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R., A. NF-B activation in cells treated with serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation. These data suggest different molecular mechanisms of activation induced by porins or by LPS. Although lipopolysaccharide (LPS) has been clearly shown to play a major role in septic shock and in the induction of cytokine production, very little is known regarding other surface bacterial components of gram-negative bacteria. It has been reported that these components also play an important role in the pathway associated with infections by gram-negative bacteria (13). LPS induces transcription Rabbit polyclonal to C-EBP-beta.The protein encoded by this intronless gene is a bZIP transcription factor which can bind as a homodimer to certain DNA regulatory regions. of several genes encoding proinflammatory mediators (21, 52). In the past few years we have studied the various immunobiological effects induced by the outer membrane pore-forming proteins compared to those induced by LPS (16C20, 30). Porins are integral components of the outer membranes of ARQ-092 (Miransertib) all gram-negative bacteria and are intimately associated with the LPS; they induce many cellular responses, including cellular activation (23) and cytokine release (17, 19, 20, 28, 30). LPS and porins are released by several bacteria during both in vitro (10) and in vivo (59) growth, and this release is significantly enhanced when the bacteria are lysed following exposure to antibiotics or human serum (10, 12, 35). Active concentrations of both LPS and porins are often reached at infection sites from either gram-negative bacteria outer membrane blebbing or bacterial lysis as a consequence of host defense (59). Intracellular signaling pathways induced by LPS stimulation have been studied in detail (54, 56); in contrast, very little is known about the signaling pathways of other components derived from gram-negative bacteria. Mitogen-activated protein kinase (MAPK) cascades are among the best known signal transduction systems and play a key role in the regulation of gene expression as well as cytoplasmic activities. MAPKs have also been shown to be involved in the regulation of cytokine responses (57). In mammalian systems, five different MAPK modules have been identified so far; single MAPK modules generally can signal independently of one another, and this specificity is manifest in distinct physiologic responses (49). MAPKs, with the exception of extracellular-signal-regulated kinase 3 (ERK3), are activated upon phosphorylation of both tyrosine and threonine residues by MAPK kinase (MEK) (49). Many different MEKs have been described, and in vitro assays indicate that each has one or at most two specific targets in the MAPK pathways: MEK1 and MEK2 act on ERK1 and ERK2, respectively. As shown in various cell types, LPS induces activation of ERK1 and ERK2 (4), c-Jun N-terminal kinases (JNKs) (25), and p38 (26). The MAPK cascade activates transcription factors such as activating protein 1 (AP-1) and nuclear factor B (NF-B). The contribution of AP-1 family members to transcriptional regulation is controlled by a ARQ-092 (Miransertib) number of well-characterized mechanisms that have been reviewed recently (3, 32, 33). The genes encoding AP-1 proteins (and for 10 min at 4C, the cell pellet was resuspended in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 U/ml) (complete medium) at a concentration of 5 106 cells/ml. Adherent macrophage monolayers were obtained by plating the cells in six-well plastic trays at 5 106 cells/well for 2 h at 37C in 5% CO2. Nonadherent cells were removed by suction, and freshly prepared complete medium was added with the indicated experimental reagents. Bacterial strain. The bacterial strain used was serovar Typhimurium ARQ-092 (Miransertib) SH5014 grown in nutrient broth (Difco, Detroit, Mich.) for 18 to 24 h at 37C under agitation. The cells were harvested at the end of the ARQ-092 (Miransertib) exponential growth phase, and outer membranes were prepared from cell envelopes according to the protocols described by Nurminen et al. (43). Preparation of porins and LPS. The porins were extracted as described by Nurminen (44). Briefly, 1 g (wet weight) ARQ-092 (Miransertib) of cell envelopes was suspended in 2% Triton X-100 in 0.01 M Tris-HCl (pH 7.5),.