Current paradigms suggest that two macrophage subsets, termed M1 and M2, are involved in inflammation and host defense. While the distinct functions of M1 and M2 macrophages have been intensively studied -the former are considered proinflammatory and the latter antiinflammatory -the determinants of their speciation are incompletely understood. Here we report our studies that identify Krüppel-like factor 4 (KLF4) as a critical regulator of macrophage polarization. Macrophage KLF4 expression was robustly induced in M2 macrophages and strongly reduced in M1 macrophages, observations that were recapitulated in human inflammatory paradigms in vivo. Mechanistically, KLF4 was found to cooperate with Stat6 to induce an M2 genetic program and inhibit M1 targets via sequestration of coactivators required for NF-κB activation. KLF4-deficient macrophages demonstrated increased proinflammatory gene expression, enhanced bactericidal activity, and altered metabolism. Furthermore, mice bearing myeloid-specific deletion of KLF4 exhibited delayed wound healing and were predisposed to developing diet-induced obesity, glucose intolerance, and insulin resistance. Collectively, these data identify KLF4 as what we believe to be a novel regulator of macrophage polarization.

URH49 is a mammalian protein that is 90% identical to the DExH/D box protein UAP56, an RNA helicase that is important for splicing and nuclear export of mRNA. Although Saccharomyces cerevisiae and Drosophila express only a single protein corresponding to UAP56, mRNAs encoding URH49 and UAP56 are both expressed in human and mouse cells. Both proteins interact with the mRNA export factor Aly and both are able to rescue the loss of Sub2p (the yeast homolog of UAP56), indicating that both proteins have similar functions. UAP56 mRNA is more abundant than URH49 mRNA in many tissues, although in testes URH49 mRNA is much more abundant. UAP56 and URH49 mRNAs are present at similar levels in proliferating cultured cells. However, when the cells enter quiescence, the URH49 mRNA level decreases 3-6-fold while the UAP56 mRNA level remains relatively constant. The amount of URH49 mRNA increases to the level found in proliferating cells within 5 h when quiescent cells are growth-stimulated or when protein synthesis is inhibited. URH49 mRNA is relatively unstable (T(1/2) = 4 h) in quiescent cells, but is stabilized immediately following growth stimulation or inhibition of protein synthesis. In contrast, there is much less change in the content or stability of UAP56 mRNA following growth stimulation. Our observations suggest that in mammalian cells, two UAP56-like RNA helicases are involved in splicing and nuclear export of mRNA. Differential expression of these helicases may lead to quantitative or qualitative changes in mRNA expression.

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The promoter of the mouse thymidylate synthase (TS) gene lacks a TATAA box and an initiator element, is bidirectional and initiates transcription at multiple start sites across broad initiation windows upstream and downstream of the 30 nt essential promoter region. The TS promoter also plays an essential role in the post-transcription regulation of TS gene expression during the G(1)-S phase transition. The goal of this study was to determine if the addition of a TATAA box or an initiator element would have a significant effect on start-site pattern, promoter bidirectionality and S phase regulation of the TS gene. A TATAA box and/or an initiator element were inserted downstream of the TS essential promoter region, and the modified promoters were used to drive expression of indicator genes. The engineered genes were transfected into cultured mammalian cells, and the effects of the mutations were determined. Addition of the TATAA box and especially the initiator element had a significant effect on the transcription start site pattern, indicating that the elements were functional. Unexpectedly, addition of one or both of these elements had no effect on promoter bidirectionality. However, inclusion of the initiator element led to a significant reduction in S phase regulation of TS mRNA levels, indicating that changes in promoter architecture can perturb normal S phase regulation of TS gene expression.