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NF-B-Inducing Kinase Regulates Cyclooxygenase 2 Gene Expression in Macrophages by Phosphorylation of PU.1¹

Anser C. Azim^*, Xuerong Wang^*, Gye Young Park^*, Ruxana T. Sadikot^*,, Hongmei Cao, Biji Mathew^*, Michael Atchison, Richard B. van Breemen, Myungsoo Joo^¶ and John W. Christman^2,*,

^* Department of Medicine and Department of Medicinal Chemistry and Pharmacognosy, Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, Chicago, IL 60612; Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612; Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; and ^¶ Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232

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-B-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-β-D-ribofuranosylbenzimidazole. We used short interfering RNA to develop a stable NIK knockdown macrophage cell line that had an 50% decrease in COX-2 protein production and decreased generation of PGD₂, 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Department of Veterans Affairs and National Institutes of Health Grants HL 075557 and HL 66196.

² Address correspondence and reprint requests to Dr. John William Christman, Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, 840 South Wood Street, Chicago, IL 60612. E-mail address: jwc{at}uic.edu

³ Abbreviations used in this paper: CKII, casein; CBP, CREB-binding protein; NIK, NF-B-inducing kinase; COX, cyclooxygenase; IKK, IB kinase; NIK, NF-B-inducing kinase; DRB, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole; ChIP, chromatin immunoprecipitation; siRNA, short interfering RNA.