RhoA and Rac mediate endothelial cell polarization and detachment induced by T-cadherin.
T-cadherin (T-cad) is an atypical GPI-anchored member of the cadherin superfamily. Ligation of T-cad receptors on endothelial cells prevents cell spreading, promotes elongation and polarization, decreases adhesion to the matrix, and facilitates migration. This study investigates involvement of Rho GTPases in T-cad signaling. Human umbilical vein endothelial cells were infected with adenoviral vectors expressing dominant-negative and/or constitutively active mutants of RhoA (N19RhoA/RhoA63), ROCK (RB/PH(TT)/CAT), and Rac1 (N17RAC). Mutant-infected and empty vector-infected cells were compared with respect to their ability to detach and polarize when plated on substratum containing recombinant T-cad protein used as a ligand mimicking homophilic T-cad interactions. ROCK involvement was also studied using specific inhibitor Y-27632. Adhesion assays, analysis of cell phenotype, and actin cytoskeleton organization using TRITC-labeled phalloidin demonstrated that T-cad-induced cell polarization includes two complementary components: RhoA/ROCK pathway is necessary for cell contraction, stress fiber assembly, and inhibition of spreading, whereas Rac is required for formation of actin-rich lamellipodia at the leading edges of polarized cells. Individual repression of either pathway only partially prevented cell polarization and detachment, while simultaneous repression of RhoA and Rac pathways fully eliminated responses to homophilic T-cad ligation. In conclusion, these data suggest that T-cad induces cell deadhesion and polarization via RhoA-ROCK- and Rac-dependent mechanisms.
Philippova
Maria
M
Department of Research, Cardiovascular Laboratories, Basel University Hospital, Basel, Switzerland.
Ivanov
Danila
D
Allenspach
Roy
R
Takuwa
Yoh
Y
Erne
Paul
P
Resink
Thérèse
T
D015820:Cadherins / Q000494:pharmacology*; D002448:Cell Adhesion; D016764:Cell Polarity; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure; D042783:Endothelial Cells / Q000502:physiology* / Q000648:ultrastructure; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D009154:Mutation; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D014471:Umbilical Veins; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D020830:rac1 GTP-Binding Protein / Q000235:genetics; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D015820:Cadherins; C099972:H-cadherin; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein