Membrane Actions of Vitamin D Metabolites 1α,25(OH)₂D ₃ and 24R,25(OH)₂D₃ Are Retained in Growth Plate Cartilage Cells from Vitamin D Receptor Knockout Mice

1α,25(OH)₂D₃ regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1α,25(OH)₂D₃ in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1α,25(OH)₂D₃ had no effect on [ ³H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [³H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [³⁵S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1α,25(OH)₂D ₃ caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1α,25(OH)₂D₃ had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1β,25(OH)₂D₃ nor 24R,25(OH)₂D₃ affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1α,25(OH)₂D₃. As noted previously for rat growth plate chondrocytes, 1α,25(OH)₂D₃ mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A₂ (PLA₂). These responses to 1α,25(OH)₂D₃ were blocked by antibodies to 1,25-MARRS, which is a [³H]-1,25(OH)₂D₃ binding protein identified in chick enterocytes. 24R,25(OH)₂D ₃ regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)₂D₃ with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)₂D ₃ dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)₂D ₃. These results provide definitive evidence that there are two distinct receptors to 1α,25(OH)₂D₃. 1α,25(OH)₂D₃-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.