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      Targeting Ras Genes in Kidney Disease


      Cardiorenal Medicine

      S. Karger AG

      Ras, Rho, Fibrosis, GTPase, Gene therapy, Renal disease

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          Certain changes in cellular function are characteristic of renal disease. Foremost among these is the excessive proliferation of cells, but other phenotypic changes include dysregulated apoptosis, migration, adhesion, contraction, secretion, and receptor expression. Recent advances in cell biology have revealed an extensive role for the small monomeric GTPases of the Ras superfamily in the control of these cellular events through intracellular signalling cascades. The specific Ras genes appear to play discrete and identifiable roles in a range of complex signalling networks. These insights lead to the possibility of targeting Ras genes in a specific manner in renal therapies. For example, the process of renal cell proliferation might be sensitive to downregulation of Harvey Ras and Kirsten Ras; targeting of Rho A and related species may modulate cell migration, fibrosis, and intrarenal vasoconstriction. Possible strategies for such modulation could include the use of RNA-interacting agents such as antisense DNA and si-RNA and the use of small molecules acting on Ras directly or on related signalling molecules such as Rho kinase and Raf kinase.

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          Most cited references 9

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          Ras and TGFβ cooperatively regulate epithelial cell plasticity and metastasis

          Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.
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            Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro.

            We recently found evidence of tubular epithelial-myofibroblast transdifferentiation (TEMT) during the development of tubulointerstitial fibrosis in the rat remnant kidney. This study investigated the mechanisms that induce TEMT in vitro. The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of recombinant transforming growth factor-beta1 (TGF-beta1). Transdifferentiation of tubular cells into myofibroblasts was assessed by electron microscopy and by expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin. NRK52E cells cultured on plastic or collagen-coated plates showed a classic cobblestone morphology. Culture in 1 ng/ml TGF-beta caused only very minor changes in morphology, but culture in 10 or 50 ng/ml TGF-beta1 caused profound changes. This involved hypertrophy, a loss of apical-basal polarity and microvilli, with cells becoming elongated and invasive, the formation of a new front-end back-end polarity, and the appearance of actin microfilaments and dense bodies. These morphological changes were accompanied by phenotypic changes. Double immunohistochemistry staining showed that the addition of TGF-beta1 to confluent cell cultures caused a loss of the epithelial marker E-cadherin and de novo expression of alpha-SMA. An intermediate stage in transdifferentiation could be seen with hypertrophic cells expressing both E-cadherin and alpha-SMA. De novo alpha-SMA expression was confirmed by Northern blotting, Western blotting, and flow cytometry. In particular, cells with a transformed morphology showed strong alpha-SMA immunostaining of characteristic microfilament structures along the cell axis. There was a dose-dependent increase in the percentage of cells expressing alpha-SMA with increasing concentrations of TGF-beta1, which was completely inhibited by the addition of a neutralizing anti-TGF-beta1 antibody. Compared with growth on plastic, cell culture on collagen-coated plates showed a threefold increase in the percentage of cells expressing alpha-SMA in response to TGF-beta1. TGF-beta1 is a key mediator that regulates, in a dose-dependent fashion, transdifferentiation of tubular epithelial cells into alpha-SMA+ myofibroblasts. This transdifferentiation is markedly enhanced by growth on collagen type I. These findings have identified a novel pathway that may contribute to renal fibrosis associated with overexpression of TGF-beta1 within the diseased kidney.
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              A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane.

              The C-terminal CAAX motif of ras proteins undergoes a triplet of posttranslational modifications that are required for membrane association. The CAAX motif lies immediately C-terminal to the hypervariable domain, a region of 20 amino acids that distinguishes the ras proteins from each other. The hypervariable domains of p21H-ras, p21N-ras, and p21K-ras(A) contain sites for palmitoylation, which we now show must combine with the CAAX motif to target specific plasma membrane localization. Within the hypervariable domain of p21K-ras(B), which is not palmitoylated, we have identified a novel plasma membrane targeting signal consisting of a polybasic domain that also acts in combination with the CAAX motif. One function of the hypervariable domains of p21ras is therefore to provide different signals for plasma membrane localization.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                April 2003
                17 November 2004
                : 93
                : 4
                : e129-e133
                Department of Renal Medicine, Guy’s King’s St. Thomas’ School of Medicine, King’s College, London, UK
                70236 Nephron Exp Nephrol 2003;93:e129–e133
                © 2003 S. Karger AG, Basel

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                Page count
                Tables: 3, References: 19, Pages: 1
                Self URI (application/pdf):

                Cardiovascular Medicine, Nephrology

                Renal disease, Rho, GTPase, Ras, Fibrosis, Gene therapy


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