GEF-H1 mediates tumor necrosis factor-alpha-induced Rho activation and myosin phosphorylation: role in the regulation of tubular paracellular permeability.
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.
Kakiashvili
Eli
E
Keenan Research Centre in the Li Ka Shing Knowledge Institute, St. Michael's Hospital and Department of Surgery, University of Toronto, Ontario M5B 1W8, Canada.
Speight
Pam
P
Waheed
Faiza
F
Seth
Romy
R
Lodyga
Monika
M
Tanimura
Susumu
S
Kohno
Michiaki
M
Rotstein
Ori D
OD
Kapus
András
A
Szászi
Katalin
K
D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D007684:Kidney Tubules / Q000293:injuries / Q000378:metabolism*; D013058:Mass Spectrometry; D008954:Models, Biological; D009218:Myosins / Q000378:metabolism*; D010539:Permeability; D010766:Phosphorylation; D064067:Rho Guanine Nucleotide Exchange Factors; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism*
C577356:ARHGEF2 protein, human; D051339:Actin Depolymerizing Factors; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D014409:Tumor Necrosis Factor-alpha; D054460:rho-Associated Kinases; D009218:Myosins