Protein Kinase C-αRegulates Toll-like Receptor 4-Mediated Inducible Nitric Oxide Synthase Expression
Toll-like Receptor 4에 의한 Inducible Nitric Oxide Synthase의 발현에서 Protein Kinase C-a의 조절 기전
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Purpose: The nitric oxide (NO) release by inducible nitric oxide synthase (iNOS) is the key events in macrophage response to lipopolysaccharide (LPS) which is suggested to be a crucial mediator for inflammatory and innate immune responses. NO is an important mediator involved in many host defense action and may also lead to a harmful host response to bacterial infection. However, given the importance of iNOS in a variety of pathophysiological conditions, control of its expression and signaling events in response to LPS has been the subject of considerable investigation. Materials and Methods: The Raw264.7 macrophage cell line was used to observe LPS-stimulated iNOS expression. The expression of iNOS is observed by Western blot analysis and real-time RT-PCR. Protein kinase C (PKC)-α overexpressing Raw264.7 cells are established to determine the involvement of PKC-α in LPS-mediated iNOS expression. NF- κB activity is measured by IκBα degradation and NF-κB luciferase activity assay. Results: We found that various PKC isozymes regulate LPS-induced iNOS expression at the transcriptional and translational levels. The involvement of PKC-α in LPS-mediated iNOS induction was further confirmed by increased iNOS expression in PKC-α overexpressing cells. NF-κB dependent transactivation by LPS was observed and PKC-α specific inhibitory peptide abolished this activation, indicating that NF-κB activation is dependent on PKC-α. Conclusion: Our data suggests that PKC-α is involved in LPS-mediated iNOS expression and that its downstream target is NF-κB. Although PKC-α is a crucial mediator in the iNOS regulation, other PKC isozymes may contribute LPS-stimulated iNOS expression. This finding is needed to be elucidated in further study.
Abstract
Purpose: The nitric oxide (NO) release by inducible nitric oxide synthase (iNOS) is the key events in macrophage response to lipopolysaccharide (LPS) which is suggested to be a crucial mediator for inflammatory and innate immune responses. NO is an important mediator involved in many host defense action and may also lead to a harmful host response to bacterial infection. However, given the importance of iNOS in a variety of pathophysiological conditions, control of its expression and signaling events in response to LPS has been the subject of considerable investigation. Materials and Methods: The Raw264.7 macrophage cell line was used to observe LPS-stimulated iNOS expression. The expression of iNOS is observed by Western blot analysis and real-time RT-PCR. Protein kinase C (PKC)-α overexpressing Raw264.7 cells are established to determine the involvement of PKC-α in LPS-mediated iNOS expression. NF- κB activity is measured by IκBα degradation and NF-κB luciferase activity assay. Results: We found that various PKC isozymes regulate LPS-induced iNOS expression at the transcriptional and translational levels. The involvement of PKC-α in LPS-mediated iNOS induction was further confirmed by increased iNOS expression in PKC-α overexpressing cells. NF-κB dependent transactivation by LPS was observed and PKC-α specific inhibitory peptide abolished this activation, indicating that NF-κB activation is dependent on PKC-α. Conclusion: Our data suggests that PKC-α is involved in LPS-mediated iNOS expression and that its downstream target is NF-κB. Although PKC-α is a crucial mediator in the iNOS regulation, other PKC isozymes may contribute LPS-stimulated iNOS expression. This finding is needed to be elucidated in further study.
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