Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion

Tight junctions control paracellular permeability and cellpolarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro modelof renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rhosignaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells thancontrol junctions, whereas cells expressing activated Rho bettermaintained junctions during ATP depletion than control cells. ATPdepletion and Rho signaling altered phosphorylation signalingmechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoaminoacid content following ATP depletion, which was restored on recovery.Expression of Rho mutant proteins in MDCK cells also altered levels ofoccludin serine/threonine phosphorylation, indicating that occludin isa target for Rho signaling. We conclude that Rho GTPase signalinginduces posttranslational effects on tight junction components. Ourdata also demonstrate that activating Rho signaling protects tightjunctions from damage during ATP depletion.