Upregulation of fibronectin expression by COX-2 is mediated by interaction with ELMO1

Abstract

Engulfment and cell motility 1 (ELMO1), a bipartite guanine nucleotide exchange factor (GEF) for the small GTPase Rac 1, was identified as a susceptibility gene for glomerular disease. Here, we reported that ELMO1 interacted with COX-2 in human mesangial cells. Furthermore, we identified ELMO1 as a posttranslational regulator of COX-2 activity. We demonstrated that COX-2 cyclooxygenase activity increased fibronectin promoter activity. The protein-protein interaction between ELMO1 and COX-2 increased the cyclooxygenase activity of COX-2 and, correspondingly, fibronectin expression. We also found that ET625, the dominant negative form of ELMO1 lacking Rac1 activity, interacted with COX-2, increased cyclooxygenase activity of COX-2 and enhanced COX-2-mediated fibronectin upregulation. To further rule out Rac1 as an ELMO1-mediated regulator of COX-2 activity, we employed the constitutive active Rac1, Rac1(Q63E), and demonstrated that Rac1 signaling has no effect on COX-2-mediated fibronectin promoter activity. These results suggest that ELMO1 contributes to the development of glomerular injury through serving as a regulator of COX-2 activity. The interaction of ELMO1 with COX-2 could play an important role in the development and progression of renal glomerular injury.

Fig. 1

ELMO1 forms a complex with COX-2 in mesangial cells. (A) HMC lysates were immunoprecipitated with either COX-2 antibody, or control IgG, and co-precipitation of endogenous ELMO1 was determined by immunoblotting. Expression of the endogenous proteins was determined by immunoblotting of TCL. (B) Flag-ELMO1 was transfected into THMC, and co-precipitation of infected COX-2 and endogenous DOCK180 were determined by immunoblotting. Expression of the endogenous proteins or overexpressed proteins was determined by immunoblotting of TCL. (C) Flag-ELMO1 and AdWTCOX-2 expressing THMC were stained with anti-Flag (red) and anti-COX-2 (green), and their chromatin was stained with DAPI (blue). Colocalization of ELMO1 and COX-2 appears in yellow in the merge panel. Data are representative of three independent experiments.

Fig. 2

Effect of COX-2 on fibronectin gene expression. (A) Immunoblotting assay for COX-2 expression in THMC infected with AdCOX-2 WT, AdCOX-2G533A and AdCOX-2G533L. β-Actin was used for protein loading control. TCL: total cell lysates. IB: immunoblotting (B) Luciferase assay with the fibronectin promoter in THMC infected with AdCOX-2 WT, AdCOX-2G533A and AdCOX-2G533L. * P < 0.01, ** P < 0.05, *** P < 0.05 by two-tailed Student t-test. (C) Luciferase assay with the fibronectin promoter in THMC under PGE₂ stimulation. * P = 0.0001, ** P < 0.001, and *** P < 0.005 by two-tailed Student t-test. All data in B and C are means ± s.e.m from at least three separate experiments, each performed in duplicate.

Fig. 3

Effect of wild-type ELMO1 and mutant ELMO1 on COX-2-mediated fibronectin gene expression. (A) Luciferase assay with the fibronectin promoter in THMC co-introduced with AdCOX-2WT, ELMO1WT or ET625. * P < 0.001, ** P < 0.05, *** P < 0.05 by two-tailed Student t-test. (B) THMC transfected with the indicated plasmids were infected with AdCOX-2WT, and lysates were immunoprecipitated with anti-Flag antibody. Precipitated COX-2 and DOCK 180 were determined by immunoblotting. Expression of the endogenous proteins or overexpressed proteins was determined by immunoblotting of TCL. Data are representative of three independent experiments. All data in A are means ± s.e.m from at least three separate experiments, each performed in duplicate.

Fig. 4

Effect of Rac1 on COX-2mediated fibronectin promoter activity. (A) Constitutive active Rac1, Rac1^Q63E, has no effects on COX-2-mediated fibronectin promoter activity. * P < 0.005, ** P = 0.8170 by two-tailed Student t-test. (B) Immunoblotting assay for GFP tagged Rac1^Q63E expression in THMC infected with AdCOX-2 WT. β-Actin was used for protein loading control. (C) Immunoblotting demonstrated that ELMO1WT and ET625 have no effects on endogenous COX-2 expression. Data are representative of three independent experiments. All data in A are means ± s.e.m from at least three separate experiments, each performed in duplicate.

Fig. 5

Effect of COX-2 on ELMO1-Rac1 activity. Rac1 activity assay demonstrated that ELMO1 and DOCK180 cotransfection increased the amount of GTP-bound Rac1, whereas AdCOX-2 infection has no effects on ELMO1/DOCK180-mediated Rac1 activity. Data are representative of three independent experiments.