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Prostaglandin E2 increases proenkephalin mRNA level in rat astrocyte-enriched culture
1Department of Pharmacology and Institute of Natural Medicine, College of Medicine, Hallym University, 1 Okchun-Dong, Chunchon, Kangwon-Do 200-702, South Korea.
Abstract:
The effect of prostaglandin E2 (PGE2) 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 PGE2 (10 microM) treatment and this increase was potentiated by the pre-treatment with cycloheximide (CHX; 15 microM) about 1.7-fold as much as PGE2 alone treated cells. The pretreatment with staurosporine (1 microM) completely inhibited the increase of PGE2-induced proENK mRNA level, although only a partial inhibition of PGE2-induced proENK mRNA level (approximately 1.5-fold) by H89 (10 microM) was observed. The increase of PGE2-induced proENK mRNA level was not affected by the pretreatment with PD98059 (1, 5, and 10 microM), omega-conotoxin GIVA (1 microM), nimodipine (1 microM), calmidazolium (1 microM), or KN-62 (1 microM). In addition to the proENK mRNA level, PGE2 also increased c-Fos (approximately 4.3-fold), Fra-1 ( approximately 3.8 fold), and Fra-2 (approximately 8.2-fold) protein levels at 4 h after drug treatment. However, c-Jun, JunB, and JunD protein levels were not affected by PGE2. Indeed, PGE2 failed to up-regulate c-jun mRNA expression as well as its protein product. Surprisingly, although three Jun proteins were not induced by PGE2, AP-1 and ENKCRE-2 DNA binding activities were increased by PGE2, (approximately 5 and approximately 2.8-fold, respectively) and which were effectively reduced by CHX (approximately 2.5 and 2-fold, respectively). In western blot analyses, PGE2 enhanced the phosphorylation of CREB (approximately 2.6-fold at 1 h), and CHX showed a potentiative effect on PGE2-induced CREB phosphorylation ( approximately 1.7 fold at 1 h) which is similar to the action on proENK mRNA regulation. Our results suggest that PGE2 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 PGE2-induced proENK mRNA level and CHX-evoked potentiation.
Insights
Prostaglandin E2 (PGE2) significantly boosts proenkephalin (proENK) mRNA in rat astrocytes. This effect is enhanced by cycloheximide (CHX) and involves CREB phosphorylation, suggesting PKA activation.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Proenkephalin (proENK) is a precursor to endogenous opioids.
- Prostaglandin E2 (PGE2) is a lipid mediator with diverse physiological roles.
- Astrocytes play crucial roles in central nervous system function and modulation.
Purpose of the Study:
- To investigate the effect of PGE2 on proENK mRNA expression in primary cultured rat astrocytes.
- To elucidate the signaling pathways involved in PGE2-mediated proENK regulation.
Main Methods:
- Primary rat astrocyte cultures were treated with PGE2 and various inhibitors.
- ProENK mRNA levels were quantified using RT-PCR.
- Protein levels of c-Fos, Fra-1, Fra-2, c-Jun, JunB, and JunD were assessed.
- DNA binding activities of AP-1 and ENKCRE-2 were measured.
- Western blot analysis was used to detect CREB phosphorylation.
Main Results:
- PGE2 significantly increased proENK mRNA levels (3.3-fold at 4h).
- Cycloheximide (CHX) potentiated PGE2-induced proENK mRNA expression and CREB phosphorylation.
- Staurosporine completely inhibited, while H89 partially inhibited, the PGE2 effect.
- PGE2 increased c-Fos, Fra-1, and Fra-2 protein levels but not c-Jun, JunB, or JunD.
- PGE2 enhanced AP-1 and ENKCRE-2 DNA binding activities and CREB phosphorylation.
Conclusions:
- PGE2 upregulates proENK mRNA expression in astrocytes, likely via protein kinase A (PKA) activation.
- CREB phosphorylation plays a role in the early stages of PGE2-induced proENK mRNA expression and CHX potentiation.
- MAPK and calcium/calmodulin-dependent protein kinase pathways are not primarily involved.