The IL-1 receptor and Rho directly associate to drive cell activation in inflammation
R. Singh, B. Wang, A. Shirvaikar, S. Khan, S. Kamat, J.R. Schelling, M. Konieczkowski, J.R. Sedor
R. Singh, B. Wang, A. Shirvaikar, S. Khan, S. Kamat, J.R. Schelling, M. Konieczkowski, J.R. Sedor
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Article

The IL-1 receptor and Rho directly associate to drive cell activation in inflammation

Abstract

IL-1–stimulated mesenchymal cells model molecular mechanisms of inflammation. Binding of IL-1 to the type I IL-1 receptor (IL-1R) clusters a multi-subunit signaling complex at focal adhesion complexes. Since Rho family GTPases coordinately organize actin cytoskeleton and signaling to regulate cell phenotype, we hypothesized that the IL-1R signaling complex contained these G proteins. IL-1 stimulated actin stress fiber formation in serum-starved HeLa cells in a Rho-dependent manner and rapidly activated nucleotide exchange on RhoA. Glutathione S-transferase (GST) fusion proteins, containing either the full-length IL-1R cytosolic domain (GST-IL-1Rcd) or the terminal 68 amino acids of IL-1R required for IL-1–dependent signal transduction, specifically coprecipitated both RhoA and Rac-1, but not p21ras, from Triton-soluble HeLa cell extracts. In whole cells, a small-molecular-weight G protein coimmunoprecipitated by anti–IL-1R antibody was a substrate for C3 transferase, which specifically ADP-ribosylates Rho GTPases. Constitutively activated RhoA, loaded with [γ-32P]GTP, directly interacted with GST-IL-1Rcd in a filter-binding assay. The IL-1Rcd-RhoA interaction was functionally important, since a dominant inhibitory mutant of RhoA prevented IL-1Rcd–directed transcriptional activation of the IL-6 gene. Consistent with our previous data demonstrating that IL-1R–associated myelin basic protein (MBP) kinases are necessary for IL-1–directed gene expression, cellular incorporation of C3 transferase inhibited IL-1R–associated MBP kinase activity both in solution and in gel kinase assays. In summary, IL-1 activated RhoA, which was physically associated with IL-1Rcd and necessary for activation of cytosolic nuclear signaling pathways. These findings suggest that IL-1–stimulated, Rho-dependent cytoskeletal reorganization may cluster signaling molecules in specific architectures that are necessary for persistent cell activation in chronic inflammatory disease.

Authors

R. Singh, B. Wang, A. Shirvaikar, S. Khan, S. Kamat, J.R. Schelling, M. Konieczkowski, J.R. Sedor

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RhoA is necessary for IL-1–mediated actin stress fiber formation. Contro...
RhoA is necessary for IL-1–mediated actin stress fiber formation. Control-scraped HeLa cells and cells scrape-loaded in the presence of C3 transferase were plated on coverslips and allowed to recover for 12 hours, as described in Methods. Serum-starved cells were stimulated with or without IL-1α for 10 minutes, and actin stress fibers were viewed by rhodamine-conjugated phalloidin. (a) Few actin stress fibers were seen in vehicle-stimulated HeLa cells. (b) Cells stimulated with IL-1 organize actin stress fibers. (c) In contrast, IL-1 failed to stimulate stress fiber formation in HeLa cells previously scrape-loaded with C3 transferase. Each photomicrograph is representative of a blinded evaluation of photographs taken from 10 fields for each condition from 4 separate experiments.