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 β-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.