GEF-H1 Mediates Tumor Necrosis Factor-α-induced Rho Activation and Myosin Phosphorylation: ROLE IN THE REGULATION OF TUBULAR PARACELLULAR PERMEABILITY

Eli Kakiashvili,Pam Speight,Faiza Waheed,Romy Seth,Monika Lodyga,Susumu Tanimura,Michiaki Kohno,Ori D Rotstein,András Kapus,Katalin Szászi

doi:10.1074/jbc.m805933200

Abstract

Tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-alpha, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-alpha on the Rho pathway in tubular cells (LLC-PK(1) and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-alpha-induced alterations of paracellular permeability. We show that TNF-alpha induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-alpha-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-alpha-induced activation of these proteins. Importantly TNF-alpha enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-alpha-induced paracellular permeability increase was absent in LLC-PK(1) cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-alpha-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.

Highlights

Tumor necrosis factor-␣ (TNF-␣)2 is a pleiotropic proinflammatory cytokine that is synthesized as a membrane protein in response to inflammation, infection, and injury [1]
In this study we have shown that TNF-␣ induces myosin light chain (MLC) and cofilin phosphorylation through the GTP exchange factors (GEFs)-H1/RhoA/Rho kinase pathway, leading to cytoskeletal remodeling in tubular epithelium
We provide evidence that the ERK pathway is upstream of TNF-␣-induced GEF-H1 and RhoA activation and that GEF-H1 phosphorylation on Thr-678 is a key event in this process

Summary

Introduction

Tumor necrosis factor-␣ (TNF-␣) is a pleiotropic proinflammatory cytokine that is synthesized as a membrane protein in response to inflammation, infection, and injury [1]. It is cleaved by the metalloprotease TNF-␣ convertase enzyme to release a 17-kDa soluble peptide (for a review, see Ref. 2). Effects of locally released TNF-␣ on the tubular epithelium could contribute to its deleterious actions, the underlying mechanisms have been incompletely explored. GEF-H1 Mediates Rho Activation in Tubular Epithelium importantly, the upstream signaling that connects the TNF receptor to activation of the Rho pathway remains completely unknown. The transepithelial resistance decrease was blocked by genistein, a general tyrosine kinase inhibitor; the exact mechanism underlying the observed permeability changes remained incompletely explored

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