derived from patients with biallelic mutations in ABCC6 show TNAP-dependent in vitro calcification.

    Fig. S5. TNAP inhibition does not alter circulating PPi concentration in mice.

    Fig. S6. TNAP inhibition had no negative effects on bone microarchitecture or mineralization in a PXE mouse model.

    Fig. S7. TNAP inhibition prevents progression of established calcification in a PXE mouse model.

    Table S1. List of patient mutations in ABCC6, ENPP1, and NT5E.

    Table S2. Number of calcified mice at 20 weeks and 1 year of age.

    Table S3. Cortical bone microarchitecture.

    Table S4. Cortical bone strength.

    Table S5. Individual-level data.

    References (4651)

  • Supplementary Material for:

    Ectopic calcification in pseudoxanthoma elasticum responds to inhibition of tissue-nonspecific alkaline phosphatase

    Shira G. Ziegler,* Carlos R. Ferreira, Elena Gallo MacFarlane, Ryan C. Riddle, Ryan E. Tomlinson, Emily Y. Chew, Ludovic Martin, Chen-Ting Ma, Eduard Sergienko, Anthony B. Pinkerton, José Luis Millán, William A. Gahl, Harry C. Dietz*

    *Corresponding author. Email: sgziegler{at}jhmi.edu (S.G.Z.); hdietz{at}jhmi.edu (H.C.D.)

    Published 7 June 2017, Sci. Transl. Med. 9, eaal1669 (2017)
    DOI: 10.1126/scitranslmed.aal1669

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Location of calcification in the fibrous capsule surrounding the vibrissae.
    • Fig. S2. Demonstration of genetic interaction between Abcc6 and Nt5e mice when aged to 1 year.
    • Fig. S3. Demonstration of efficient liver-specific deletion of Abcc6 in mice.
    • Fig. S4. Primary dermal fibroblasts derived from patients with biallelic mutations in ABCC6 show TNAP-dependent in vitro calcification.
    • Fig. S5. TNAP inhibition does not alter circulating PPi concentration in mice.
    • Fig. S6. TNAP inhibition had no negative effects on bone microarchitecture or mineralization in a PXE mouse model.
    • Fig. S7. TNAP inhibition prevents progression of established calcification in a PXE mouse model.
    • Table S1. List of patient mutations in ABCC6, ENPP1, and NT5E.
    • Table S2. Number of calcified mice at 20 weeks and 1 year of age.
    • Table S3. Cortical bone microarchitecture.
    • Table S4. Cortical bone strength.
    • References (4651)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Individual-level data.

    [Download Table S5]

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