Extracellular signal-regulated kinase and GEF-H1 mediate depolarization-induced Rho activation and paracellular permeability increase.
Plasma membrane depolarization activates the Rho/Rho kinase (ROK) pathway and thereby enhances myosin light chain (MLC) phosphorylation, which in turn is thought to be a key regulator of paracellular permeability. However, the upstream mechanisms that couple depolarization to Rho activation and permeability changes are unknown. Here we show that three different depolarizing stimuli (high extracellular K(+) concentration, the lipophilic cation tetraphenylphosphonium, or l-alanine, which is taken up by electrogenic Na(+) cotransport) all provoke robust phosphorylation of ERK in LLC-PK1 and Madin-Darby canine kidney (MDCK) cells. Importantly, inhibition of ERK prevented the depolarization-induced activation of Rho. Searching for the underlying mechanism, we have identified the GTP/GDP exchange factor GEF-H1 as the ERK-regulated critical exchange factor responsible for the depolarization-induced Rho activation. This conclusion is based on our findings that 1) depolarization activated GEF-H1 but not p115RhoGEF, 2) short interfering RNA-mediated GEF-H1 silencing eliminated the activation of the Rho pathway, and 3) ERK inhibition prevented the activation of GEF-H1. Moreover, we found that the Na(+)-K(+) pump inhibitor ouabain also caused ERK, GEF-H1, and Rho activation, partially due to its depolarizing effect. Regarding the functional consequences of this newly identified pathway, we found that depolarization increased paracellular permeability in LLC-PK1 and MDCK cells and that this effect was mitigated by inhibiting myosin using blebbistatin or a dominant negative (phosphorylation incompetent) MLC. Taken together, we propose that the ERK/GEF-H1/Rho/ROK/pMLC pathway could be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and regulate paracellular transport in the tubular epithelium.
St. Michael's Hospital, Toronto, ON, Canada.
Faiza
F
Waheed
Pam
P
Speight
Glenn
G
Kawai
Qinghong
Q
Dan
András
A
Kapus
Katalin
K
Szászi
D000409:Alanine / Q000378:metabolism; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D018374:LLC-PK1 Cells; D008564:Membrane Potentials; D018994:Myosin Light Chains / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D010539:Permeability; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D002070:Butadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D020662:Guanine Nucleotide Exchange Factors; D006576:Heterocyclic Compounds, 4 or More Rings; D018994:Myosin Light Chains; D009570:Nitriles; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D047428:Protein Kinase Inhibitors; C113580:U 0126; C472645:blebbistatin; D010042:Ouabain; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins; D020741:rho GTP-Binding Proteins; D000254:Sodium-Potassium-Exchanging ATPase; D000409:Alanine; D011188:Potassium; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium; C013289:tetraphenylphosphonium