Membrane actions of vitamin D metabolites 1α,25(OH)₂D₃ and 24R,25(OH)₂D₃ are retained in growth plate cartilage cells from vitamin D receptor knockout mice

Abstract

1alpha,25(OH)(2)D(3) 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 1alpha,25(OH)(2)D(3) 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. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-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. 1alpha,25(OH)(2)D(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, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) 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)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-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.

Terminal Differentiation of Chick Embryo Chondrocytes Requires Shedding of a Cell Surface Protein That Binds 1,25-Dihydroxyvitamin D3

“…We have shown here that, similarly to rat growth plate chondrocytes at proliferative stages (36), late differentiation of cranial cells still can be restrained by 1,25-dihydroxyvitamin D3. It has been shown previously (37) that rat costal chondrocytes are capable of producing this vitamin D metabolite, which eliminates the necessity of availability of the systemic vitamin D hormone.…”

Inorganic phosphate modulates responsiveness to 24,25(OH)2D3 in chondrogenic ATDC5 cells

“…This was unanticipated since studies using rat [Schwartz et al, 1988[Schwartz et al, , 1995 and mouse [Boyan et al, 2003] costochondral growth plate chondrocytes have shown that resting zone cells are the primary target for 24R,25(OH) 2 D 3 , whereas prehypertrophic and hypertrophic chondrocytes are primary targets for 1a,25(OH) 2 D 3 . Moreover, 20 mM Pi induced sensitivity of the ATDC5 cells to 10 À7 M 24R,25(OH) 2 D 3 , which is the concentration at which costochondral resting zone cells exhibit maximal responses to the seco-steroid [Boyan et al, 2003] and similar to the level of endogenous 24R,25(OH) 2 D 3 produced by these cells when stimulated in culture . h on day 10 and day 11 and RT-PCR was performed to assess mRNA expression of collagen types I, II, and X and also SOX9, COMP, and aggrecan (AGG).…”

“…Previous work examining mouse and rat growth plates has shown that two metabolites of vitamin D,24,25(OH) [Boyan et al, , 2003. Chondrocytes from the resting zone exhibit specific sensitivity to 24,25(OH) 2 D 3 , whereas cells in the growth zone no longer exhibit the same responses to 24,25(OH) 2 D 3 but have acquired specific sensitivity to 1,25(OH) 2 D 3 .…”

Rapid steroid hormone actions via membrane receptors

“…As global knockout of Pdia3 is embryologically lethal, the ability to determine its full impact is hampered. However, chondrocytes isolated from costochondral cartilage of VDR knockout mice and Ca 2+ influx studies in the guts of these mice demonstrate that 1α,25(OH) 2 D 3 still elicits rapid activation of PKC and a biological response [38,39]. Studies using Pdia3 conditional knockout mice [40], Pdia3-silenced osteoblasts [41], and Pdia3 ± heterozygote mice [42] have demonstrated a loss of rapid activation of PKC and release of PGE 2 , decreased osteopontin production and alkaline phosphatase activity, and bone and cartilage abnormalities.…”