1α,25-(OH)2D3 regulates 25-hydroxyvitamin D3 24R-hydroxylase activity in growth zone costochondral growth plate chondrocytes via protein kinase C

Zvi Schwartz, H. A. Pedrozo, V. L. Sylvia, R. Gomez, David D Dean, B. D. Boyan

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1α- 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 1α,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1α,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 1α,25-(OH)2D3 or 24R,25-(OH)2D3 for 24 hours, and 1α- 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 10-10-10-8 M 1α,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1α,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 1α,25-(OH)2D3 to the response to 1 β,25-(OH)2D3. 1α,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 1α,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1 1α,25-(OH)2D3 caused an increase; 1β,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours, 1α-hydroxylase activity was unaffected by either metabolite at 24 hours, 1α,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1α,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1α,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.

Original languageEnglish (US)
Pages (from-to)365-372
Number of pages8
JournalCalcified Tissue International
Volume69
Issue number6
DOIs
StatePublished - 2001

Fingerprint

Calcifediol
Growth Plate
Chondrocytes
Mixed Function Oxygenases
Protein Kinase C
Growth
Messenger RNA
Membranes
Enterocytes
Cycloheximide
Proteins

Keywords

  • 1α-hydroxylase
  • 24R-hydroxylase
  • 25-hydroxyvitamin D
  • Chondrocytes
  • Cyp24
  • Cyp27B1

ASJC Scopus subject areas

  • Endocrinology

Cite this

1α,25-(OH)2D3 regulates 25-hydroxyvitamin D3 24R-hydroxylase activity in growth zone costochondral growth plate chondrocytes via protein kinase C. / Schwartz, Zvi; Pedrozo, H. A.; Sylvia, V. L.; Gomez, R.; Dean, David D; Boyan, B. D.

In: Calcified Tissue International, Vol. 69, No. 6, 2001, p. 365-372.

Research output: Contribution to journalArticle

Schwartz, Zvi ; Pedrozo, H. A. ; Sylvia, V. L. ; Gomez, R. ; Dean, David D ; Boyan, B. D. / 1α,25-(OH)2D3 regulates 25-hydroxyvitamin D3 24R-hydroxylase activity in growth zone costochondral growth plate chondrocytes via protein kinase C. In: Calcified Tissue International. 2001 ; Vol. 69, No. 6. pp. 365-372.
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abstract = "Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1α- 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 1α,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1α,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 1α,25-(OH)2D3 or 24R,25-(OH)2D3 for 24 hours, and 1α- 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 10-10-10-8 M 1α,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1α,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 1α,25-(OH)2D3 to the response to 1 β,25-(OH)2D3. 1α,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 1α,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1 1α,25-(OH)2D3 caused an increase; 1β,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours, 1α-hydroxylase activity was unaffected by either metabolite at 24 hours, 1α,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1α,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1α,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.",
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T1 - 1α,25-(OH)2D3 regulates 25-hydroxyvitamin D3 24R-hydroxylase activity in growth zone costochondral growth plate chondrocytes via protein kinase C

AU - Schwartz, Zvi

AU - Pedrozo, H. A.

AU - Sylvia, V. L.

AU - Gomez, R.

AU - Dean, David D

AU - Boyan, B. D.

PY - 2001

Y1 - 2001

N2 - Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1α- 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 1α,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1α,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 1α,25-(OH)2D3 or 24R,25-(OH)2D3 for 24 hours, and 1α- 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 10-10-10-8 M 1α,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1α,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 1α,25-(OH)2D3 to the response to 1 β,25-(OH)2D3. 1α,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 1α,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1 1α,25-(OH)2D3 caused an increase; 1β,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours, 1α-hydroxylase activity was unaffected by either metabolite at 24 hours, 1α,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1α,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1α,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.

AB - Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1α- 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 1α,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1α,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 1α,25-(OH)2D3 or 24R,25-(OH)2D3 for 24 hours, and 1α- 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 10-10-10-8 M 1α,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1α,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 1α,25-(OH)2D3 to the response to 1 β,25-(OH)2D3. 1α,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 1α,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1 1α,25-(OH)2D3 caused an increase; 1β,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours, 1α-hydroxylase activity was unaffected by either metabolite at 24 hours, 1α,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1α,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1α,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.

KW - 1α-hydroxylase

KW - 24R-hydroxylase

KW - 25-hydroxyvitamin D

KW - Chondrocytes

KW - Cyp24

KW - Cyp27B1

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