Regulation of phospholipase D (PLD) in growth plate chondrocytes by 24R,25-(OH)2D3 is dependent on cell maturation state (resting zone cells) and is specific to the PLD2 isoform

V. L. Sylvia, Z. Schwartz, F. Del Toro, P. DeVeau, R. Whetstone, R. R. Hardin, D. D. Dean, B. D. Boyan

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Many of the effects of 1α,25-(OH)2D3 and 24R,25-(OH)2D3 on costochondral chondrocytes are mediated by the protein kinase C (PKC) signal transduction pathway. 1α,25-(OH)2D3 activates PKC in costochondral growth zone chondrocytes through a specific membrane receptor (1α,25-mVDR), involving rapid increases in diacylglycerol via a phospholipase C (PLC)-dependent mechanism. 24R,25-(OH)2D3 activates PKC in resting zone chondrocytes. Although diacylglycerol is increased by 24R,25-(OH)2D3, PLC is not involved, suggesting a phospholipase D (PLD)-dependent mechanism. Here, we show that resting zone and growth zone cells express mRNAs for PLD1a, PLD1b, and PLD2. Both cell types have PLD activity, but levels are higher in resting zone cells. 24R,25-(OH)2D3, but not 24S,25-(OH)2D3 or 1α,25-(OH)2D3, stimulates PLD activity in resting zone cells within 3 min via nongenomic mechanisms. Neither 1α,25-(OH)2D3 nor 24R,25-(OH)2D3 affected PLD in growth zone cells. Basal and 24R,25-(OH)2D3-stimulated PLD were inhibited by the PLD inhibitors wortmannin and EDS. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase), PKC, phosphatidylinositol-specific PLC (PI-PLC), and phosphatidylcholine-specific PLC (PC-PLC) had no effect on PLD activity. Thus, 24R,25-(OH)2D3 stimulates PLD, and PI 3-kinase, PI-PLC and PKC are not involved, whereas PLD is required for stimulation of PKC by 24R,25-(OH)2D3. Pertussis toxin, GDPβS, and GTPγS had no effect on 24R,25-(OH)2D3-dependent PLD when added to cell cultures, indicating that G-proteins are not involved. These data show that PKC activation in resting zone cells is mediated by PLD and suggest that a functional 24R,25-(OH)2D3-mVDR is required. The results also support the conclusion that the 24R,25-(OH)2D3-responsive PLD is PLD2, since this PLD isoform is G-protein-independent.

Original languageEnglish (US)
Article number14704
Pages (from-to)209-221
Number of pages13
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1499
Issue number3
DOIs
StatePublished - 2001

Keywords

  • 24R,25-Dihydroxycholecalciferol
  • Alkaline phosphatase
  • Cell maturation
  • Chondrocyte culture
  • Phospholipase D
  • Protein kinase C
  • Signal transduction

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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