NF-kappaB-inducing kinase regulates cyclooxygenase 2 gene expression in macrophages by phosphorylation of PU.1.
Selective expression of cyclooxygenase 2 (COX-2) by macrophages could have an important role in the pathobiology of inflammation. We reported a functional synergism between PU.1 and other transcription factors that contributes to COX-2 gene expression in macrophages. PU.1 resides in the nuclear compartment and is activated by phosphorylation to bind to cognate DNA elements containing a 5'-GGAA/T-3' motif, but the involved kinase has not been discovered. We tested the hypothesis that NF-kappaB-inducing kinase (NIK) regulates COX-2 gene expression in macrophages through inducible phosphorylation of PU.1. Our initial experiments showed an in vitro protein-protein binding interaction between myc-NIK and GST-PU.1. Purified myc-NIK had a strong in vitro kinase activity for purified GST-PU.1, and this activity and production of COX-2 protein is blocked by treatment with a nonspecific kinase inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. We used short interfering RNA to develop a stable NIK knockdown macrophage cell line that had an approximately 50% decrease in COX-2 protein production and decreased generation of PGD(2), and this was correlated with decreased binding of activated PU.1 to the COX-2 promoter in response to treatment with endotoxin. These findings suggest a novel role for NIK in mediating COX-2 gene expression in endotoxin-treated macrophages by a mechanism that involves phosphorylation of PU.1.
Azim
Anser C
AC
Department of Medicine, University of Illinois, Chicago, IL 60612, USA.
Wang
Xuerong
X
Park
Gye Young
GY
Sadikot
Ruxana T
RT
Cao
Hongmei
H
Mathew
Biji
B
Atchison
Michael
M
van Breemen
Richard B
RB
Joo
Myungsoo
M
Christman
John W
JW
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D004731:Endotoxins / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000276:immunology / Q000378:metabolism*; D000097782:NF-kappaB-Inducing Kinase; D000099232:Proto-Oncogene Protein Spi-1
D051546:Cyclooxygenase 2; D004731:Endotoxins; D008070:Lipopolysaccharides; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000097782:NF-kappaB-Inducing Kinase; D000099232:Proto-Oncogene Protein Spi-1; C077623:Ptgs2 protein, mouse