Alkaline phosphatase is the marker enzyme for matrix vesicles, extracellular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma × fibrosarcoma hybrids in which alkaline phosphatase expression was greatly reduced, a phenomenon known as extinction. In the present study, we used two cell hybrids, LTA‐1 and LTA‐5, constructed from a human osteoblast‐like osteosarcoma, TE85, and a mouse fibrosarcoma, La−t−, to examine the differential distribution of alkaline phosphatase between matrix vesicles and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasma membranes was extinguished, enzyme activity in matrix vesicles from LTA‐1 hybrid cells was 34.2% of that present in matrix vesicles from the TE85 parent cells and 200 times that found in La−t− matrix vesicles. Matrix vesicles from LTA‐5 had alkaline phosphatase levels similar to La−t−. When other membrane enzymes (phospholipase A2, 5′‐nucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle and plasma membrane levels were similar to those of TE85 and significantly higher than in La−t− membrane fractions. Northern analysis detected mRNA for alkaline phosphatase in TE85 cells, but not in the hybrids or La−t− cells. In contrast, reverse transcription‐polymerase chain reaction (RT‐PCR) revealed alkaline phosphatase mRNA in the hybrid cells, but at very low levels. Taken together, the data indicate that regulation of plasma membrane and matrix vesicle alkaline phosphatase is independent and suggest that matrix vesicle biogenesis is independent and distinct from that of plasma membrane biogenesis. Analysis of 1B‐ and 1L‐type alkaline phosphatase mRNA by RT‐PCR showed that alternate promoter usage of the alkaline phosphatase gene was not responsible 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.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Orthopedics and Sports Medicine