Related Experiment Videos
Induction of inducible nitric oxide synthase gene expression by Pokeweed mitogen
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Abstract:
The present study has characterized the expression of iNOS gene in Pokeweed mitogen (PWM)-driven murine macrophage RAW 264.7 cells. PWM significantly induced nitric oxide production in a dose-dependent manner. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that the inducible nitric oxide synthase gene expression is increased by PWM treatment. Since iNOS transcription has recently been shown to be under the control of the nuclear factor (NF)-kappaB/Rel family of transcription factors, the effects of PWM on NF-kappaB/Rel activation were examined using a transient transfection assay and an electrophoretic mobility shift assay (EMSA). Transient expression assays with NF-kappaB/Rel binding sites linked to the chloramphenicol acetyltransferase gene suggest that the PWM-induced increase in transcription is mediated by the NF-kappaB/Rel transcription factor complex. Using DNA fragments containing the NF-kappaB/Rel binding sequence, PWM was shown to activate the protein/DNA binding of NF-KB/Rel to its cognate site as measured by EMSA. Supershift EMSA showed the presence of p50 and c-Rel protein in the complex at the kappaB site. Western blot analysis of isolated nuclear fractions, using p65 and c-Rel-specific antibodies, provided further evidence that c-Rel is increased by PWM treatment. N-Tosyl-1-phenylalanine chloromethyl ketone, a potent inhibitor of NF-kappaB/Rel activation, inhibited PWM-induced nitrite generation in a dose-dependent manner. Collectively, the results of these experiments indicate that c-Rel is positively regulated by PWM to assist in the initiation of iNOS gene expression.
Insights
Pokeweed mitogen (PWM) boosts nitric oxide production by activating the nuclear factor kappa B (NF-κB)/Rel pathway in macrophages. This study shows c-Rel activation by PWM is key for initiating inducible nitric oxide synthase (iNOS) gene expression.
Area of Science:
- Immunology
- Molecular Biology
Background:
- Nitric oxide (NO) is a critical signaling molecule in immune responses.
- Inducible nitric oxide synthase (iNOS) produces NO and its gene expression is tightly regulated.
- The nuclear factor kappa B (NF-κB)/Rel family of transcription factors plays a role in iNOS regulation.
Purpose of the Study:
- To investigate the role of Pokeweed mitogen (PWM) in regulating iNOS gene expression in RAW 264.7 macrophages.
- To elucidate the specific NF-κB/Rel family members involved in PWM-mediated iNOS induction.
Main Methods:
- Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to measure iNOS gene expression.
- Transient transfection assays and electrophoretic mobility shift assays (EMSA) to assess NF-κB/Rel activation.
- Western blot analysis to detect specific transcription factor protein levels.
- Inhibition studies using N-Tosyl-1-phenylalanine chloromethyl ketone (TPCK).
Main Results:
- PWM significantly increased nitric oxide production and iNOS gene expression in a dose-dependent manner.
- PWM activated NF-κB/Rel transcription factors, as evidenced by transient transfection and EMSA.
- Supershift EMSA and Western blot confirmed the involvement of p50 and c-Rel proteins in the NF-κB complex.
- TPCK, an NF-κB inhibitor, dose-dependently reduced PWM-induced nitrite generation.
Conclusions:
- Pokeweed mitogen (PWM) activates the NF-κB/Rel pathway, specifically involving c-Rel.
- PWM positively regulates c-Rel to initiate inducible nitric oxide synthase (iNOS) gene expression in macrophages.
- This study clarifies a key molecular mechanism underlying PWM-driven immune responses.