Rho/Rho Kinase Pathway Regulates Maintenance of the Differentiated Tubular Epithelial Cell Phenotype on Laminin-1

Abstract

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 µg/ml) or the Rho kinase inhibitor Y27632 (10 µM) 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 β-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. Conclusion: 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.

Autotaxin and lysophosphatidic acid stimulate intestinal cell motility by redistribution of the actin modifying protein villin to the developing lamellipodia

“…This is consistent with previous reports which have demonstrated that the effects of Rho family proteins on cell migration depend on the cell type and environment specific migration [29,30]. Studies done with epithelial cells have reported either no effect of RhoA on cell migration or have reported inhibition of epithelial cell migration by RhoA [31,32]. Likewise, we found no effect of Rac inhibition or of overexpression of constitutively active Rac 1 on either basal or LPA-induced epithelial cell migration.…”

Inhibition of Rho Kinase Regulates Specification of Early Differentiation Events in P19 Embryonal Carcinoma Stem Cells

“…Furthermore, the extensive intracellular remodelling that takes place during differentiation is accompanied by extensive changes in gene expression. Rho-associated protein kinases (ROCKs) are downstream effectors of Rho GTPase, which function in numerous physiological processes including the reorganization of cytoskeletal elements [30] – [32] . In addition to this role, our results would indicate that ROCK inhibition blocks the differentiation of endoderm, whereas it also attenuates the induction of P19 cells into mesoderm or ectoderm, but only in the presence of DMSO or RA and LIF, respectively.…”

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

“…Thus, the exact role of ROCKs in cell migration is controversial and might be cell-type dependent. In addition, the effects of pharmacological inhibition of ROCKs on the migration of specific cell lines derived from renal tubular epithelium have been shown to depend on the type of extracellular matrix to which the cells adhered [ 30 ], indicating that ROCK inhibition exerts differential as well as cell type-dependent effects on cell migration. Furthermore, in a study using clinical samples collected from patients with esophageal cancer, ROCK inhibition suppressed the invasion and metastasis of esophageal cancer cells [ 31 ], suggesting the importance of ROCKs in esophageal cancer progression.…”