1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 differentially affect the specific activity of alkaline phosphatase (ALPase) and phospholipase-A2 (PLA2) of plasma membranes and extracellular matrix vesicles produced by costochondral reserve zone and growth zone cartilage chondrocytes in culture. In the present study, growth zone and cartilage and reserve zone matrix vesicles and plasma membranes were isolated from confluent chondrocyte cultures and incubated with hormone for 3 and 24 h in vitro. Addition of 1,25-(OH)2D3 to GC matrix vesicles and plasma membranes resulted in dose-dependent increases in ALPase and PLA2 specific activities in both membrane fractions. Addition of 24,25-(OH)2D3 to RC membrane fractions stimulated matrix vesicle ALPase at 10(-7) and 10(-8) M and plasma membrane ALPase at 10(-8) M only. However, 24,25-(OH)2D3 inhibited matrix vesicle and plasma membrane PLA2 activity. The effects of the vitamin D metabolites were noticed after both 3 and 24 h. Neither hormone metabolite had any effect on these enzymes in membrane fractions from cultures of neonatal rat muscle mesenchymal cells, which do not calcify their matrix in vivo. These data suggest that 1,25-(OH)2D3 and 24,25-(OH)2D3 can directly affect chondrocyte membrane enzymes without genomic influence or protein synthesis and that membrane response depends on the stage of chondrocyte differentiation. Changes in PLA2 activity may change membrane fluidity and may be a mechanism by which the hormones affect cell membranes.

Previous studies have suggested that vitamin D metabolites directly influence the differentiation and maturation of chondrocytes in calcifying cartilage. Recently, this laboratory has shown that the response of chondrocyte plasma membrane and matrix vesicle enzymes to 1,25-(OH)2D3 and 24,25-(OH)2D3 is both cell and membrane specific. The current study demonstrates that cell replication and matrix protein synthesis are also modulated by vitamin D. Confluent, third-passage growth zone (GC) and resting zone (RC) costochondral chondrocytes were incubated in medium containing 10(-13)-10(-7) M 1,25-(OH)2D3 or 10(-12)-10(-6) M 24,25-(OH)2D3. The amount of collagenase-digestible protein (CDP) secreted into the media was inversely proportional to the concentration of fetal bovine serum (FBS). At 10% FBS, greater than 80% of the CDP was incorporated into the matrix. 1,25-(OH)2D3 stimulated CDP and percentage collagen synthesis by GC cells but had no effect on the synthesis of noncollagenous protein (NCP). 1,25-(OH)2D3 inhibited CDP and percentage collagen synthesis by RC cells but did not alter NCP synthesis. [3H]thymidine incorporation was inhibited in both cell types, whether confluent or subconfluent cultures were examined. At 10(-6) and 10(-7) M 24,25-(OH)2D3, there was a significant decrease in CDP production and percentage collagen synthesis by RC cells but no effect on NCP. However, at 10(-9) and 10(-10) M hormone there was an increase in NCP production but no effect on CDP, resulting in a decrease in percentage collagen synthesis. CDP and NCP production were unaffected by 24,25-(OH)2D3 in GC cells. High concentrations of hormone inhibited [3H]thymidine incorporation in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

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