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

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          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 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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          Most cited references33

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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.
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                Author and article information

                Journal
                NEP
                Nephron Physiol
                10.1159/issn.1660-2137
                Nephron Physiology
                S. Karger AG
                1660-2137
                2006
                September 2006
                28 September 2006
                : 104
                : 2
                : p95-p106
                Affiliations
                aDepartment of Medicine I, Cologne General Hospital, Merheim Medical Center, and bCenter for Molecular Medicine Cologne CMMC, University of Cologne, Cologne, Germany
                Article
                94573 Nephron Physiol 2006;104:p95–p106
                10.1159/000094573
                16847378
                b5433fa7-d5d3-47e0-970f-337dde875008
                © 2006 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                History
                : 14 January 2005
                : 04 February 2006
                Page count
                References: 51, Pages: 1
                Categories
                Original Paper

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

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