1alpha,25-(OH)2D3 regulates 25-hydroxyvitamin D3 24R-hydroxylase activity in growth zone costochondral growth plate chondrocytes via protein kinase C.
Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1alpha- and 24R-hydroxylase activities and metabolize 25-(OH)D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 in a cell maturation-specific and time-dependent manner. This study examined the hypothesis that 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1alpha,25-(OH)2D3 exerts its effects via mechanisms involving protein kinase C (PKC) mediated pathways. Rat costochondral growth zone chondrocytes were treated with 10(-9) - 10(-7) M 1alpha,25-(OH)2D3 or 24R,25-(OH)2D, for 24 hours, and 1alpha- and 24R-hydroxylase activities in cell homogenates determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3. Metabolite production by intact cells was determined at 6 and 24 hours. Involvement of PKC was assessed using chelerythrine, and the requirement for protein synthesis was assessed using cycloheximide. In addition, the effect of 10(-10) - l0(-8) M 1alpha,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1alpha,25-(OH)2D3 (1,25-mVDR), which exerts its effects via PKC, was assessed by blocking the 1,25-mVDR with an antibody (Ab99) generated against the 1,25-mVDR in chick enterocyte membranes. Specificity of the 1,25-(OH)2D3-dependent effect on 24,25-(OH)2D3 production was determined by comparing the response to 1alpha,25-(OH)2D3 to the response to 1beta,25-(OH)2D3. 1alpha,25-(OH)2D3 increased 24R-hydroxylase specific activity in a dose-dependent manner, 24,25-(OH)2D3 production by intact cells was also increased. The effect of 1alpha,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1alpha,25-(OH)2D3 caused an increase; 1beta,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours. 1alpha-hydroxylase activity was unaffected by either metabolite at 24 hours. 1alpha,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1alpha,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1alpha,25(OH)2D3-dependent stimulation of 24R-hydroxylase. These results suggest an interrelationship between the 1,25-mVDR and gene expression via the nuclear VDR (nVDR) and/or a PKC-mediated mechanism in modulating 24R-hydroxylase in growth zone chondrocytes.
Schwartz
Z
Z
Department of Orthopaedics, The University of Texas Health Science Center, San Antonio 78229-3900. USA
Pedrozo
H A
HA
Sylvia
V L
VL
Gomez
R
R
Dean
D D
DD
Boyan
B D
BD
D015650:24,25-Dihydroxyvitamin D 3 / Q000494:pharmacology; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002356:Cartilage / Q000166:cytology / Q000201:enzymology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System; D004305:Dose-Response Relationship, Drug; D006132:Growth Plate / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D012333:RNA, Messenger; D015650:24,25-Dihydroxyvitamin D 3; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D011493:Protein Kinase C; D002117:Calcitriol