Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligandreceptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin⅐protein complexes of high specific radioactivity. The binding of endotoxin⅐MD-2 to the TLR4 ectodomain (TLR4 ECD ) and transfer of endotoxin from CD14 to MD-2/TLR4 ECD were demonstrated using HEK293T-conditioned medium containing TLR4 ECD ؎ MD-2. These interactions are specific, of high affinity (K D < 300 pM), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a M r ϳ 190,000 complex composed of endotoxin, MD-2, and TLR4 ECD . CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin⅐MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin⅐protein complexes with TLR4 ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.Essential arms of the innate immune system are the Tolllike receptors (TLRs).2 These receptors link recognition of unique microbial molecules to activation of host defense effector systems by rapidly triggering pro-inflammatory responses (1). Potent host responses toward many Gramnegative bacteria (GNB) are mediated by recognition and response to unique glycolipids (lipopoly-or lipooligosaccharides LOS, endotoxin) of the GNB outer membrane by TLR4. TLR4 does not function alone but requires the accessory protein MD-2, which binds non-covalently to the N-terminal ectodomain of TLR4 (2-6). Maximally potent endotoxin-induced cell activation also requires the extracellular lipopolysaccharide-binding protein (LBP) and membrane (m) or soluble (s) extracellular CD14 (4, 7-9). The sequential action of LBP, CD14, secreted or TLR4-associated MD-2, and TLR4 confers the extraordinary sensitivity of mammalian cells to many GNB endotoxins. This ordered action implies differences in endotoxin binding specificity, with LBP having the highest affinity for endotoxin organized at lipid/water interfaces (e.g. purified endotoxin aggregates and endotoxin in the GNB outer membrane), CD14 for LBP-modified endotoxin-rich interfaces, MD-2 for monomeric endotoxin⅐CD14 and TLR4, apparently, for endotoxin presented as a monomeric complex with MD-2 (8). Together, these proteins can convert one GNB (containing ϳ10 6 endotoxin molecules) to 10 6 TLR4-activating monomeric endotoxin⅐protein complexes (i.e. endotoxin⅐CD14 or endotoxin⅐MD-2), greatly amplifying host responsiveness to endotoxin. At pM concentrations, monomeric complexes of endotoxin⅐CD14 or endotoxin⅐MD-2 activate, respectively, mammalian cells expressing MD-2/TLR4 or TLR4 alone, triggering robust cell activation through engagement of Ͻ10 3 TLR4 molecules.Despite the ability of endotoxin⅐CD14 and endotoxin⅐ MD-2 to activate cells at pM concentrations (half-maximal cell acti...
Beta transforming growth factor (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta-2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta-2 was much less active than TGF beta-1 for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H-thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta-1 and TGF beta-2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvILu), and in AEC, and for stimulation of 3H-thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta-1 and TGF beta-2 inhibited cell proliferation in MvILu with no significant differences in the ED50 (31 +/- 8 pg/ml vs 23 +/- 7). TGF beta-2 was much less potent than TGF beta-1 in inhibiting DNA synthesis in AEC (6310 +/- 985 pg/ml vs 101 +/- 34). The reduced specific activity of TGF beta-2 was also observed in adrenal capillary endothelial cells. Both beta-1 and beta-2 stimulated proliferation of CBC (ED50 26 +/- 2 pg/ml vs 10 +/- 4). We also examined the specificity of the MvILu and AEC inhibition assays. Epidermal growth factor (EGF), platelet derived growth factor (PDGF), acidic and basic fibroblast growth factors (FGF), skeletal growth factor (SGF)/insulin-like growth factor-II (IGF-II), and insulin-like growth factor-I (IGF-I) did not inhibit DNA synthesis in either assay system. However, when the growth factors were added to maximal inhibiting concentrations of TGF beta-1, both acidic and basic FGF significantly reduced TGF beta-1 inhibition in AEC. We conclude that (1) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta-1, (2) inhibition of DNA synthesis in MvILu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta-1 from beta-2, (3) the relative inhibition of DNA synthesis in MvILu and AEC may provide a means to quantitatively estimate TGF beta-1 and TGF beta-2, and (4) both TGF beta-1 and TGF beta-2 are potent mitogens for chicken embryonic calvarial bone cells.
Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E·CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E·CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E·CD14 ± TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe121 and Tyr131 are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E·MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E·CD14 ± TLR4. E·MD-2F126A binds TLR4 with high affinity (Kd ∼ 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E·MD-2. These findings reveal roles of Phe121 and Tyr131 in TLR4-independent interactions of human MD-2 with E·CD14 and, together with Phe126, in activation of TLR4 by bound E·MD-2. These findings strongly suggest that the structural properties of E·MD-2, not E alone, determine agonist or antagonist effects on TLR4.
Arachidonic acid metabolism by lipoxygenases and cytochrome P450 monooxygenases produces regioisomeric hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs), which serve as components of cell signaling cascades. Intracellular fatty acid-binding proteins (FABPs) may differentially bind these nonprostanoid oxygenated fatty acids, thus modulating their metabolism and activities. Vascular cells, which express heart FABP (H-FABP), utilize oxygenated fatty acids for regulation of vascular tone. Therefore, the relative affinities of H-FABP for several isomeric series of these compounds were measured by fluorescent displacement of 1-anilinonaphthalene-8-sulfonic acid (ANS). In general, H-FABP rank order affinities (arachidonic acid > EETs > HETEs > DHETs) paralleled reversed-phase high-performance liquid chromatography retention times, indicating that the differences in H-FABP affinity were determined largely by polarity. H-FABP displayed a similar rank order of affinity for compounds derived from linoleic acid. H-FABP affinity for 20-HETE [apparent dissociation constant (K(d)') of 0.44 microM] was much greater than expected from its polarity, indicating unique binding interactions for this HETE. H-FABP affinity for 5,6-EET and 11,12-EET (K(d)' of approximately 0.4 microM) was approximately 20-fold greater than for DHETs (K(d)' of approximately 8 microM). The homologous proteins, liver FABP and intestinal FABP, also displayed selective affinity for EET versus DHET. Thus, FABP binding of EETs may facilitate their intracellular retention whereas the lack of FABP affinity for DHETs may partially explain their release from cells. The affinity of H-FABP for EETs suggests that this family of intracellular proteins may modulate the metabolism, activities, and targeting of these potent eicosanoid biomediators.
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