J Korean Assoc Oral Maxillofac Surg.  2008 Feb;34(1):28-35.

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

• Lee JG
• Chin BR
• Baek SH

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.

Keyword

lipopolysaccharide; Inducible nitric oxide synthase; protein kinase C; NF-kappaB

MeSH Terms

Bacterial Infections
Blotting, Western
Cell Line
I-kappa B Proteins
Immunity, Innate
Isoenzymes
Luciferases
Macrophages
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Protein Kinase C
Protein Kinase C-alpha
Protein Kinases
Toll-Like Receptors
Transcriptional Activation
I-kappa B Proteins
Isoenzymes
Luciferases
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Protein Kinase C
Protein Kinase C-alpha
Protein Kinases
Toll-Like Receptors