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Arkadia-Smad7-mediated Positive Regulation of TGF-β Signaling in Renal Fibrosis

Author: LiXiaoZhao
Tutor: LiuFuYou
School: Central South University
Course: Internal Medicine
Keywords: Arkadia Smurf2 Smad7 TGF-β/Smad signaling transduction pathway Tubulointerstitial fibrosis Smurf2 Renal tubular epithelial cell transdifferentiation
CLC: R692
Type: PhD thesis
Year: 2007
Downloads: 400
Quote: 2
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Abstract


Background Extensive studies suggest that transforming growth factor-β1(TGF-β1) plays a significant role in the pathogenesis of tubulointerstitial fibrosis, which is implicated in tubular epithelial to mesenchymal transition(EMT). Smads are critical intracellular mediators in TGF-βsignaling pathway. Smad7 prevents or attenuates TGF-βsignaling through multiple mechanisms, including interaction with activated TGF-βtypeⅠreceptor(TβRI) and prevention of R-Smad phosphorylation. Studies reported that the expression level of Smad7 was markedly decreased in tissue and organ fibrosis, and also demonstrates that up-regulated Smad7 protein expression by transfection of the Smad7 gene in vivo and in vitro can reverse or block fibrogenesis. These studies suggest that Smad7 plays a key role in the regulation of Smad-mediated TGF-βsignaling during progression of fibrosis in various diseases.Recent studies have showed that ubiquitin-proteasome pathway has an important influence on TGF-βsignaling through regulating Smads degradation, including Smad7. In the process of degradation, E3 ubiquitin ligases, such as Smurfs and Arkadia, paly an important role. At the same time, Smad7 has been shown to function as an adaptor protein that recruits ubiquitin ligases, termed Smurfs, to the TGF-βreceptor complex to promote its degradation through proteasomal pathways. In contrast, Arkadia, one of the E3 ubiquitin ligases, degrades SmadT, but not TGF-βreceptors, resulting in amplification of TGF-βsignaling in vitro. In view of Arkadia is of relative specificity and uniqueness in degrading Smad7, the major focus of this study is to understand the role of Arkadia and Smad7 in the pathogenesis of renal tubulointerstitial fibrosis and tubular EMT.Chapter one Arkadia-Smad7-mediated positive regulation of TGF-βsignaling in a rat modal of tubulointerstitial fibrosisObjective In this study, we focused on Arkadia, Smurf2' dissertation">Smurf2, Smad7 and TβRI, principal molecules in the regulation of TGF-βsignaling, to understand the regulatory mechanism of ubiquitin-proteasomal degradation of TGF-βsignaling in the pathogenesis of renal fibrosis.Methods A unilateral ureteral obstruction(UUO) model was induced in male Sprague-Dawley rats by ligation of the left ureter as described. Sham-operated rats(n=4) were used as controls. Rats were sacrificed at 3, 7, 14, or 28 days after surgery (each group; n=6) and the obstructed kidneys were harvested and subjected to the studies. Renal lesions and the expression of Arkadia, Smurf2, Smad7, TβRI, TGF-β1 and TypeⅠcollagen(COL-1) were detected by Western blot, immunoprecipitation, immunohistochemistry, and/or RT-PCR.Results The results showed progressive tubular dilatation or atrophy, interstitial mononuclear cell infiltration and tubulointerstitial fibrosis in the obstructed kidneys of UUO rats compared with the sham-operated kidneys. These pathological changes were associated with a significant and time-dependent increase in TGF-β1 mRNA and COL-1 mRNA expression levels.Western blot showed that Arkadia, Smurf2 and TβRI were expressed weakly in the sham-operated kidneys, however, the expressions of them were gradually increased in the obstructed kidneys. Arkadia mRNA expression was also increased in the obstructed kidneys compared with the sham-operated kidneys. Immunohistochemical staining and Western blot showed that