Inhibition of Rho-kinase abrogates migration of human transitional cell carcinoma cells: results of an in vitro study.
BACKGROUND
Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting.


METHODS
Intracellular calcium concentration ([Ca(2+)](i)) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[(3)H]inositol- (0.5 μCi/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used.


RESULTS
Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca(2+)](i), cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the G(i) inhibitor pertussis toxin. Agonists potently stimulated migration of T24 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration.


CONCLUSIONS
G protein-coupled receptors potently stimulated cell migration in T24 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer.
Institute for Pharmacology, University of Essen Medical School, Essen, Germany. frankvomdorp @ web.de
Frank
F
vom Dorp
Harald
H
Sanders
Christof
C
Boergermann
Gerd
G
Lümmen
Herbert
H
Rübben
Karl H
KH
Jakobs
Martina
M
Schmidt
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000199:Actins / Q000737:chemistry; D000577:Amides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002295:Carcinoma, Transitional Cell / Q000201:enzymology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D016257:Fura-2 / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D066298:In Vitro Techniques; D008246:Lysophospholipids / Q000737:chemistry / Q000494:pharmacology; D009154:Mutation; D037342:Pertussis Toxin / Q000494:pharmacology; D010590:Phalloidine / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000199:Actins; D000577:Amides; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D008246:Lysophospholipids; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C031815:toxB protein, Clostridium difficile; C108830:Y 27632; D010590:Phalloidine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D037342:Pertussis Toxin; D054460:rho-Associated Kinases; D010738:Type C Phospholipases; D020741:rho GTP-Binding Proteins; C032881:lysophosphatidic acid; C049347:fasudil; D002118:Calcium; D016257:Fura-2