Abstract
We reported previously that fMLP stimulates NF-kappaB activation, and this function of fMLP requires small GTPase RhoA in human peripheral blood monocytes (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we present evidence that RhoA associates specifically with the guanine nucleotide exchange factor Lbc in human peripheral blood monocytes stimulated with fMLP and that Lbc specifically catalyzes the guanine nucleotide exchange activity of RhoA in human peripheral blood monocytes. Cotransfection of the monocytic THP1 cells with lbc with a kappaB promoter reporter plasmid results in a marked increase in NF-kappaB-mediated reporter gene expression. Finally, Lbc-enhanced NF-kappaB activation is inhibited by a RhoA inhibitor, C3 transferase from Clostridium botulinum. A dominant-negative form of RhoA (T19N) also inhibited Lbc-enhanced reporter gene expression in a kappaB-dependent manner. These results indicate that guanine nucleotide exchange factor Lbc is a novel signal transducer for RhoA-mediated NF-kappaB activation in human peripheral blood monocytes stimulated with bacterial products.
Highlights
Leukocyte infiltration is characteristic of inflammatory reaction to tissue injuries caused by trauma, invasion of foreign particles, ischemia-reperfusion, cancer, autoimmune diseases, and other conditions
We found that RhoA associates with the guanine nucleotide exchange factor Lbc in monocytes stimulated with fMLP and that Lbc catalyzes the guanine nucleotide exchange activity of RhoA
We examined whether the biological activity of the Lbc in monocytes is based on its ability to interact with low molecular weight GTP-binding proteins or whether Lbc can functionally bind to Rho family GTP-binding proteins in vivo
Summary
Aliquots (20 l) of the GDP-loaded RhoA were mixed with 20 l of Lbc-containing monocyte immunoprecipitates (anti-Lbc antibody) or control monocyte immunoprecipitates (control antibody) in the reaction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 100 M AMP-PNP, 0.5 mg/ml bovine serum albumin, and 5 M [35S]GTP␥S) to initiate the exchange reaction at room temperature. Aliquots (20 l) of the [3H]GDP-loaded RhoA were mixed with 20 l of Lbc-containing monocyte immunoprecipitates (anti-Lbc antibody) or control monocyte immunoprecipitates (control antibody) in the reaction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 100 M AMP-PNP, 0.5 mg/ml bovine serum albumin, and 1 mM GTP) to initiate the exchange reaction at room temperature. Luciferase activity was determined by using the luciferase assay kit (Promega) and the Monolight 2010 luminometer (Analytical Luminescence, San Diego, CA)
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