Interleukin 12 (IL-12) is an inducible cytokine composed of 35-and 40-kDa subunits that is critical for promoting T helper type 1 development and cell-mediated immunity against pathogens. The 40-kDa subunit, expressed by activated macrophages and B cells, is induced by several pathogens in vivo and in vitro and is augmented or inhibited by gamma interferon (IFN-␥) or IL-10, respectively. Control of IL-12 p40 expression is therefore important for understanding resistance and susceptibility to a variety of pathogens, including Leishmania major and perhaps human immunodeficiency virus. In this report, we provide the first characterization of IL-12 p40 gene regulation in macrophages. We localize inducible activity of the promoter to the sequence ؊122 GGGGAATTTTA؊132 not previously recognized to bind Rel family transcription factors. We demonstrate binding of this sequence to NF-B (p50/p65 and p50/c-Rel) complexes in macrophages activated by several p40-inducing pathogens and provide functional data to support a role for NF-B family members in IL-12 p40 activation. Finally, we find that IFN-␥ treatment of cells enhances this binding interaction, thus potentially providing a mechanism for IFN-␥ augmentation of IL-12 production by macrophages.Interleukin 12 (IL-12) production by macrophages is critical in induction of T helper type 1 (Th1) cells during initial immune responses to pathogens (5,15,21,32,45). Th1 cells produce gamma interferon (IFN-␥) and IL-2, which promote macrophage activation and cytolytic T-cell maturation, thus generating effective cell-mediated responses to intracellular pathogens (33, 37). The macrophage is the principal source of IL-12 production in responses to certain intracellular pathogens, such as Listeria monocytogenes (20,21,48). Recently, Chehimi and colleagues described diminished IL-12 production by macrophages in human immunodeficiency virus-infected individuals (2) and proposed that this may contribute to reduced cell-mediated immunity seen in AIDS (6, 7). Thus, understanding IL-12 production by macrophages, and its inhibition in settings of disease, could contribute to immune response-based therapies or vaccine designs.IL-12 is an inducible, heterodimeric, disulfide-linked cytokine composed of 35-and 40-kDa subunits encoded by separate genes (5,19,24,43,49). Expression of the 35-kDa subunit is constitutive and ubiquitous. In contrast, the 40-kDa subunit is expressed only by macrophages and B cells; it is strongly induced by several bacterial stimuli and is considered the regulatory component for IL-12 expression (11). Further, we and others have found that some cytokines, notably IFN-␥ and IL-10, can exert their effects on T-cell responses by augmenting or inhibiting macrophage production of 21,26). To date, the transcriptional regulation of p40 gene induction by bacteria and by cytokines is uncharacterized.In this report, we demonstrate several important features of IL-12 p40 gene regulation in macrophages. First, we localize the inducible promoter activity for the p40 gene to a nov...
Mutation of the XRCC4 gene in mammalian cells prevents the formation of the signal and coding joints in the V(D)J recombination reaction, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation. However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be. Here we show that DNA ligase IV co-immunoprecipitates with XRCC4 and that these two proteins specifically interact with one another in a yeast two-hybrid system. Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV. We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4-DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends.
Abstract. Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH: terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2-terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the HI subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT-4 and was sequestered more from the cell surface than was the wildtype HI protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.
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