THE STIMULATION OF RAT ASTROCYTES WITH PHORBOL-12-MYRISTATE-13-ACETATE INCREASES THE PROENKEPHALIN MESSENGER-RNA - INVOLVEMENT OF PROTOONCOGENES

The effect of phorbol-12-myristate-13-acetate (PMA) on the regulation of proenkephalin (proENK) mRNA level, ENKCRE-2 or AP-1 DNA binding act ivity, and the mRNA and protein levels of proto-oncogenes (c-fos, fra- 1, and c-jun) in primary cultured rat astrocytes were studied. The pro ENK mRNA level was elevated at 4 h after the treatment of PMA (2.5 mu M) without altering the intracellular proENK protein level, and this i ncrease was attenuated by pre-treatment with cycloheximide (CHX; 15 mu M), a protein synthesis inhibitor. Both AP-1 and ENKCRE-2 DNA binding activities were markedly increased at 1-4 h by PMA treatment and thes e PMA-induced responses were inhibited by pre-treatment with CHX, show ing that the increase of proENK mRNA level was well correlated with th e 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-trea tment 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 l evel and, especially, PMA-induced increase of fra-1 mRNA level was fur ther enhanced by CHX treatment at 4 h. Furthermore, western immunoblot assay showed that PMA caused induction of c-Fos, Fra-1, and c-Jun pro tein levels. PMA-induced increases of proto-oncoproteins levels were a lso inhibited by CHX treatment. The results suggest that newly synthes ized 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 cult ured rat astrocytes. Phospho-CREB protein appears not to be involved i n the regulation of PMA-induced proENK gene expression. (C) 1998 Elsev ier Science B.V.