The stimulation of rat astrocytes with phorbol-12-myristate-13-acetate increases the proenkephalin mRNA: involvement of proto-oncogenes.
The effect of phorbol-12-myristate-13-acetate (PMA) on the regulation of proenkephalin (proENK) mRNA level, ENKCRE-2 or AP-1 DNA binding activity, and the mRNA and protein levels of proto-oncogenes (c-fos, fra-1, and c-jun) in primary cultured rat astrocytes were studied. The proENK mRNA level was elevated at 4 h after the treatment of PMA (2.5 microM) without altering the intracellular proENK protein level, and this increase was attenuated by pre-treatment with cycloheximide (CHX; 15 microM), a protein synthesis inhibitor. Both AP-1 and ENKCRE-2 DNA binding activities were markedly increased at 1-4 h by PMA treatment and these PMA-induced responses were inhibited by pre-treatment with CHX, showing that the increase of proENK mRNA level was well correlated with the AP-1 and ENKCRE-2 DNA binding activities. In contrast, although the phospho-CREBP level was also increased by PMA at 0.5-1 h, the pre-treatment with CHX further increased the PMA-induced phospho-CREBP level. In addition, PMA caused the induction of c-fos, c-jun and fra-1 mRNA level and, especially, PMA-induced increase of fra-1 mRNA level was further enhanced by CHX treatment at 4 h. Furthermore, western immunoblot assay showed that PMA caused induction of c-Fos, Fra-1, and c-Jun protein levels. PMA-induced increases of proto-oncoproteins levels were also inhibited by CHX treatment. The results suggest that newly synthesized AP-1 proteins, such as c-Fos, Fra-1, and c-Jun may play important roles in the regulation of PMA-induced proENK gene expression in cultured rat astrocytes. Phospho-CREB protein appears not to be involved in the regulation of PMA-induced proENK gene expression.
Won
J S
JS
Department of Pharmacology and Institute of Natural Medicine, College of Medicine, Hallym University, 1 Okchun-Dong, Chunchon, Kangwon-Do, 200-702, South Korea.
Song
D K
DK
Kim
Y H
YH
Huh
S O
SO
Suh
H W
HW
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004745:Enkephalins / Q000235:genetics*; D004789:Enzyme Activation; D009419:Nerve Tissue Proteins / Q000235:genetics; D010766:Phosphorylation; D011493:Protein Kinase C / Q000187:drug effects*; D011498:Protein Precursors / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011519:Proto-Oncogenes / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D017362:Cyclic AMP Response Element-Binding Protein; D004745:Enkephalins; D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C029992:proenkephalin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate