In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor B (NF-B) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-B-mediated transactivation induced by physiological stimuli such as tumor necrosis factor ␣, interleukin-1␤, and overexpression of NF-B-inducing kinase. TLS augmented NF-B-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-␤ gene. These results suggest that TLS acts as a coactivator of NF-B and plays a pivotal role in the NF-B-mediated transactivation. Nuclear factor B (NF-B)1 is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including cytokines, cell adhesion molecules and human immunodeficiency virus (1-5). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, RelA (p65), RelB, NFkB1 (p50/105), and NFkB2 (p52/p100). These proteins share a conserved 300-amino acid region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (1, 2, 4, 5). In most cells, Rel family members form hetero-and homodimers with distinct specificities in various combinations. p65, RelB, and c-Rel are transcriptionally active members of the NF-B family, whereas p50 and p52 primarily serve as DNA binding subunits (1, 2, 4, 5). These proteins play fundamental roles in immune and inflammatory responses and in the control of cell proliferation (4, 6 -9). A common feature of the regulation of NF-B is the sequestration in the cytoplasm as an inactive complex with a class of inhibitory molecules known as IBs (2, 10). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1 (IL-1), and tumor necrosis factor ␣ (TNF-␣) results in phosphorylation, ubiquitination, and degradation of the IB proteins (5, 11, 12). The degradation of IB proteins exposes the nuclear localization sequence in the remaining NF-B dimers, followed by the rapid translocation of NF-B to the nucleus where it activates the target genes by binding to the DNA regulatory element (1, 2, 4, 5).The protein regions responsible for the transcriptional activation (called "transactivation domain") of p65, RelB, and c-Rel have been mapped in their unique C-terminal regions. p65 contains at least two independent transactivation domains within its C-terminal 120 amino acids (Fig. 1A) (13-16). One of these transactivation doma...

NF-B-inducing kinase (NIK) has been shown to play an essential role in the NF-B activation cascade elicited by lymphotoxin ␤ receptor (LT␤R) signaling. However, the molecular mechanism of this pathway remains unclear. In this report we demonstrate that both NIK and IB kinase ␣ (IKK␣) are involved in LT␤R signaling and that the phosphorylation of the p65 subunit at serine 536 in its transactivation domain 1 (TA1) plays an essential role. We also found that NF-B could be activated in the LT␤R pathway without altering the level of the phosphorylation of IB and nuclear localization of p65. By using a heterologous transactivation system in which Gal4-dependent reporter gene is activated by the Gal4 DNA-binding domain in fusion with various portions of p65, we found that TA1 serves as a direct target in the NIK-IKK␣ pathway. In addition, mutation studies have revealed the essential role of Ser-536 within TA1 of p65 in transcriptional control mediated by NIK-IKK␣. Furthermore, we found that Ser-536 was phosphorylated following the stimulation of LT␤R, and this phosphorylation was inhibited by the kinase-dead dominant-negative mutant of either NIK or IKK␣. These observations provide evidence for a crucial role of the NIK-IKK␣ cascade for NF-B activation in LT␤R signaling.

We examined the role of p38 mitogen-activated protein (MAP) kinase in the tumor necrosis factor(IL-6) and interleukin-8 (IL-8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL-6 and IL-8 without affecting nuclear translocation of nuclear factor U UB (NF-U UB) or IL-6 and IL-8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.z 2000 Federation of European Biochemical Societies.

The inflaatory cytokine interleukin 1,B(IL-113) induces both cyclooxygenase (COX) and nitric oxide synthase (NOS) with increases in the release of prostaglandin (PG) IL-1 and TNF increase nitric oxide (NO) release in macrophages (11, 12) and mesangial cells (13). The free radical NO has emerged as an important signal and effector molecule in mammalian physiology (11,12), including neurotransmission, vasodilation, and inflammation. NO is synthesized from the guanidino nitrogen of L-Arg by the catalytic reaction of NO synthase (NOS) (11,12,14,15 (21) and vascular endothelium (22)(23)(24). The cDNA for iNOS has also been cloned from a macrophage cell line (16-18).Thus inflammatory cytokines including IL-1 and TNF drive both COX and NOS pathways. These pathways share a number of similarities. A variety of cells and tissues that produce PGs simultaneously release NO in response to cytokines or other activators. Both of them are paracrine modulators of the cell functions and mediate intracellular signals via cyclic nucleotides (i.e., cAMP or cGMP). NO and some PGs dilate vascular smooth muscle and inhibit platelet aggregation (11,12). In addition, NOS and COX require heme as a cofactor (25-27) and have constitutive and cytokineinducible forms.Many effects of NO are mediated by cGMP. NO activates the soluble guanylate cyclase and increases a second messenger, cGMP, by binding to the heme moiety of the enzyme (11,12). However, it is likely that guanylate cyclase is not the only molecular target of NO. NO inhibits several enzymes in the mitochondrial electron transport system and aconitase in the citric acid cycle by interacting with iron-sulfur centers of these enzymes (12). Likewise, NO has been shown to stimulate COX activity possibly via the heme component, which binds to the active site of the COX enzyme (28-31). Thus, a close interaction between NOS and COX pathways has become evident. However, the effect of PGs on the NOS pathway has not been fully explored. Thus we determined the effects of COX inhibition and PGs on iNOS induction and expression in rat mesangial cells. MATERIALS AND METHODSMaterials. Human recombinant IL-1,B (50 half-maximal units/ng) and restriction enzymes were purchased from Boehringer Mannheim. Primers and cDNA [for the polymerase chain reaction (PCR)] for mouse iNOS and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Clontech. NG-Monomethyl-L-arginine (LNMMA), aminoguanidine (AG), indomethacin (Indo), PGE2, forskolin (FSK), sulfanilamide, and naphthylethylenediamine dihydrochloride were from Sigma. A stable analogue of PGI2, carba prostacyclin, was from Cayman Chemicals (Ann Arbor, MI).