The stimulation of rat astrocytes with phorbol-12-myristate-13-acetate increases the proenkephalin mRNA: involvement of proto-oncogenes

Abstract

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 μM) without altering the intracellular proENK protein level, and this increase was attenuated by pre-treatment with cycloheximide (CHX; 15 μM), 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.