The effects of 17β-estradiol on chondrocyte differentiation are modulated by vitamin D₃ metabolites

Both 17β-estradiol (17β) and the vitamin D metabolites, 1,25- (OH)₂D₃(1,25) and 24,25-(OH)₂D₃ (24,25), regulate endochondral bone formation in vivo and in vitro. The effects of 17β are sex-specific and cell maturation-dependent. Similarly, the effects of 1,25 and 24,25 are cell maturation-dependent, with 1,25 affecting growth zone chondrocytes (GC) and 24,25 affecting resting zone chondrocytes (RC). This study examined whether the response of chondrocytes to 17β is altered after pretreatment with 1,25 or 24,25. Cells were isolated from the costochondral cartilage of male or female rats. Confluent, fourth-passage GC and RC cultures were pretreated with 1,25 or 24,25, respectively, for 24 or 48 h followed by treatment with 17β for an additional 24 h. At harvest, cell proliferation ([³H]-thymidine incorporation), differentiation (alkaline phosphatase specific activity [ALPase]), general metabolism ([³H]-uridine incorporation), and proteoglycan production ([³⁵S]-sulfate incorporation) were determined. 1,25 enhanced the inhibitory effect of 17β on [³H]-thymidine incorporation by female GC cells; in contrast, no effect was observed in GC cells obtained from male rats. When male RC cells were treated with 17β, [³H]-thymidine incorporation was inhibited; however, when these cells were pretreated with 24,25 for 48 h, 17β stimulated [³H]-thymidine incorporation 24,25 had no effect on 17β-dependent [³H]-thymidine incorporation by female RC cells. 17β stimulated ALPase in female GC cells, but had no effect on male GC cells. 1,25 pretreatment of female GC cells inhibited the stimulatory effect of 17β on ALPase, but had no effect on ALPase in male GC cultures. 17β had no effect on male RC cell ALPase and stimulated ALPase in female RC cells. This was not affected by pretreatment with 24,25. Pretreatment with 1,25 increased the basal level of sulfate incorporation only in female GC. No effect was found in RC cells. These results indicate that pretreatment of rat costochondral chondrocytes with vitamin D metabolites modulate the effect of 17β. Although the effect of vitamin D metabolites alone on these chondrocytes is maturation-dependent and not sex-specific, the influence of preincubation with vitamin D metabolites on the effect of 17β is hormone- specific, sex-specific, and maturation-dependent.