Induction of alkaline phosphatase in primary cultures of epiphyseal growth plate chondrocytes by a serum-derived factor.

Abstract

Alkaline phosphatase (AP) activity in epiphyseal growth plate cartilage increases markedly during differentiation of the chondrocytes, and reaches high levels in the zone of hypertrophy where vascular penetration and provisional mineralization begin. A proteinaceous factor has been discovered in serum that stimulates the expression of AP in chicken growth plate chondrocytes when these cells are grown in serum-free media. Sera from a variety of vertebrate species (goat, fetal bovine, horse, human, and chicken) all contained detectable levels of the inducing activity. The chondrocyte AP-induction factor (CAP-IF) from fetal bovine serum was precipitated with ammonium sulfate between 33% and 50% saturation, and purified by dye-ligand affinity chromatography. The active fraction, which eluted from an Affi-Gel Blue column between 0.10 and 0.15 M NaCl, was further resolved on a QMA anion exchange column. The most active and almost homogeneous fraction contained primarily a 64.5 kDa protein; about 3 micrograms/ml medium induced 50% of the maximal level of AP induction. CAP-IF is stable to heat (100 degrees C for 3 min) and dithiothreitol (50 mM) treatment, and is only mildly inactivated by 2 h treatment with trypsin. CAP-IF caused no significant effect on cell division as measured by 3H-thymidine uptake. Time-course studies revealed that at least 18-24 h exposure of the chondrocytes to CAP-IF is required to produce major increases in AP activity. Longer exposure time generally further increases the response. Cycloheximide almost completely blocked the increase in AP activity, indicating that de novo protein synthesis is required for induction.