the expression level of Smad7 protein was moderate and persistent in sham-operated kidneys, and gradually but significantly decreased in a time-dependent manner in obstructed kidneys of UUO rats, but the Smad7 mRNA expression was increased in the obstructed kidneys compared with the sham-operated kidneys.Immunoprecipitation showed that the complex composed of Arkadia-Smad7 and the complex of Smurf2-Smad7 were noted in UUO kidneys and Sham-operated kidneys. In addition, the complex composed of Smurf2-TβRI was noted, however, the complex composed of Arkadia -TβRI were not detected in UUO kidneys and sham-operated kidneys.Conclusion Arkadia-Smad7-mediated positive regulation of TGF-βsignaling may contribute to the progression of renal tubulointerstitial fibrosis. Chapter two Arkadia-Smad7-mediated positive regulation of TGF-βsignaling in renal tubular epithelial to mesenchymal transitionObjective The major focus of this study is to understand the role of Arkadia and Smad7 in the pathogenesis of renal tubular epithelial to mesenchymal transition by TGF-β1 using Arkadia-siRNA and Lactacystin.Methods we treated normal human renal tubular epithelia cell(HKC) with TGF-β1 in the presence or absence of Arkadia-siRNA and Lactacystin. Then the expression of Arkadia(Arkadia mRNA), Smad7(Smad7 mRNA), Smurfs, TβRI, Col-l(Col-1 mRNA),α-SMA(α-SMA mRNA) and E-cadherin(E-cadherin mRNA) in HKC were detected by Western-blot, RT-PCR and/or immunocytochemistry. To explore the interaction of Arkadia/Smurf2 with Smad7/TβRI in HKC, Cell lysates were subjected to Smad7/TβRI immunoprecipitation, followed by Arkadia, Smurf2 and Smad7/TβRI immunoblotting. Results Arkadia and its mRNA were expressed weakly in control HKC, however, they were gradually increased in HKC stimulated with TGF-β1. Western blot showed that the expression level of Smad7 protein was moderate and persistent in control HKC and gradually but significantly decreased in a time-dependent manner in HKC stimulated with TGF-β1. In contrast to Smad7 protein, the Smad7 mRNA expression was increased in HKC stimulated with TGF-β1 compared with control HKC. Western blot showed that Smurf2 and TβRI were expressed weakly in control HKC, however, the expressions of them were gradually increased in HKC stimulated with TGF-β1.Immunoprecipitation showed that both the complex composed of Arkadia-Smad7 and the complex of Smurf2-Smad7 were noted in HKC stimulated with TGF-β1 and control HKC. In addition, the complex composed of Smurf2-TβRI was noted, however, the complex composed of TβRI and Arkadia were not detected in HKC stimulated with TGF-β1 and control HKC.Western blot and RT-PCR showed that the expression of Arkadia and its mRNA were inhibited by Arkadia-siRNA or Lactacystin. In addition, Similar to Western blot, immunocytochemistry demonstrated that Smad7 protein was increased in HKC stimulated with TGF-β1 in the presence of Arkadia-siRNA or Laetacystin compared with HKC stimulated with TGF-β1. RT-PCR showed that both Arkadia-siRNA and Lactacystin did not significantly affect the expression of Smad7 mRNA in HKC treated with TGF-β1. Similar to the control siRNA and sham plasmid, Arkadia-siRNA also did not significantly affect the expression of TβRI, but Lactacystin inhibited the expression of TβRI induced by TGF-β1 in HKC. The expression ofα-SMA and COL-1 were decreased in HKC stimulated with TGF-β1 in the presence of Arkadia-siRNA or Lactacystin compared with HKC stimulated with TGF-β1, but the expression of E-cadherin were elevated in HKC stimulated with TGF-β1 in the presence of Arkadia-siRNA or Lactacystin compared with HKC stimulated with TGF-β1.Conclusion Arkadia-Smad7-mediated positive regulation of TGF-βsignaling may contribute to renal tubular epithelial to mesenchymal transition

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease
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