Enhancer-Mediated Control of Macrophage-Specific Arginase I Expression

Abstract

Arginase I expression in the liver must remain constant throughout life to eliminate excess nitrogen via the urea cycle. In contrast, arginase I expression in macrophages is silent until signals from Th2 cytokines such as IL-4 and IL-13 are received and the mRNA is then induced four to five orders of magnitude. Arginase I is hypothesized to play a regulatory and potentially pathogenic role in diseases such as asthma, parasitic, bacterial, and worm infections by modulating NO levels and promoting fibrosis. We show that Th2-inducible arginase I expression in mouse macrophages is controlled by an enhancer that lies −3 kb from the basal promoter. PU.1, IL-4-induced STAT6, and C/EBPβ assemble at the enhancer and await the effect of another STAT6-regulated protein(s) that must be synthesized de novo. Identification of a powerful extrahepatic regulatory enhancer for arginase I provides potential to manipulate arginase I activity in immune cells while sparing liver urea cycle function.

Nuclear Receptor Nr4a2 Promotes Alternative Polarization of Macrophages and Confers Protection in Sepsis

“…STAT6 also acts in synergy with KLF4 to regulate arginase 1 expression; however, arginase 1 expression in response to Bacillus Calmette-Guérin (BCG) is STAT6-independent and depends only upon CCAAT/enhancer-binding protein ␤ transcriptional activity (13,49). PU.1 is also known to bind to the arginase 1 promoter at two sites, one in the enhancer region 3 kb upstream of transcription start site and another 700 bp upstream of the basal promoter (48,50). Furthermore, arginase 1 is known to be a PPAR-responsive gene (51).…”

A Distal Enhancer in Il12b Is the Target of Transcriptional Repression by the STAT3 Pathway and Requires the Basic Leucine Zipper (B-ZIP) Protein NFIL3

“…In some experiments, cDNAs encoding C/EBP␤ and arginase-1 were used under the same conditions. From the final 50 l of IVTT product, we used 6 l in DNA binding assays as described previously (34). Briefly, IVTT reactions were blocked with 2 g of poly(dI-dC) on ice for 20 min, followed by binding to 32 P-labeled and annealed primers at RT for 30 min.…”

“…Enhancer fragments of various lengths were cloned into the reporter vector using the same strategy. Luciferase reporter stable lines were derived by transfecting RAW264.7 macrophages as described (34). Cells were selected with G418 and expanded.…”

“…To assay luciferase reporter lines, cells were plated in 12-well plates at 2 ϫ 10 6 cells/well and stimulated with LPS (100 ng/ml) or LPS plus IL-10 (10 ng/ml) for 2, 4, or 8 h followed by lysis and standard luciferase assays. In some experiments, luciferase reporter constructs were used in transient assays as described (34).…”

General Nature of the STAT3-Activated Anti-Inflammatory Response

“…Plasmids containing the Epo Rleptin R hybrid (ELR) or ELR mutant constructs were linearized with BglII. RAW cells were transfected by electroporation as described previously (22). Cells were plated into 10-cm dishes with 10 ml of complete RPMI 1640 containing 10% FBS (HyClone) and 1% penicillin-streptomycin (Invitrogen Life Technologies) and incubated at 37°C overnight.…”