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. 2013 Jan;28(1):81-91.
doi: 10.1002/jbmr.1733.

Pharmacological inhibition of PHOSPHO1 suppresses vascular smooth muscle cell calcification

Tina Kiffer-Moreira  1 , Manisha C Yadav, Dongxing Zhu, Sonoko Narisawa, Campbell Sheen, Boguslaw Stec, Nicholas D Cosford, Russell Dahl, Colin Farquharson, Marc F Hoylaerts, Vicky E Macrae, José Luis Millán
Affiliations

Pharmacological inhibition of PHOSPHO1 suppresses vascular smooth muscle cell calcification

Tina Kiffer-Moreira et al. J Bone Miner Res. 2013 Jan.

Abstract

Medial vascular calcification (MVC) is common in patients with chronic kidney disease, obesity, and aging. MVC is an actively regulated process that resembles skeletal mineralization, resulting from chondro-osteogenic transformation of vascular smooth muscle cells (VSMCs). Here, we used mineralizing murine VSMCs to study the expression of PHOSPHO1, a phosphatase that participates in the first step of matrix vesicles-mediated initiation of mineralization during endochondral ossification. Wild-type (WT) VSMCs cultured under calcifying conditions exhibited increased Phospho1 gene expression and Phospho1(-/-) VSMCs failed to mineralize in vitro. Using natural PHOSPHO1 substrates, potent and specific inhibitors of PHOSPHO1 were identified via high-throughput screening and mechanistic analysis and two of these inhibitors, designated MLS-0390838 and MLS-0263839, were selected for further analysis. Their effectiveness in preventing VSMC calcification by targeting PHOSPHO1 function was assessed, alone and in combination with a potent tissue-nonspecific alkaline phosphatase (TNAP) inhibitor MLS-0038949. PHOSPHO1 inhibition by MLS-0263839 in mineralizing WT cells (cultured with added inorganic phosphate) reduced calcification in culture to 41.8% ± 2.0% of control. Combined inhibition of PHOSPHO1 by MLS-0263839 and TNAP by MLS-0038949 significantly reduced calcification to 20.9% ± 0.74% of control. Furthermore, the dual inhibition strategy affected the expression of several mineralization-related enzymes while increasing expression of the smooth muscle cell marker Acta2. We conclude that PHOSPHO1 plays a critical role in VSMC mineralization and that "phosphatase inhibition" may be a useful therapeutic strategy to reduce MVC.

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Conflict of interest statement

Disclosure

All the authors state that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1. Expression of Phospho1 in VSMCs and its role in VSMC mineralization
Normalized Phospho1 mRNA expression as a function of the calcification time in cultured WT murine (A) calvarial osteoblasts and (B) aortic VSMCs. (C) Calcium deposition in WT and Phospho1-/- murine aortic VSMCs after 28 days of culture. (D) Alizarin Red staining of WT and Phospho1-/- murine aortic VSMCs after 28 days of culture. (E) Normalized Phospho1 and Alpl expression in Enpp1-/- aortic VSMCs versus WT VSMCs, after 21 days in culture under mineralizing conditions. Results are presented as mean ± S.E.M. (*p < 0.05, **p < 0.01, ***p < 0.001).
Fig. 2
Fig. 2. Mode of action of PHOSPHO1 inhibition by lansoprazole and MLS-0263839
Double reciprocal plots of PHOSPHO1 activity versus the concentration of the PHOSPHO1 substrate P-Etn (32 – 170 μmol/L), in the presence of increasing concentrations of lansoprazole (A) and MLS-0263839 (B). Results are presented as mean ± S.E.M.
Fig. 3
Fig. 3. Selectivity of PHOSPHO1 and TNAP inhibitors
Point experiments with the PHOSPHO1 inhibitors MLS-0390838 and MLS-0263939 and TNAP inhibitor MLS-0038949 at 0 (100% control, open bar), 1 (light gray bars), 10 (dark gray bars) and 30 (black bars) μmol/L, illustrating specificity during hydrolysis by TNAP of the substrates pNPP (A, 10 mmol/L Pnpp in 50 mmol/L Tris-HCl buffer, pH 7.5, containing 1 mmol/L MgCl2, 20 μmol/L zinc acetate) and PPi (B, 60 μmol/L PPi in 50 mmol/L Tris-HCl buffer, pH 7.5, containing 1 mmol/L MgCl2, 20 μmol/L ZnAc), and by PHOSPHO1 for the substrates P-Etn (C, 62.5 μmol/L P-Etn in 20 mmol/L MES-NaOH, pH 6.7, 0.01% (w/v) BSA, 0.0125% (v/v) Tween 20, 2 mmol/L MgCl2) and P-Cho (D, 62.5 μmol/L P-Cho in MES-NaOH, pH 6.7, 0.01% (w/v) BSA, 0.0125% (v/v) Tween 20, 2 mmol/L MgCl2).
Fig. 4
Fig. 4. Mode of action of TNAP inhibition by MLS-0038949
Double reciprocal plots of TNAP activity versus the concentration of the TNAP substrate pNPP (0.055 – 1 mmol/L) at pH 7.4, in the presence of increasing concentrations of MLS-0038949 and re-plot of the y-intercepts versus the concentration of MLS-0038949 (A). Residual TNAP activity during dose-dependent inhibition by MLS-0038949 (B) and levamisole (C) of hTNAP and its three active site mutants (Tyr371Ala, Glu108Phe and His434Glu).
Fig. 5
Fig. 5. Modeling of docked PHOSPHO1 and TNAP inhibitors
Computer docking of PHOSPHO1 inhibitors MLS-0263839 (in blue) and MLS-0390838 (in purple) in comparison to lansoprazole (in yellow) into the active site of the modeled structure of PHOSPHO1 (A) and of the potent uncompetitive TNAP inhibitor MLS-0038949 (in blue) in the active site of the modeled structure of TNAP (B). Insets: close-up views of binding inhibitors.
Fig. 6
Fig. 6. Inhibition of matrix mineralization
Alizarin Red-staining (A570 nm) of 21 day cultures of WT VSMC in the presence of inorganic phosphate (A) and in the presence of β-glycerophosphate for WT (B), Enpp1-/- (C) and Phospho1-/- (D) VSMCs. The cells were cultured with 30 μmol/L of the TNAP inhibitor MLS-0038949 and the PHOSPHO1 inhibitors lansoprazole and MLS-0263839, either alone or combined, as indicated. Calcium deposition in 21-day VSMC cultures was evaluated by staining cell layers with Alizarin Red.
Fig. 7
Fig. 7. Gene expression changes in WT VSMCs treated with inhibitors
WT VSMCs were cultured for 21 days in the presence of TNAP inhibitor MLS-0038949 or PHOSPHO1 inhibitor MLS-0263839, either alone or combination, as indicated. The mRNA expression was determined by qPCR for Alpl (A), Ennp1 (B), Phospho1 (C), Runx2 (D), Spp1 (E), Bglap (F), Col1a1 (G), and Acta2 (H).
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