Recombinant CC16 protein inhibits the production of pro-inflammatory cytokines via NF-κB and p38 MAPK pathways in LPS-activated RAW264.7 macrophages.
Accumulating evidence indicates that Clara cell protein-16 (CC16) has anti-inflammatory functions, although the involved molecular pathways have not been completely elucidated. Here, we evaluated the effect of recombinant rat CC16 (rCC16) on the expression of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8 in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells) and explored the underlying molecular mechanisms. It was found that rCC16 inhibited LPS-induced TNF-α, IL-6, and IL-8 expression at both the messenger ribonucleicacid (mRNA) level and protein level in a concentration-dependent manner, as demonstrated by real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Such suppressive effects were accompanied by the inhibition of transcriptional activity and the deoxyribonucleic acid binding activity of nuclear factor (NF)-κB but not activator protein (AP)-1. Western blot analysis further revealed that rCC16 inhibited the increase of nuclear NF-κB and the reduction of cytosolic NF-κB, the phosphorylation and reduction of NF-κB inhibitory protein IκBα, and the p38 mitogen-activated protein kinase (MAPK)-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. Furthermore, rCC16 was found to have no effect on the phosphorylation of c-Jun N-terminal kinase, c-Jun, or the nuclear translocation of c-Jun. In addition, reduction of TNF-α, IL-6, and IL-8 were reversed when the level of endogenous uteroglobin-binding protein was reduced by RNA interference in rCC16- and LPS-treated RAW264.7 cells. Our data suggest that rCC16 suppresses LPS-mediated inflammatory mediator TNF-α, IL-6, and IL-8 production by inactivating NF-κB and p38 MAPK but not AP-1 in RAW264.7 cells.
Department of Respiratory, The First Hospital, Shanxi Medical University, Taiyuan 030001, China.
Min
M
Pang
School of Basic Medicine, Shanxi Medical University, Taiyuan 030001, China.
Yangyang
Y
Yuan
Department of Respiratory, The First Hospital, Shanxi Medical University, Taiyuan 030001, China.
Dong
D
Wang
Department of Respiratory, The First Hospital, Shanxi Medical University, Taiyuan 030001, China.
Ting
T
Li
Department of Respiratory, The First Hospital, Shanxi Medical University, Taiyuan 030001, China.
Dan
D
Wang
Department of Epidemiology, School of Public Health, Shanxi Medical University, Taiyuan 030001, China.
Xiaohong
X
Shi
Center of Laboratory Animal, Shanxi Medical University, Taiyuan 030001, China.
Min
M
Guo
Center of Laboratory Animal, Shanxi Medical University, Taiyuan 030001, China.
Chunfang
C
Wang
Department of Respiratory, The First Hospital, Shanxi Medical University, Taiyuan 030001, China.
Xinri
X
Zhang
School of Basic Medicine, Shanxi Medical University, Taiyuan 030001, China.|Centre for Transplantation and Renal Research, Westmead Millennium Institute, University of Sydney, Sydney NSW 2145, Australia.
Guoping
G
Zheng
School of Basic Medicine, Shanxi Medical University, Taiyuan 030001, China.
Baofeng
B
Yu
School of Basic Medicine, Shanxi Medical University, Taiyuan 030001, China.
Hailong
H
Wang
D000818:Animals; D016207:Cytokines / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects / Q000276:immunology; D007249:Inflammation / Q000276:immunology* / Q000517:prevention & control; D018836:Inflammation Mediators / Q000276:immunology*; D008070:Lipopolysaccharides; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000276:immunology; D051379:Mice; D016328:NF-kappa B / Q000276:immunology*; D000067996:RAW 264.7 Cells; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000235:genetics; D014598:Uteroglobin / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000276:immunology*
D016207:Cytokines; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011994:Recombinant Proteins; C556389:SCGB1A1 protein, human; D014598:Uteroglobin; D048051:p38 Mitogen-Activated Protein Kinases
NF-κB; p38 MAPK; pro-inflammatory cytokines; recombinant rat CC16; uteroglobin-binding protein