Spatial Compartmentalization of Tumor Necrosis Factor (TNF) Receptor 1-dependent Signaling Pathways in Human Airway Smooth Muscle Cells

Abstract

Tumor necrosis factor (TNF)-α-induced activation of RhoA, mediated by TNF receptor 1 (TNFR1), is a prerequisite step in a pathway that leads to increased 20-kDa light chain of myosin (MLC20) phosphorylation and airway smooth muscle contraction. In this study, we have investigated the proximal events in TNF-α-induced RhoA activation. TNFR1 is localized to both lipid raft and nonraft regions of the plasma membrane in primary human airway smooth muscle cells. TNF-α engagement of TNFR1 recruited the adaptor proteins TRADD, TRAF-2, and RIP into lipid rafts and activated RhoA, NF-κB, and MAPK pathways. Depletion of cholesterol from rafts with methyl-β-cyclodextrin caused a redistribution of TNFR1 to nonraft plasma membrane and prevented ligand-induced RhoA activation. By contrast, TNF-α-induced activation of NF-κB and MAPKs was unaffected by methyl-β-cyclodextrin indicating that, in airway smooth muscle cells, activation of these pathways occurred independently of lipid rafts. Targeted knockdown of caveolin-1 completely abrogated TNF-α-induced RhoA activation, identifying this raft-resident protein as a positive regulator of the activation process. The signaling adaptors TRADD and RIP were also found to be necessary for ligand-induced RhoA activation. Taken together, our results suggest that in airway smooth muscle cells, spatial compartmentalization of TNFR1 provides a mechanism for generating distinct signaling outcomes in response to ligand engagement and define a mechanistic role for lipid rafts and caveolin-1 in TNF-α-induced activation of RhoA.