Identification of Mouse MD-2 Residues Important for Forming the Cell Surface TLR4-MD-2 Complex Recognized by Anti-TLR4-MD-2 Antibodies, and for Conferring LPS and Taxol Responsiveness on Mouse TLR4 by Alanine-Scanning Mutagenesis

Abstract

The expression of MD-2, which associates with Toll-like receptor (TLR) 4 on the cell surface, confers LPS and LPS-mimetic Taxol responsiveness on TLR4. Alanine-scanning mutagenesis was performed to identify the mouse MD-2 residues important for conferring LPS and Taxol responsiveness on mouse TLR4, and for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 Ab MTS510. Single alanine mutations were introduced into mouse MD-2 (residues 17–160), and the mutants were expressed in a human cell line expressing mouse TLR4. Mouse MD-2 mutants, in which a single alanine mutation was introduced at Cys37, Leu71, Leu78, Cys95, Tyr102, Cys105, Glu111, Val113, Ile117, Pro118, Phe119, Glu136, Ile138, Leu146, Cys148, or Thr152, showed dramatically reduced ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, and the mutants also showed reduced ability to confer LPS and Taxol responsiveness. In contrast, mouse MD-2 mutants, in which a single alanine mutation was introduced at Tyr34, Tyr36, Gly59, Val82, Ile85, Phe126, Pro127, Gly129, Ile153, Ile154, and His155 showed normal ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, but their ability to confer LPS and Taxol responsiveness was apparently reduced. These results suggest that the ability of MD-2 to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510 is essential for conferring LPS and Taxol responsiveness on TLR4, but not sufficient. In addition, the required residues at codon numbers 34, 85, 101, 122, and 153 for the ability of mouse MD-2 to confer LPS responsiveness are partly different from those for Taxol responsiveness.

Paclitaxel (Taxol) upregulates expression of functional interleukin-6 in human ovarian cancer cells through multiple signaling pathways

“…At pharmacologically low concentrations (exemplified by 1 mM in this study), the JNK signaling pathway was the major stimulator of IL-6 gene regulation. However, at suprapharmacologically high concentrations (exemplified by 50 mM in this study), paclitaxel exerted LPS-like effects likely through the TLR4 signaling pathway as reported previously (Kawasaki et al, 2000(Kawasaki et al, , 2003Perera et al, 2001). Elucidation of the signaling networks that are differentially initiated by low concentrations or by high concentrations of paclitaxel and the verification of specific inhibitors against key molecules inside the networks may help us in developing a better chemotherapeutic strategy in treatment of ovarian cancer with paclitaxel.…”

“…Taken together, these results indicated that two pathways ( Figure 7) are involved in the upregulation of IL-6 by treatment with paclitaxel: (a) at low, pharmacological concentrations (exemplified by 1 mM in this study), the JNK signaling pathway was the major stimulator of IL-6 gene regulation, and (b) at high, suprapharmacological concentrations (exemplified by 50 mM in this study), paclitaxel exerted LPS-like effects, which were most likely carried out through the TLR4 signaling pathway as reported previously (Kawasaki et al, 2000(Kawasaki et al, , 2003Perera et al, 2001). …”

“…Results of this study demonstrated that paclitaxel in this range of concentrations activated IL-6 through the activation of JNK signaling pathway but not through the LPS-like activity of paclitaxel. First, the 1 mM concentration of paclitaxel used in this study was much lower than that reportedly (3-140 mM) effective for upregulation of IL1alpha, IL-1 beta, IL-8 and TNF-alpha (Ding et al, 1990;Lee et al, 1996;Lanni et al, 1997;Kawasaki et al, 2000Kawasaki et al, , 2003Perera et al, 2001;Takeda et al, 2003). Second, no upregulation of TLR4, MyD88, or NF-IL6 in terms of mRNA levels in BR cells treated with 1 mM paclitaxel was noted (Figure 6a).…”

“…Lipopolysaccharidelike effects of paclitaxel were thought to be conveyed through TNF receptor (Ding et al, 1990) or TLR-4 (Kawasaki et al, 2000(Kawasaki et al, , 2003Perera et al, 2001). Microtubule cytoskeleton dysfunction has been shown to activate NF-kB (Rosette and Karin, 1995; Das and Mechanisms of IL-6 upregulation by paclitaxel T-H Wang et al White, 1997).…”

“…More recently, Toll-like receptor 4 (TLR4) on murine macrophage has been shown to be the receptor for paclitaxel (Kawasaki et al, 2000(Kawasaki et al, , 2003Perera et al, 2001). Paclitaxel, notably at 5-30 mM, was thus proposed as a TLR4 ligand (Takeda et al, 2003).…”