Zinc protoporphyrin inhibition of lipopolysaccharide-, lipoteichoic acid-, and peptidoglycan-induced nitric oxide production through stimulating iNOS protein ubiquitination.
In the present study, zinc protoporphyrin (ZnPP), but not ferric protoporphyrin (FePP), tin protoporphyrin (SnPP), or zinc chloride (ZnCl(2)), at the doses of 0.5, 1, and 2 microM, dose-dependently inhibited lipopolysaccharide- (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages in a serum-free condition. NO inhibition and HO-1 induction by ZnPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). A decrease in the iNOS/NO ratio and an increase in HO-1 protein by ZnPP were identified in three different conditions including ZnPP pretreatment, ZnPP co-treatment, and ZnPP post-treatment with LPS and LTA. Activation of c-Jun N-terminal kinases (JNKs) and extracellular regulated kinases (ERKs) were detected in LPS-, LTA-, and PGN-treated RAW264.7 cells, and iNOS/NO production was blocked by adding the JNK inhibitor, SP600125, but not the ERK inhibitor, PD98059. However, ZnPP addition potentiated ERK and JNK protein phosphorylation stimulated by LPS, LTA, and PGN. Increases in total protein ubiquitination and ubiquitinated iNOS proteins were detected in ZnPP-treated macrophages elicited by LPS according to Western and immunoprecipitation/Western blotting assays, respectively. The decrease in LPS-induced iNOS protein by ZnPP was reversed by adding the proteasome inhibitors MG132 and lactacystin. The reduction in HO-1 protein induced by ZnPP via transfection of HO-1 small interfering RNA did not affect the inhibitory effect of ZnPP against LPS-induced iNOS/NO production and protein ubiquitination induced by ZnPP in macrophages. Data of the present study provide the first evidence to support ZnPP effectively inhibiting inflammatory iNOS/NO production through activation of protein ubiquitination in a HO-1-independent manner in macrophages.
Section of Hematology-Oncology, Department of Internal Medicine, Taipei Municipal Wan Fang Hospital, Taipei Medical University, Taipei 110, Taiwan.
Jyh-Ming
JM
Chow
Hui-Yi
HY
Lin
Shing-Chuan
SC
Shen
Ming-Shun
MS
Wu
Cheng-Wei
CW
Lin
Wen-Ta
WT
Chiu
Chien-Huang
CH
Lin
Yen-Chou
YC
Chen
D000818:Animals; D002460:Cell Line; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000502:physiology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010457:Peptidoglycan / Q000494:pharmacology; D011524:Protoporphyrins / Q000494:pharmacology*; D013682:Teichoic Acids / Q000037:antagonists & inhibitors*; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology
D008070:Lipopolysaccharides; D010457:Peptidoglycan; D011524:Protoporphyrins; D013682:Teichoic Acids; C017803:zinc protoporphyrin; D009569:Nitric Oxide; C009900:lipoteichoic acid; D052247:Nitric Oxide Synthase Type II