P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells |
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Authors: | Raihana Zaka David Stokes Arnold S Dion Anna Kusnierz Fei Han and Charlene J Williams |
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Institution: | (1) Division of Rheumatology, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA;(2) College of Graduate Studies, Thomas Jefferson University, Philadelphia, PA 19107, USA |
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Abstract: | Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive
ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition
of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank,
result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result
in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a
mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing
Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes,
and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR
and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase
and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization
phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently
displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced
lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity
in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells
overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank.
Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an
empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function
with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression
of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic
calcium phosphate crystals. |
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