Prostaglandin E 2 increases proenkephalin mRNA level in rat astrocyte-enriched culture

Abstract

The effect of prostaglandin E 2 (PGE 2) on proenkephalin (proENK) mRNA expression in primary cultured rat astrocytes was studied. The proENK mRNA level was significantly increased about 3.3-fold 4 h after PGE 2 (10 μM) treatment and this increase was potentiated by the pre-treatment with cycloheximide (CHX; 15 μM) about 1.7-fold as much as PGE 2 alone treated cells. The pretreatment with staurosporine (1 μM) completely inhibited the increase of PGE 2-induced proENK mRNA level, although only a partial inhibition of PGE 2-induced proENK mRNA level (≈1.5-fold) by H89 (10 μM) was observed. The increase of PGE 2-induced proENK mRNA level was not affected by the pretreatment with PD98059 (1, 5, and 10 μM), ω-conotoxin GIVA (1 μM), nimodipine (1 μM), calmidazolium (1 μM), or KN-62 (1 μM). In addition to the proENK mRNA level, PGE 2 also increased c-Fos (≈4.3-fold), Fra-1 (≈3.8 fold), and Fra-2 (≈8.2-fold) protein levels at 4 h after drug treatment. However, c-Jun, JunB, and JunD protein levels were not affected by PGE 2. Indeed, PGE 2 failed to up-regulate c- jun mRNA expression as well as its protein product. Surprisingly, although three Jun proteins were not induced by PGE 2, AP-1 and ENKCRE-2 DNA binding activities were increased by PGE 2, (≈5 and ≈2.8-fold, respectively) and which were effectively reduced by CHX (≈2.5 and 2-fold, respectively). In western blot analyses, PGE 2 enhanced the phosphorylation of CREB (≈2.6-fold at 1 h), and CHX showed a potentiative effect on PGE 2-induced CREB phosphorylation (≈1.7 fold at 1 h) which is similar to the action on proENK mRNA regulation. Our results suggest that PGE 2 increases proENK mRNA expression via activating serine/threonine protein kinase such as PKA, but not calcium/calmodulin dependent protein kinase and MAPK. In addition, phosphorylation of CREB rather than the increase of AP-1 may have a possible role at least early stage in PGE 2-induced proENK mRNA level and CHX-evoked potentiation.