Spatial compartmentalization of tumor necrosis factor (TNF) receptor 1-dependent signaling pathways in human airway smooth muscle cells. Lipid rafts are essential for TNF-alpha-mediated activation of RhoA but dispensable for the activation of the NF-kappaB and MAPK pathways

Abstract

Tumor necrosis factor (TNF)-alpha-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-alpha-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-alpha engagement of TNFR1 recruited the adaptor proteins TRADD, TRAF-2, and RIP into lipid rafts and activated RhoA, NF-kappaB, and MAPK pathways. Depletion of cholesterol from rafts with methyl-beta-cyclodextrin caused a redistribution of TNFR1 to nonraft plasma membrane and prevented ligand-induced RhoA activation. By contrast, TNF-alpha-induced activation of NF-kappaB and MAPKs was unaffected by methyl-beta-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-alpha-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-alpha-induced activation of RhoA.

FIGURE 1. TNFRs co-localize with lipid rafts in human airway smooth muscle cells

A, lipid rafts were detected by confocal microscopy after labeling human airway smooth muscle cells with CTxB-Alexa 488, which binds to the raft-associated glycosphingolipid GM1. B, TNFRs on the surface of the same cells were labeled with biotin-TNF-α and detected with TRITC-avidin. C, the merged image of A and B revealed considerable co-localization of TNFRs and lipid rafts, which was more clearly seen at higher magnification (D). Scale bar = 0.5 μm (A–C) and 0.1 μm (D).

FIGURE 2. TNFR1 is located in lipid rafts prepared under different conditions

Human airway smooth muscle cells extracted with cold Triton X-100 (A), Brij 98 at 37 °C (B), or in the absence of detergent (C) were fractionated by sucrose density gradient centrifugation. Equal volumes of the 12 collected fractions from each gradient were separated by SDS-PAGE and analyzed by immunoblotting. Lipid rafts were identified by their low buoyant density and the expression of raft proteins caveolin-1 and flotillin-1 and the raft-associated glycosphingolipid GM1. RhoGDI and the transferrin receptor (TfR) were used as markers of nonraft fractions.

FIGURE 3. TNF-α induces recruitment of signaling adaptors and RhoA into lipid rafts

Human airway smooth muscle cells were extracted with cold Triton X-100 and fractionated by sucrose density gradient centrifugation (A) or stimulated with TNF-α for the indicated times (B) before isolation of Triton X-100-soluble (S) and Triton X-100-insoluble, octyl glucoside-soluble raft fractions (R). Equal volume samples were separated by SDS-PAGE and analyzed with specific antibodies for the expression of TNFR1, signaling adaptors, signaling intermediates, and RhoA as indicated. Lipid rafts were identified by the expression of the raft-resident protein, caveolin-1, whereas soluble fractions expressed RhoGDI. The cytoskeletal protein, smooth muscle actin (sm-actin), was restricted to soluble fractions.

FIGURE 4. Disruption of lipid rafts inhibits TNF-α-induced RhoA activation

A, human airway smooth muscle cells were treated with (+) or without (−) 10 mm MCD for 1 h at 37 °C prior to extraction with cold Triton X-100 and sucrose density gradient fractionation. The distribution of TNFR1, RhoA, and the raft-resident proteins flotillin-1 and caveolin-1 was determined by immunoblotting. B, cells, treated with (+) or without (−) 10 mm MCD for 30 min at 37 °C, were stimulated with TNF-α (200 ng/ml) for 5 min at 37 °C, extracted, and analyzed by immunoblotting for TNFR1 and RhoA expression and TNF-α binding. Activated RhoA (RhoA-GTP) was isolated using a glutathione S-transferase fusion protein containing the Rho binding domain of Rhotekin as described under “Experimental Procedures.” C, human airway smooth muscle cells were surface-biotinylated with sulfo-NHS-biotin prior to treatment with (+) or without (−) MCD as in B. Cell extracts were immunoprecipitated (IP) with control (con Ig) or anti-TNFR1 antibody, and cell surface-associated TNFR1 was detected by blotting with HRP-streptavidin. To control for protein input, total cell extracts (total) were analyzed by immunoblotting for expression of TNFR1. D, cells treated with (+) or without (−) MCD as in B were stimulated for 5 min at 37 °C with TNF-α (200 ng/ml), PDGFBB (50 ng/ml), or S1P (1 μm). Cell extracts were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against the indicated proteins. RhoA-GTP was isolated as described in B.

FIGURE 5. TNF-α-induced RhoA activation is dependent on caveolin-1

A, soluble (S) and raft (R) fractions of sucrose density gradient fractionated Triton X-100 extracts of airway smooth muscle cells were immunoprecipitated with control (con Ig) or anti-TNFR1 antibody, separated by SDS-PAGE, and immunoblotted with antibodies against TNFR1 and caveolin-1. The input panel shows the proteins present in 1% of cell extracts used for immunoprecipitation. Human airway smooth muscle cells were transfected with control or caveolin-1 siRNA (100 nm). 72 h after transfection, cells were stimulation with TNF-α (200 ng/ml) (B) or S1P (1 μm) (C ) for 5 min at 37 °C. Cell lysates were fractionated by SDS-PAGE and immunoblotted with antibodies against the indicated proteins. RhoA-GTP was isolated as described in the legend to Fig. 4.

FIGURE 6. Caveolin-1 is not required for raft localization of TNFR1, signaling adaptors, or RhoA

A, human airway smooth muscle cells, treated with either control or caveolin-1 siRNA (100 nm), were fractionated into soluble (S) and raft (R) fractions as described in the legend to Fig. 3. Equal volumes of cell extracts were analyzed by immunoblotting for the indicated proteins. B, HepG2 cells, which do not express caveolin-1, were extracted with cold Triton X-100 and fractionated by sucrose density gradient centrifugation. Equal volumes of soluble (S) and raft (R) fractions were separated by SDS-PAGE and analyzed for the indicated proteins by immunoblotting. C, HepG2 cells were treated with TNF-α (200 ng/ml) at 37 °C for the indicated times. Cell extracts were analyzed for the indicated proteins, and RhoA-GTP was isolated as described in the legend to Fig. 4.

FIGURE 7. TNF-α-induced RhoA activation is dependent on TRADD and RIP but independent of TRAF-2

Human airway smooth muscle cells were transfected with control siRNA or siRNA targeted against either TRADD (A), TRAF-2 (B), or RIP (C ). 72 h after transfection, cells were treated with (+) or without (−) TNF-α (200 ng/ml) for 5 min at 37 °C. Cell extracts were analyzed for the expression of adaptor proteins, phosphorylated IKK and IκB, and for activated RhoA as described in the legend to Fig. 4.