Anthrax Edema Toxin Inhibits Endothelial Cell Chemotaxis via Epac and Rap1

Jia Hong,Robert C Doebele,Mark W Lingen,Lawrence A Quilliam,Wei-Jen Tang,Marsha Rich Rosner

doi:10.1074/jbc.m700128200

Abstract

Angiogenesis involves the assembly of endothelial cells into capillaries from a pre-existing vasculature. Because abnormal angiogenesis is a hallmark of many cancers, it is critical to find factors that control this process. Endothelial cells are enriched in the anthrax receptor; we therefore determined the effect of anthrax edema toxin (ET), an adenylyl cyclase, on chemotaxis. cAMP generated by ET does not block proliferation or survival but causes cytoskeletal changes and inhibits chemotaxis by primary human microvascular endothelial cells (HMVECs). These effects are due to the action of a downstream cAMP effector, Epac, a guanine nucleotide exchange-activating protein for Rap1 (RAP1-GEF). ET induces transcription of Epac-related activators of Rap1, Epac2 (RapGEF4), and MR-GEF/RapGEF5. Similar to ET, activated Epac or Rap1 induces cytoskeletal changes and blocks chemotaxis in human endothelial cells. These results identify Epac and Rap1 as key regulators of signaling cascades leading to endothelial cell chemotaxis.

Highlights

The results we have obtained indicate that treatment of endothelial cells with either edema toxin, an adenylyl cyclase, or activators of Epac, a guanine nucleotide exchange factor (GEF) for Rap1, causes cytoskeletal changes and inhibition of vascular endothelial growth factor (VEGF)-induced chemotaxis in response to angiogenic factors in human microvascular endothelial cells
The link between edema toxin (ET) and Epac is strengthened by the fact that Epac is activated by cAMP, the only known product of ET, and because ET rapidly induces an increase in the transcription of Rap1GEFs, thereby amplifying the signaling through Rap1
The difference in these results may be due to the different chemoattractants (VEGF versus factors released by wounding in culture) that are reflective of these two different processes as well as the specific cells used

Summary

EXPERIMENTAL PROCEDURES

Reagents—PA was purchased from List Biological Laboratories (Campbell, CA). Alexa Fluor 594 phalloidin was from Molecular Probes (Eugene, Oregon). Cells were treated as indicated and actin stress fibers stained with Alexa Fluor 594 phalloidin (3 units/ml) as previously described [17]. Endothelial Cell Migration Assay—The endothelial cell migration assay was performed as previously described [19]. Microarrays—Subconfluent HMVECs (1 ϫ 106 in T25 flask) were starved overnight in EBM2 medium containing 0.1% BSA and treated with various reagents as indicated. Assays-on-demandTM for Epac, Epac, RapGEF5, and GAPDH were purchased from Applied Biosystems. Analysis was performed using RQ Study software, version 1.2.3 (Applied Biosystems, Foster City, CA). Rap Pulldown Assays—HMVECs were grown to subconfluency in 100-mm plates, starved in EBM2 medium plus 0.1% BSA overnight, and treated as indicated. Rap pulldown assays were performed by using glutathione S-transferase-RalGDSRas-binding domain (RBD) beads as described previously [23].

RESULTS

HMVECs with forskolin plus a phosphodiesterase inhibitor

DISCUSSION