Abstract
Protein kinase C (PKC)-α, -βI, and -δ are known to be involved in the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. The role of mitogen-activated protein kinases (MAPK) p44/42 and p38 in the LPS effect was studied further. LPS-mediated NO release and the inducible form of NO synthase expression were inhibited by the p38 inhibitor, SB 203580, but not by the MAPK kinase inhibitor, PD 98059. Ten-minute treatment of cells with LPS resulted in the activation of p44/42 MAPK, p38, and c-Jun NH2-terminal kinase. Marked or slight activation, respectively, of p44/42 MAPK or p38 was also seen after 10-min treatment with 12-O-tetradecanoylphorbol-13-acetate, but c-Jun NH2-terminal kinase activation did not occur. Tyrosine kinase inhibitor, genestein, attenuated the LPS-induced activation of both p44/42 MAPK and p38, whereas the PKC inhibitors, Ro 31-8220 and calphostin C, or long-term treatment with 12-O-tetradecanoylphorbol-13-acetate resulted in inhibition of p44/42 MAPK activation, but had only a slight effect on p38 activation, indicating that LPS-mediated PKC activation resulted in the activation of p44/42 MAPK. Nuclear factor-κB (NF-κB)-specific DNA-protein-binding activity in the nuclear extracts was enhanced by 10-min, 1-h, or 24-h treatment with LPS. Analysis of the proteins involved in NF-κB binding showed translocation of p65 from the cytosol to the nucleus after 10-min treatment with LPS. The onset of NF-κB activation correlated with the cytosolic degradation of both inhibitory proteins of NF-κB, IκB-α and IκB-β. IκB-α was resynthesized rapidly after loss (1-h LPS treatment), whereas IκB-β levels were not restored until after 24-h treatment. SB 203580 but not PD 98059 inhibited the LPS-induced stimulation of NF-κB DNA-protein binding. Thus, activation of p38 but not p44/42 MAPK by LPS resulted in the stimulation of NF-κB-specific DNA-protein binding and the subsequent expression of inducible form of NO synthase and NO release in RAW 264.7 macrophages.
Footnotes
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Send reprint requests to: Ching-Chow Chen, Institute of Pharmacology, College of Medicine, National Taiwan University, No.1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan. E-mail:ccchen{at}ha.mc.ntu.edu.tw
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This work was supported by a research grant from the National Science Council of Taiwan.
- Abbreviations:
- NO
- nitric oxide
- iNOS
- inducible NO synthase
- NF-κB
- nuclear factor-κB
- LPS
- lipopolysaccharide
- MAPK
- mitogen-activated protein kinase
- JNK
- c-Jun NH2-terminal kinase
- PDTC
- pyrrolidine dithiocarbamate
- EMSA
- electrophoretic mobility-shift assay
- MBP
- myelin basic protein
- PKC
- protein kinase C
- PI-PLC
- phosphatidylinositol-phospholipase C
- PC-PLC
- phosphatidylcholine-PLC
- MEK
- MAPK kinase
- Received July 28, 1998.
- Accepted November 25, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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