[Regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by protein kinase C].
OBJECTIVE
To explore the molecular mechanism of inducible nitric oxide synthase (iNOS) gene expression induced by lipopolysaccharide (LPS) in monocytes or macrophages.


METHODS
Luciferase report gene vector pGL2iNOSLuc driven by murine iNOS promoter region was constructed using gene recombination technique. RAW264.7 cells were cultured and transfected with luciferase report gene vector pGL2iNOSLuc. Twenty four hours later, PKC inhibitor H-7, calcium channel blocker verapamil, was added into the culture. LPS or PKC activator PMA was added 2 hours later to stimulate the cells. The activity of beta-galactosidase and luciferase was examined and the relative luciferase activity, representing the iNOS transactivity, was calculated. Modified Griess method was used to measure the concentration of NO(2)(-)/NO(3)(-) in the culture so as to evaluate the effect of PKC inhibitor on NO production induced by LPS. RT-PCR was used to study the effect of LPS on iNOS gene expression in RAW264.7 cells.


RESULTS
Thirty minutes after stimulation of RAW264.7 cells by LPS, the activity of PKC in cytomembrane increased and the activity of PKC in cytoplasm decreased significantly in comparison with the baseline levels (P < 0.01). The NO concentration was 30.1 micro mol/L +/- 5.4 micro mol/L 12 hours after the stimulation of LPS on RAW264.7 cells, and was much more higher 24 and 48 hours after, all significantly higher than before (all P < 0.01). The NO concentration in the supernatant with H-7 at any time point was significantly lower than those in corresponding samples without H-7 (all P < 0.01). After stimulation of LPS and PMA, the relative luciferase activity was 25.3 +/- 4.6, significantly higher than the baseline level (P < 0.01). H-7 and verapamil significantly inhibited the LPS-induced iNOS transactivity (both P < 0.01). iNOS mRNA expression was increased greatly, and H-7 significantly inhibited this expression 12, 24, and 48 hours after LPS stimulation.


CONCLUSIONS
In RAW264.7 cells, LPS stimulation increases intracellular calcium and activates PKC, thus inducing iNOS gene expression that contributes to the production of NO, which is an important signaling mechanism of NO production in septic shock.
Du
Shaohui
S
Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518033, China.
Zhang
Yue
Y
Wei
Zhijun
Z
Huang
Jie
J
Liu
Xin
X
Chen
Weiming
W
Liu
Zhifeng
Z
Qin
Qinghe
Q
Jiang
Yong
Y
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D008156:Luciferases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C