Involvement of phosphatidylcholine hydrolysis by phospholipase C in prostaglandin F2alpha-induced 1,2-diacylglycerol formation in osteoblast-like MC3T3-E1 cells

J Bone Miner Metab. 2004;22(3):198-206. doi: 10.1007/s00774-003-0470-2.

Abstract

We previously demonstrated that a prostaglandin F2alpha (PGF2alpha)-induced, sustained increase in 1,2-diacylglycerol (DAG) production was important for proliferation in osteoblast-like MC3T3-E1 cells. The 1,2-DAG formation is mediated by various enzymes, such as phos-phoinositide (PI)-specific phospholipase C (PLC), phospholipase D (PLD), and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). In the present study, to elucidate the mechanism of the 1,2-DAG formation, we have examined the PGF2alpha-induced production of [(3)H]phosphorylcholine, a product of PC-PLC activity, in [(3)H]choline-labeled MC3T3-E1 cells. The PGF2alpha-induced [(3)H]phosphorylcholine production was inhibited by genistein, a potent protein tyrosine kinase inhibitor, and increased by vanadate, a potent protein tyrosine phosphatase inhibitor. However, there were no effects after treatment with protein kinase C (PKC) inhibitors, the guanosine triphosphate (GTP) binding protein activator, NaF/AlCl(3), a Ca(2+)-ionophore, or the potent activator of PKC, phorbol 12-myristate 13-acetate (PMA), suggesting that a tyrosine kinase(s) was involved in the PGF2alpha-induced [(3)H]phosphorylcholine formation. Furthermore, a PGF2alpha analogue, 16-(3-trifluoromethylphenoxy)-Omega-tetranor-trans-Delta(2) PGF2alpha methyl ester (ONO-995), stimulated the proliferation of MC3T3-E1 cells to a level similar to that seen with PGF2alpha, and also caused phosphorylcholine and 1,2-DAG generation. However, neither an increase in intracellular free calcium ion ([Ca(2+)]i) levels by PI-PLC, nor phosphatidylethanol formation (and choline production) by PC-PLD were observed. From these results, we conclude that PGF2alpha-induced 1,2-DAG accumulation was mediated mainly via tyrosine kinase(s)-dependent PC hydrolysis by PLC activity in osteoblast-like MC3T3-E1 cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Calcium / metabolism
  • Cell Line
  • Diglycerides / biosynthesis*
  • Dinoprost / analogs & derivatives
  • Dinoprost / pharmacology*
  • GTP-Binding Proteins / metabolism
  • Hydrolysis
  • Mice
  • Osteoblasts / drug effects*
  • Osteoblasts / enzymology
  • Osteoblasts / metabolism
  • Phosphatidylcholines / biosynthesis
  • Phosphatidylcholines / metabolism*
  • Protein Kinases / metabolism
  • Thymidine
  • Type C Phospholipases / metabolism*

Substances

  • 1,2-diacylglycerol
  • Diglycerides
  • Phosphatidylcholines
  • Dinoprost
  • Protein Kinases
  • Type C Phospholipases
  • GTP-Binding Proteins
  • Calcium
  • Thymidine