Stability of phospholipase D in primary astrocytes

Abstract

Induction of expression and proteolytic breakdown of phospholipase D (PLD) isoforms in primary astrocyte cultures have been investigated. Astrocytes express both PLD1 and 2 and are dependent on PLD activity for cell proliferation [K. Kotter, J. Klein, J. Neurochem. 73 (1999) 2517]. Competitive RT-PCR analysis demonstrated a higher level of PLD1 mRNA than PLD2 mRNA (8.9 vs. 0.9 amol/mug RNA, respectively). Treatment of astroglial cultures with the phorbol ester, 4beta-phorbol-12beta,13alpha-dibutyrate (0.1 muM), for 24-48 h selectively induced PLD1b but not PLD1a or 2 expression as shown by PCR and Western blot; the effect was sensitive to Go 6976. In cells transiently permeabilized with streptolysin-O, antisense oligonucleotides directed against PLD1 or 2 entered the cytoplasm as shown by immunofluorescence experiments but did not affect astroglial proliferation within 2-6 days. Treatment of the cultures with cycloheximide revealed that PLD1 and 2 proteins had biological half-lives of 2 3 days (PLD2) and 46 days (PLD1), respectively. It has been concluded that astroglial PLD1b is up-regulated by phorbol esters via protein kinase C activation. Down-regulation of PLD isoforms is prevented by extended biological half-lives of the PLD proteins. (C) 2002 Elsevier Science (USA). All rights reserved.