Rho/Rho kinase pathway regulates maintenance of the differentiated tubular epithelial cell phenotype on laminin-1.
BACKGROUND
Maintenance of a polarized tubular epithelium by appropriate intracellular signaling and extracellular matrix is critical both in normal renal function as well as in acute and chronic tubular injury. We examined the hypothesis that maintenance of a differentiated epithelial phenotype on the basement membrane glycoprotein laminin-1 is controlled by the Rho/Rho kinase pathway.


METHODS
Using the tubular epithelial cell lines LLC-PK1 and MDCK which were cultured on laminin-1 vs. collagen IV, we analyzed cell morphology and motility (cohort migration assay) as well as expression of differentiation and dedifferentiation markers (immunofluorescence microscopy).


RESULTS
Cohort migration of LLC-PK1 cells was significantly slowed down on laminin-1 (10.7 +/- 2.2 m.u. (migratory units)) compared with collagen IV (16.6 +/- 2.3 m.u.; BSA control: 2.8 +/- 2.5 m.u.). Inhibition of the Rho/Rho kinase pathway by C3 exotoxin (1 mug/ml) or the Rho kinase inhibitor Y27632 (10 microM) significantly augmented cohort migration on laminin-1 (14.5 +/- 1.4 and 16.0 +/- 1.8 m.u. vs. 10.7 +/- 2.2 m.u.). In parallel to the increased migratory activity, inhibition of the Rho/Rho kinase pathway resulted in a more mesenchymal phenotype of LLC-PK1 cells on laminin-1 with increased formation of lamellopodia and filopodia, distinct loss of focal contacts and stress fibers, upregulation of the dedifferentiation marker vimentin, and loss of cell-cell contacts with translocation of beta-catenin from the adherens junctions to the cytosol and nucleus. Similarly, cohort migration of MDCK cells was retarded on laminin-1 when compared with collagen IV, and addition of the Rho kinase inhibitor Y27632 resulted in enhanced motility and a change in cell morphology.


CONCLUSIONS
The study demonstrates that the Rho/Rho kinase pathway is required to maintain a non-migratory epithelial phenotype of cultured renal tubular LLC-PK1 and MDCK cells on the basement membrane glycoprotein laminin-1.
Reuters
Irith
I
Department of Medicine I, Cologne General Hospital, Merheim Medical Center, Cologne, Germany.
Weber
Manfred
M
Schulze-Lohoff
Eckhard
E
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002465:Cell Movement; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007797:Laminin / Q000008:administration & dosage*; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013552:Swine; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D007797:Laminin; C121346:laminin 1; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins