Functional assay of the mutant tissue-nonspecific alkaline phosphatase gene using U2OS osteoblast-like cells.

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization. A defect in the TNAP gene causes hypophosphatasia, which is characteristic of systemic skeletal hypomineralization. To determine the mineralizing ability of the mutant proteins, we developed a functional assay that uses U2OS osteoblast-like cells. Expression plasmids containing TNAP mutant cDNAs were constructed and introduced into U2OS cells, which are derived from a human osteosarcoma and exhibit very low alkaline phosphatase (ALP) activity and disabled mineralization. U2OS cells, in which active TNAP cDNAs were introduced, expressed high ALP activity and mineralized their circumstance when they were cultured with beta-glycerophosphate. The ALP activity in these U2OS cells corresponded to the activity reported for COS cells in which active TNAP cDNA was introduced. An in vitro mineralization assay of U2OS cells transfected with moderate allele cDNAs showed that approximately 35% of TNAP enzymatic activity may be the threshold value for mineralization. In addition, U2OS cells transfected with wild-type TNAP and polymorphism TNAP cDNA showed PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) induction as in SaOS-2 cells. In summary, the introduction of active TNAP cDNA into U2OS cells allowed these cells to mineralize, and this technique may be a useful functional assay of TNAP mutant proteins.