Vitamin D3 metabolites affect the proliferation and differentiation of cartilage cells. Previous reports have shown that rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1, 25-dihydroxyvitamin D3 [1, 25-(OH)2D3], whereas those from the resting zone (RC) respond to 24, 25-(OH)2D3. The aim of the present study was to determine whether 24, 25-(OH)2D3 induces differentiation of RC cells into a 1, 25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24, 25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1, 25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24, 25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1, 25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24, 25-(OH)2D3, treatment with 1, 25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1, 25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1, 25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24, 25-(OH)2D3 caused these cells to lose their response to 1, 25-(OH)2D3. These results indicate that 24, 25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1, 25-(OH)2D3. 24, 25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1, 25-(OH)2D3 responsiveness. These observations support the hypothesis that 24, 25-(OH)2D3 plays a significant role in cartilage development.
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