Protein Kinase C-alpha Regulates Toll-like Receptor 4-Mediated Inducible Nitric Oxide Synthase Expression

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)-alpha overexpressing Raw264.7 cells are established to determine the involvement of PKC-alpha in LPS-mediated iNOS expression. NF-kappaB activity is measured by IkappaBalpha degradation and NF-kappaB 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-alpha in LPS-mediated iNOS induction was further confirmed by increased iNOS expression in PKC-alpha overexpressing cells. NF-kappaB dependent transactivation by LPS was observed and PKC-alpha specific inhibitory peptide abolished this activation, indicating that NF-kappaB activation is dependent on PKC-alpha. CONCLUSION: Our data suggests that PKC-alpha is involved in LPS-mediated iNOS expression and that its downstream target is NF-kappaB. Although PKC-alpha 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.