Signaling flux redistribution at toll-like receptor pathway junctions

Abstract

Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus. Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR) 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS) stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR) at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and Ifit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxcl10 and Ifit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed.

Figure 1. Schematic presentation of TLR4 pathway and temporal profiles of experimental and simulated Tnf and Socs3.

(A) Following LPS stimulation, TLR4 signaling bifurcates into MyD88-dependent (brown) and TRAM-dependent (blue) pathways, which activates target genes such as Tnf and Socs3, and Cxcl10 and Ifit2, respectively. Black arrows indicate cross talks between MyD88-dependent and TRAM-dependent pathways. The two junctions analyzed in this study are indicated by stars (TLR4 to MyD88/TRAM and TRIF to TRAF6/RIP1/TBK1). Dashed line between TLR4 and TRAM represents indirect activation of TRAM for its recruitment to the TLR4 ( and supplementary Table SI). (B) Tnf and (C) Socs3 in vivo mRNA levels after LPS treatment in wildtype (blue) and MyD88 KO (red, dotted) macrophages measured by qRT-PCR. Values are an average of six independent cultures and shown as means±SEM. In silico simulated expression (arbitrary units) of (D) Tnf and (E) Socs3 in the presence (blue) and absence (red, dotted) of MyD88 upon TLR4 activation. **p<0.01 vs. wildtype.

Figure 2. In silico simulations of TRAM-dependent pathway molecules upon TLR4 activation.

Simulation profiles (arbitrary units) of (A) TRAM activation, (B) IRF3 activation, and (C) Cxcl10 induction in the wildtype (blue), knockout (red, dotted), and two-fold overexpression (green) of MyD88. (D) Schematic of SFR. (Top) Wildtype. Fluxes propagate through both the MyD88-dependent and TRAM-dependent pathways. (Middle) MyD88 KO. More fluxes propagate or overflows through the TRAM-dependent pathway resulting in increased Cxcl10 induction. (Bottom) MyD88 overexpression by two-fold.

Figure 3. Enhanced TRAM-dependent pathway in the absence of MyD88.

Macrophages were treated with LPS (100 ng/ml) for indicated periods. Cell lysates were analyzed for (A) IRF3, JNK, ERK, p38 phosphorylation and NF-κB (degradation of IκBα) using Western blot analysis with Actin as a loading control. (B) Cxcl10 and (C) Ifit2 mRNAs levels in wildtype (blue) and MyD88 KO (red, dotted) macrophages using qRT-PCR. Six independent cultures were analyzed and shown as means±SEM. †p = 0.064.

Figure 4. Competition at TLR4 and SFR in TRAF6 KO.

(A) MyD88 and TRAM compete for TLR4 in GST pull-down assay. GST or the GST-tagged TLR4 were expressed in HEK293T cells with Myc-tagged MyD88 or Flag-tagged TRAM. After GST-pull-down, Western blotting was performed. (B–E) Enhanced TRAM-dependent pathway in the absence of TRAF6. In silico expression of (B) Tnf and (C) Cxcl10, and in vivo expression of (D) Tnf and (E) Cxcl10 mRNA in wildtype (blue) and TRAF6 KO (green, dotted) macrophages. Four independent cultures were analyzed. means±SEM. *p<0.05, **p<0.01 vs. wildtype. Paired student's t-test was used for (E).

Figure 5. Schematic representation of Signaling Flux Redistribution (SFR).

(A) removal of MyD88 results in enhancement of TRAM-dependant pathway, (B) removal of TRAF6 results in enhancement of TRAM-dependant pathway downstream of TRIF, (C) overexpression of MyD88 downregulates TRAM-dependant pathway, (D) removal of TRAM does not enhance the MyD88-dependant pathway due to upstream intermediates. * signaling molecules/events upstream of TRAM , , .

Figure 6. Proposed mechanisms of action for SFR.

(A) Competition: Molecules X and Y compete to bind with molecule Z. X and Y share binding sites at Z. (B) Steric hindrance: When X binds to Z, the complex prevents the binding of Y to another binding site at Z (C) Conformational change: When X binds to Z, structural changes to Z lowers the affinity of Y binding to Z.