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Cyclooxygenase 2 (COX-2) gene activation is regulated by cyclic adenosine monophosphate
Abstract:
Prostaglandin E2 production by tissue-fixed macrophages (Mphi) after severe injury contributes to an enhanced susceptibility to infection and sepsis. The purpose of this study was to investigate the effect of cyclic adenosine monophosphate (cAMP) on prostaglandin (PGE2) production and cyclooxygenase II (COX-2) gene activation in LPS-stimulated macrophages (Mphi). RAW264.7 cells, a mouse Mphi cell line, were exposed to various concentrations of dibutyryl cAMP +/- lipopolysaccharide (10 microg/mL) stimulation. Total Mphi ribonucleic acid (RNA) was harvested for the determination of COX-2 messenger RNA (mRNA) with mouse complementary deoxyribonucleic acid (cDNA) by Northern blot assay. Mphi supernatant was collected for the measurement of tumor necrosis factor (TNF) by L929 bioassay and PGE2 by enzyme-linked immunosorbent assay (ELISA), respectively. Mphi NFkappaB activity was determined by electrophoretic mobility shift assays (EMSA). Dibutyryl cAMP significantly inhibited TNF production by LPS-stimulated Mphi. Dibutyryl cAMP (1 mM) alone induced PGE2 production. Dibutyryl cAMP (100 microM and 1 mM) also augmented PGE2 production by LPS-stimulated Mphi. Dibutyryl cAMP had similar effect on Mphi COX-2 mRNA expression and NFkappaB activity. Our data demonstrate that cAMP modulates Mphi TNF production and upregulates COX-2 gene and PGE2 production.
Insights
Cyclic adenosine monophosphate (cAMP) influences macrophage function. This study shows cAMP modulates tumor necrosis factor (TNF) production and upregulates prostaglandin E2 (PGE2) and cyclooxygenase II (COX-2) gene expression in macrophages.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Prostaglandin E2 (PGE2) production by macrophages (Mphi) following severe injury increases susceptibility to infection and sepsis.
- Understanding the regulation of PGE2 production is crucial for developing therapeutic strategies against post-injury complications.
Purpose of the Study:
- To investigate the effect of cyclic adenosine monophosphate (cAMP) on prostaglandin E2 (PGE2) production.
- To determine the impact of cAMP on cyclooxygenase II (COX-2) gene activation in lipopolysaccharide (LPS)-stimulated macrophages (Mphi).
Main Methods:
- RAW264.7 macrophage cell line treated with dibutyryl cAMP and LPS.
- Analysis of COX-2 messenger RNA (mRNA) via Northern blot.
- Measurement of tumor necrosis factor (TNF) and PGE2 levels using bioassay and ELISA.
- Assessment of NF-kappaB activity using electrophoretic mobility shift assays (EMSA).
Main Results:
- Dibutyryl cAMP significantly inhibited LPS-induced TNF production.
- Dibutyryl cAMP alone induced PGE2 production and augmented LPS-stimulated PGE2 production.
- cAMP treatment increased Mphi COX-2 mRNA expression and NF-kappaB activity.
Conclusions:
- Cyclic adenosine monophosphate (cAMP) plays a significant role in modulating macrophage (Mphi) inflammatory responses.
- cAMP upregulates COX-2 gene expression and PGE2 production in macrophages.
- These findings suggest cAMP as a potential target for managing inflammatory conditions associated with infection and sepsis.