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.
Highlights
The TLR4 is a transmembrane receptor for LPS, found on the outer membrane of Gram-negative bacteria [1,2]
Simulating the MyD88-dependent pathway We updated our previous TLR4 model [9] incorporating several new features into the topology; i) crosstalk mechanisms from TRIF to TRAF6 and TRIF to RIP1 for NF-kB (p65/p50) and MAP kinases activation, ii) the addition of MKK1/2-ERKAP-1 activation pathway, and iii) phosphorylation of the IkB/cRel/p50 complex by TBK1 leading to NF-kB (c-Rel/p50) activation (Figure 1A and supplementary Table SI)
The in silico model begins with LPS-activated TLR4, which results in signal flux propagation through the MyD88-dependent and TRAM-dependent pathways (Figure 1A)
Summary
The TLR4 is a transmembrane receptor for LPS, found on the outer membrane of Gram-negative bacteria [1,2]. The TRAMdependent pathway, on the other hand, predominantly induces type I interferons (IFNs) and chemokines such as IP-10 (encoded by Cxcl10) and interferon (IFN)-induced proteins through activation of interferon regulatory factor (IRF) 3 or 7 and NFkB [4]. From in silico MyD88 KO or TRAF6 KO, we simulated enhanced activation of alternative TRAM-dependent pathway through the re-channelling of signal transduction or SFR, which occurs when molecules at pathway junction are removed. These findings were validated through in vivo and in vitro experiments. SFR may explain the mechanistic basis for unexpected alterations in cellular signaling, for example, due to gain or loss-of-function mutations found in human diseases [11,12]
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