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Effects of pH on the Production of Phosphate and Pyrophosphate by Matrix Vesicles’ Biomimetics

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Abstract

During endochondral bone formation, chondrocytes and osteoblasts synthesize and mineralize the extracellular matrix through a process that initiates within matrix vesicles (MVs) and ends with bone mineral propagation onto the collagenous scaffold. pH gradients have been identified in the growth plate of long bones, but how pH changes affect the initiation of skeletal mineralization is not known. Tissue-nonspecific alkaline phosphatase (TNAP) degrades extracellular inorganic pyrophosphate (PPi), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), while contributing Pi from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP, and PPi was studied at pH 8 and 9 and compared to the data determined at pH 7.4. While catalytic efficiencies in general were higher at alkaline pH, PPi hydrolysis was maximal at pH 8 and indicated a preferential utilization of PPi over ATP at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph containing 1 mM ATP as substrate and amorphous calcium phosphate or calcium–phosphate–phosphatidylserine complexes as nucleators. Propagation of mineralization was significantly more efficient at pH 7.5 and 8 than at pH 9. Since a slight pH elevation from 7.4 to 8 promotes considerably more hydrolysis of ATP, ADP, and AMP primarily by TNAP, this small pH change facilitates mineralization, especially via upregulated PPi hydrolysis by both NPP1 and TNAP, further elevating the Pi/PPi ratio, thus enhancing bone mineralization.

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Acknowledgments

We thank Professor Mathieu Bollen (Leuven, Belgium) for providing the NPP2/NPP1 expression construct. This work was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Grants DE12889, AR53102, and AR47908 from the National Institutes of Health. A. M. S. S. was the recipient of studentships from CAPES and FAPESP.

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Authors and Affiliations

  1. Department of Chemistry, FFCLRP-USP, Av. Bandeirantes, 3900, Ribeirão Preto, SP, 14040-901, Brazil

    Ana Maria S. Simão, Maytê Bolean & Pietro Ciancaglini

  2. Sanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA

    Ana Maria S. Simão, José Luis Millán & Pietro Ciancaglini

  3. Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium

    Marc F. Hoylaerts

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  1. Ana Maria S. Simão
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  2. Maytê Bolean
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  3. Marc F. Hoylaerts
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  4. José Luis Millán
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  5. Pietro Ciancaglini
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Correspondence to Pietro Ciancaglini.

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Simão, A.M.S., Bolean, M., Hoylaerts, M.F. et al. Effects of pH on the Production of Phosphate and Pyrophosphate by Matrix Vesicles’ Biomimetics. Calcif Tissue Int 93, 222–232 (2013). https://doi.org/10.1007/s00223-013-9745-3

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