OSTEOSARCOMA HYBRIDS CAN PREFERENTIALLY TARGET ALKALINE-PHOSPHATASE ACTIVITY TO MATRIX VESICLES - EVIDENCE FOR INDEPENDENT MEMBRANE BIOGENESIS

Alkaline phosphatase is the marker enzyme for matrix vesicles, extrace llular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma x fibrosarcoma hyb rids in which alkaline phosphatase expression was greatly reduced, a p henomenon known as extinction. In the present study, we used two cell hybrids, LTA-1 and LTA-5, constructed from a human osteoblast-like ost eosarcoma, TE85, and a mouse fibrosarcoma, La(-)t(-), to examine the d ifferential distribution of alkaline phosphatase between matrix vesicl es and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasm a membranes was extinguished, enzyme activity in matrix vesicles from LTA-1 hybrid cells was 34.2% of that present in matrix vesicles from t he TE85 parent cells and 200 times that found in La(-)t(-) matrix vesi cles. Matrix vesicles from LTA-5 had alkaline phosphatase levels simil ar to La(-)t(-). When other membrane enzymes (phospholipase A(2), 5'-n ucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle a nd plasma membrane levels were similar to those of TE85 and significan tly higher than in La(-)t(-) membrane fractions. Northern analysis det ected mRNA for alkaline phosphatase in TE85 cells, but not in the hybr ids or La(-)t(-) cells. In contrast, reverse transcription-polymerase chain reaction (RT-PCR) revealed alkaline phosphatase mRNA in the hybr id cells, but at very low levels. Taken together, the data indicate th at regulation of plasma membrane and matrix vesicle alkaline phosphata se is independent and suggest that matrix vesicle biogenesis is indepe ndent and distinct from that of plasma membrane biogenesis. Analysis o f 1B- and 1L-type alkaline phosphatase mRNA by RT-PCR showed that alte rnate promoter usage of the alkaline phosphatase gene was not responsi ble for the differential localization of this enzyme in matrix vesicle . Thus, it is likely that matrix vesicle and plasma membrane alkaline phosphatase are regulated differently at a post-transcriptional level.