title: Membrane actions of vitamin D metabolites 1alpha,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.
authors.affiliation: Georgia Institute of Technology, Atlanta, Georgia, USA. Barbara.Boyan@bme.gatech.edu
authors.forename: Barbara D
authors.initials: BD
authors.lastname: Boyan
authors.forename: V L
authors.initials: VL
authors.lastname: Sylvia
authors.forename: N
authors.initials: N
authors.lastname: McKinney
authors.forename: Z
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism*; D000818:Animals; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D004195:Disease Models, Animal; D006132:Growth Plate / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D012279:Rickets / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013431:Sulfates / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: C116441:2-methyl-1,25-dihydroxyvitamin D3; D011509:Proteoglycans; D018167:Receptors, Calcitriol; D013431:Sulfates; D014807:Vitamin D; D015650:24,25-Dihydroxyvitamin D 3; D011493:Protein Kinase C; D010741:Phospholipases A; D010738:Type C Phospholipases; D010739:Phospholipase D