Stability of phospholipase D in primary astrocytes.
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. Kötter, J. Klein, J. Neurochem. 73 (1999) 2517]. Competitive RT-PCR analysis demonstrated a higher level of PLD1 mRNA than PLD2 mRNA (8.9 vs. 0.9amol/microg RNA, respectively). Treatment of astroglial cultures with the phorbol ester, 4beta-phorbol-12beta,13alpha-dibutyrate (0.1 microM), for 24-48h selectively induced PLD1b but not PLD1a or 2 expression as shown by PCR and Western blot; the effect was sensitive to Gö 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 4-6 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.
Jin
Shenchu
S
Department of Pharmacology, Johannes Gutenberg University of Mainz, Obere Zahlbacher Str. 67, D-55101 Mainz, Germany.
Schatter
Beate
B
Weichel
Oksana
O
Walev
Ivan
I
Ryu
SungHo
S
Klein
Jochen
J
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D001921:Brain / Q000201:enzymology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D007527:Isoenzymes; D020319:Oligodeoxyribonucleotides, Antisense; D011500:Protein Synthesis Inhibitors; D015240:Phorbol 12,13-Dibutyrate; D003513:Cycloheximide; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1