0022-1767/91/1475-1694$02.00/0 THE JOURNAL OF IMMUNOLOGY Copyright 0 1991 by The Amerlcan Association of Immunologlsts Vol. 147. 1694-1700. No. 5, September 1, 1991 Prlnted Ln U.S.A. ENHANCERSANDTRANSCRIPTION FACTORS CONTROLLING THE IN PRIMARY MACROPHAGES INDUCIBILITY OF THE TUMOR NECROSIS FACTOR-cx PROMOTER C. DROUET,’” A. N. SHAKHOV,*+ AND C. V. JONGENEEL2* From the *Ludwig Institute for Cancer Research, Lausanne Branch, CH-1066 Epalinges, Switzerland. and ‘Engelhardt Institute of Molecular Biology, USSR Academy of Sciences, MOSCOW B-224. USSR In macrophages, the TNF-a promoter is specifi- cally induced by bacterial endotoxin, and provides a good model for gene regulation during bacterial infections. We have analyzed the protein-binding characteristics and enhancer activity of four &-like enhancers and of a MHC class 11-like Y box found in the mouse TNF-a promoter. In addition to members of the NF-KB/rel transcription factor family, at least two of the KB sites also bound a nuclear protein identified as NF-GMa, a factor that binds to pro- moter sequences from many cytokines. When in- serted upstream of an enhancer-less promoter, two of the KB sites were active as LPS-inducible enhan- cers in primary macrophages, whereas the other two were not. Mutations in nucleotides known to con- tact nuclear factors severely reduced affinity of the KB sites for NF-KB. Introduction of the same muta- tions into a construct containing 1059 bp of the TNF-a promoter coupled to a CAT reporter gene resulted in a stepwise reduction in inducibility by LPS; mutation of all four sites (11 bp of 1059) re- duced inducibility by 90%. providing compelling evi- dence for the role of transcription factors belonging to the NF-KBlrel family in the activation of the TNF- a promoter. The TNF-a Y box bound an abundant nuclear factor, but had no detectable activity in our assays, either as an enhancer or as a mutation- sensitive controlling element. Macrophages play a key role in the initiation of a nor- mal inflammatory response through their ability to syn- thesize multiple cytokines upon exposure to bacterial products, in particular theendotoxin or LPS component of Gram-negative cell walls (1). The genes coding for many of these cytokines have been characterized over the last few years; however, surprisingly little is known about the molecular mechanisms of LPS-mediated gene induction in macrophages, particularly at the transcrip- tional level. The synthesis of TNF-a is very strongly and specifically Recelved for publicatlon February 1 1, 1991. Accepted for publication June 11, 1991. The costs of publication of thls article were defrayed In part by the payment of page charges. This article must therefore be hereby marked aduertfsement In accordance with 18 U.S.C. Section 1734 solely to indl- cate thls fact. Training Program).Present address INSERM U238, DRF/LBIO/ICH, CENG ‘Recipient of a fellowship of the European Communities (Cancer 85X. F-38041 Grenoble Cedex. France. Address correspondence and reprint requests to Dr. C. Vlctor Jonge- neel. Ludwlg Institute for Cancer Research, Chemin des Boveresses 155. CH-1066Epalinges, Switzerland. induced by LPS, and overproduction of TNF-a by macro- phages is thought tobe a major cause of endotoxic shock (2). Increased transcription accounts for a significant portion of the induction of TNF-a production, although posttranscriptional events also play a major role (3-6). When mouse TNF-a promoter/CAT hybrid constructs are transfectedintoprimarymacrophages,sequences ex- tending more than 450 bp upstream of the mRNA initia- tion site are necessary for full inducibility byLPS (7). Loss of inducibility in a 5’ deletion series correlates with the deletion of sites capable of binding the NF-KB tran- scription factor,and one of the KB binding sites is able to function as an LPS-inducible enhancer. Moreover, LPS is a very potent inducer of NF-KB in macrophages (7-9). These results strongly suggested a role for NF-KB in the LPS-mediated induction of TNF-a transcription. How- ever, a recent study of the regulation of the human TNF- a promoter in transformed cell lines found no evidence for a n LPS-inducible enhancer activity of its NF-KB bind- ing sites (1 0). Recently, a motif related to but distinct from the KB enhancer was reported to be involved in the LPS induci- bility of the gene coding for G-CSF, another cytokine produced by activatedmacrophages (1 1). This motif, which was called cytokine- 1 or GPE- 1, acts as a TNF- inducible enhancer in embryonic fibroblasts (1 2). and is found inthe promoters of many cytokines (13). Although its sequence bears a strong resemblance to enhancers of the KB family, it binds a protein (NF-GMa)distinct from NF-KB (12). The KB sites that we identified in the TNF-a promoter also fit the consensus for the CK-1 motif. There- fore, NF-GMa may contribute to the LPS inducibility of the TNF-a promoter. Our previous results suggested that a sequence identi- cal to the Y box of MHC class 11 promoters may play a role in the regulation of TNF-a transcription (7). The Y box is a member of the “CCAAT box” family of upstream regu- latory elements, and binds one of the CCAAT-binding factors, CP-1/NF-Y (14). The Y box is thought to play a role in the baseline expressionof MHC class I1 promoters rather than in their inducibility. The experiments described in this paper attempt to assess the relative contributions of DNA sequences bind- ing NF-KB, NF-GMa, and NF-Y to the LPS inducibility of the TNF-a promoter. MATERIALS AND METHODS Artlflcial enhancer constructs. Double-stranded oligonucleotides generated ends (Fig. 2) were introduced into pBLCAT2, a vector that containing 5’ overhangs compatible with restriction endonuclease- 1694