Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury

Abstract

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK10 and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations.

Mycophenolic Acid Inhibits Cell Proliferation and Extracellular Matrix Synthesis in Rat Vascular Smooth Muscle Cells Through Direct and Indirect Inhibition of Cellular Reactive Oxygen Species

“…Cellular guanosine content is an important regulator of small GTPases family activation including rac1 [31,32]. MPA inhibition on PDGF-induced rac1 activation is in-line with the observed effects of other IMPDH inhibitors such as tiazofurin [33] and ribavirin [34] on small GTPases.…”

Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis

“…GST-PBD (for Rac1 and Cdc42) and GST-RBD (for Rho) pull-down binding assays were performed as previously described (Hallett et al, 2003).…”

HMG-CoA reductase inhibitors up-regulate anti-aging klotho mRNA via RhoA inactivation in IMCD3 cells

“…Likewise, FPP and GGPP also prevented the upregulation of klotho expression by atorvastain. In addition, to examine whether the Rho/Rho kinase system mediated the effect of atorvastatin on klotho mRNA expression, IMCD3 cells were cultured with Y-27632, a pharmacological inhibitor of Rho kinase [22,23]. Treatment with Y-27632 (10 Amol/l) increased the expression level of klotho mRNA by 2.4 fold (Fig.…”