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Requirement of nuclear factor kappaB for the constitutive expression of nitric oxide synthase-2 and cyclooxygenase-2
N A Callejas1, M Casado, L Boscá
1Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense de Madrid, Spain.
Abstract:
Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), decreasing the levels of the corresponding mRNAs when the cells were maintained in culture. The sustained expression of COX-2 and NOS-2 in trophoblasts was dependent on the activation of nuclear factor kappaB (NF-kappaB) since proteasome inhibitors and antioxidants that abrogated NF-kappaB activity suppressed the induction of both genes. The time-dependent fall of the mRNA levels of NOS-2 and COX-2 paralleled the inhibition of NF-kappaB, determined by electrophoretic mobility shift assays, and the increase of the IkappaBalpha and IkappaBbeta inhibitory proteins. Isolated trophoblasts synthesized reactive oxygen intermediates (ROI), a process impaired after culturing the cells, and that might be involved in the NF-kappaB activation process. Moreover, treatment of recently isolated cells with ROI scavengers suppressed the expression of COX-2 and NOS-2. Challenge of trophoblasts with interleukin-1beta up-regulated the expression of both proteins, an effect that was potentiated by lipopolysaccharide. These results indicate that the physiological expression of NOS-2 and COX-2 in trophoblasts involves a sustained activation of NF-kappaB which inhibition abrogates the inducibility of both genes.
Insights
Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), regulated by nuclear factor kappaB (NF-kappaB) activation. This study reveals NF-kappaB
Area of Science:
- Immunology
- Cell Biology
- Reproductive Biology
Background:
- Trophoblasts are crucial for placental development and function.
- Nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2) are key enzymes in trophoblast physiology.
- Understanding the regulation of NOS-2 and COX-2 in trophoblasts is vital for reproductive health.
Purpose of the Study:
- To investigate the regulatory mechanisms of NOS-2 and COX-2 expression in freshly isolated trophoblasts.
- To determine the role of nuclear factor kappaB (NF-kappaB) and reactive oxygen intermediates (ROI) in trophoblast gene expression.
- To explore the effects of inflammatory stimuli on NOS-2 and COX-2 expression.
Main Methods:
- Primary trophoblast cell culture and isolation.
- Quantitative mRNA analysis and protein expression studies.
- Electrophoretic mobility shift assays (EMSA) for NF-kappaB activity assessment.
- Treatment with proteasome inhibitors, antioxidants, ROI scavengers, interleukin-1beta, and lipopolysaccharide.
Main Results:
- Freshly isolated trophoblasts express NOS-2 and COX-2, with decreasing levels upon culture.
- Sustained NOS-2 and COX-2 expression is dependent on nuclear factor kappaB (NF-kappaB) activation.
- Reactive oxygen intermediates (ROI) are involved in NF-kappaB activation and subsequent NOS-2/COX-2 expression.
- Interleukin-1beta and lipopolysaccharide up-regulate NOS-2 and COX-2 expression.
Conclusions:
- Physiological expression of NOS-2 and COX-2 in trophoblasts requires sustained NF-kappaB activation.
- Inhibition of NF-kappaB abrogates the inducibility of NOS-2 and COX-2 in trophoblasts.
- ROI play a significant role in mediating NF-kappaB-dependent gene expression in trophoblasts.