TREATMENT OF RESTING ZONE CHONDROCYTES WITH 24,25-DIHYDROXYVITAMIN D-3[24,25-(OH)(2)D-3] INDUCES DIFFERENTIATION INTO A 1,25-(OH)(2)D-3-RESPONSIVE PHENOTYPE CHARACTERISTIC OF GROWTH ZONE CHONDROCYTES

Vitamin D-3 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 D-3 [1,25-(OH)(2)D-3], whereas those from the resting zone (RC) respond to 24,25-(OH)(2)D-3. The aim of the present study was to determine whether 24,25-(OH)(2)D-3 induces differentiation of RC cells into a 1,25-(OH)(2)D-3-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)(2)D-3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)(2)D-3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [H-3]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [H-3]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [H-3]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as (SO4)-S-35 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)(2)D-3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)(2)D-3 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)(2)D-3, treatment with 1,25-(OH)(2)D-3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)(2)D-3 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)(2)D-3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)(2)D-3 caused these cells to lose their response to 1,25-(OH)(2)D-3. These results indicate that 24,25-(OH)(2)D-3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)(2)D-3 24,25-(OH)(2)D-3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)(2)D-3 responsiveness. These observations support the hypothesis that 24,25-(OH)(2)D-3 plays a significant role in cartilage development.