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


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          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 µ M) 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.

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          BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.

          Bone morphogenic protein (BMP)-7 is a 35-kDa homodimeric protein and a member of the transforming growth factor (TGF)-beta superfamily. BMP-7 expression is highest in the kidney, and its genetic deletion in mice leads to severe impairment of eye, skeletal and kidney development. Here we report that BMP-7 reverses TGF-beta1-induced epithelial-to-mesenchymal transition (EMT) by reinduction of E-cadherin, a key epithelial cell adhesion molecule. Additionally, we provide molecular evidence for Smad-dependent reversal of TGF-beta1-induced EMT by BMP-7 in renal tubular epithelial cells and mammary ductal epithelial cells. In the kidney, EMT-induced accumulation of myofibroblasts and subsequent tubular atrophy are considered key determinants of renal fibrosis during chronic renal injury. We therefore tested the potential of BMP-7 to reverse TGF-beta1-induced de novo EMT in a mouse model of chronic renal injury. Our results show that systemic administration of recombinant human BMP-7 leads to repair of severely damaged renal tubular epithelial cells, in association with reversal of chronic renal injury. Collectively, these results provide evidence of cross talk between BMP-7 and TGF-beta1 in the regulation of EMT in health and disease.
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            The Small GTPases Rho and Rac Are Required for the Establishment of Cadherin-dependent Cell–Cell Contacts

            Cadherins are calcium-dependent cell–cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell–cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell–cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell–cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell– cell contact formation and the remodelling of actin filaments in epithelial cells.
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              The role of epithelial-to-mesenchymal transition in renal fibrosis.

              Epithelial-to-mesenchymal transition (EMT) involving injured epithelial cells plays an important role in the progression of fibrosis in the kidney. Tubular epithelial cells can acquire a mesenchymal phenotype, and enhanced migratory capacity enabling them to transit from the renal tubular microenvironment into the interstitial space and escape potential apoptotic cell death. EMT is a major contributor to the pathogenesis of renal fibrosis, as it leads to a substantial increase in the number of myofibroblasts, leading to tubular atrophy. However, recent findings suggest that EMT involving tubular epithelial cell is a reversible process, potentially determined by the surviving cells to facilitate the repopulation of injured tubules with new functional epithelia. Major regulators of renal epithelial cell plasticity in the kidney are two multifunctional growth factors, bone morphogenic protein-7 (BMP-7) and transforming growth factor beta1 (TGF-beta1). While TGF-beta1 is a well-established inducer of EMT involving renal tubular epithelial cells, BMP-7 reverses EMT by directly counteracting TGF-beta-induced Smad-dependent cell signaling in renal tubular epithelial cells. Such antagonism results in the repair of injured kidneys, suggesting that modulation of epithelial cell plasticity has therapeutic advantages.

                Author and article information

                Nephron Physiol
                Nephron Physiology
                S. Karger AG
                September 2006
                28 September 2006
                : 104
                : 2
                : p95-p106
                aDepartment of Medicine I, Cologne General Hospital, Merheim Medical Center, and bCenter for Molecular Medicine Cologne CMMC, University of Cologne, Cologne, Germany
                94573 Nephron Physiol 2006;104:p95–p106
                © 2006 S. Karger AG, Basel

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                : 14 January 2005
                : 04 February 2006
                Page count
                References: 51, Pages: 1
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/94573
                Self URI (text/html): https://www.karger.com/Article/FullText/94573
                Self URI (journal page): https://www.karger.com/SubjectArea/Nephrology
                Original Paper

                Cardiovascular Medicine,Nephrology
                Proximal tubular cells,Epithelial cell migration,Rho/Rho kinase pathway,Laminin


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