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The Protein Journal

, Volume 34, Issue 1, pp 29–34 | Cite as

Molecular Characterisation of Group IVA (Cytosolic) Phospholipase A2 in Murine Osteoblastic MC3T3-E1 Cells

  • Hans Jörg Leis
  • Werner Windischhofer
Article

Abstract

Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell activity, is preceded by release of the precursor arachidonic acid from phospholipid stores. The main routes of arachidonate liberation may involve phospholipase enzymes such as group IVA phospholipase A2 which is believed to be the main effector in many cell system due to its preference for arachidonate-containing lipids. MC3T3-E1 cells are non-transformed osteoblasts and are widely used as an in vitro model of osteoblast function. In these cells there is still no clarity about the main release pathway of arachidonic acid. Besides cytosolic phospholipase A2, phospholipase C and D pathways may play a key role in arachidonate release. Despite the crucial role of osteoblastic prostgalandin synthesis information on the occurrence of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. We have characterised group IVA phospholipase A2 at the mRNA in these cells as a constitutively expressed enzyme which is cytosolic and translocates to the membrane upon endothelin-1 stimulation. Using immunopurification combined with Western blotting and high-resolution mass spectrometry, the enzyme was also identified at the protein level. Using specific gene silencing we were able to show that osteoblastic cytosolic phospholipase A2 is crucially involved in ET-1-induced prostaglandin formation.

Keywords

cPLA2 Bone Osteoblast MC3T3-E1 

Abbreviations

PGE2

Prostaglandin E2

ET-1

Endothelin-1

IP

Immunoprecipitation

MS

Mass spectrometry

PL

Phospholipase

cPLA2

Group IVA calcium-dependent cytosolic phospholipase A2 (pla2g4a)

AA

Arachidonic acid

PA

Phosphatidic acid

COX

Cyclooxygenase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

SDS

Sodium dodecylsulphate

PAGE

Polyacrylamide gel electrophoresis

RT-PCR

Reverse transcriptase-polymerase chain reaction

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Research Unit of Analytical Mass Spectrometry, Cell Biology and Biochemistry of Inborn Errors of Metabolism, University Hospital of Youth and Adolescence MedicineMedical University of GrazGrazAustria

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