Patel Sharmila, Takagi Ken-ichro, Suzuki Jun, Imaizumi Atsushi, Kimura Tsuyoshi, Mason Roger M, Kamimura Takashi, Zhang Zhi
Teijin Biomedical Laboratory, Medical Research Council Technology, 1-3 Burtonhole Lane, London, NW7 1AD, UK.
J Am Soc Nephrol. 2005 Jul;16(7):1977-84. doi: 10.1681/ASN.2004110943. Epub 2005 May 18.
ESRD is characterized by an interstitial infiltrate of inflammatory cells in association with tubular atrophy, epithelial mesenchymal transdifferentiation (EMT), and interstitial fibrosis. Human proximal tubular epithelial cells (HK2 cells) undergo EMT in response to activated PBMC conditioned medium (aPBMC-CM), showing acquisition of a fibroblastoid morphology, increased fibronectin-EDA (EDA) expression, loss of junctional E-cadherin localization, and cytokeratin 19 (K19) expression. The signaling pathway(s) that regulates EMT in response to aPBMC-CM is not well understood. This study shows that aPBMC-CM induces a rapid activation of RhoA, Rac1, and Cdc42 activity in HK2 cells from 15 min to 48 h. Moreover, infection with adenovirus expressing constitutively active RhoA, Rac1, and Cdc42 significantly increased the expression of EDA and downregulated expression of E-cadherin and K19. Dominant negative RhoA expression suppressed aPBMC-CM-induced upregulation of EDA but did not restore the expression of E-cadherin and K19. Constitutively active RhoA activated the Rho kinase and its downstream effectors, whereas constitutively active Rac1 and Cdc42 activated the P21-activated protein kinase in epithelial cells. In further experiments, HK2 cells were treated with toxin B, exoenzyme C3, Y-27632, and HA1077. These strategies, inhibiting the Rho/Rho kinase pathway, as well as the Rac1/Cdc42/P21-activated protein kinase pathway, blocked transdifferentiation of HK2 cells in response to aPBMC-CM. To conclude, these results indicate that aPBMC-CM activates RhoA, Rac1, and Cdc42 and their downstream effectors, leading to HK2 cells undergoing transdifferentiation. Therefore, activation of small RhoGTPases is a key step in the mechanism of EMT and likely to be a contributor to tubulointerstitial fibrosis.
终末期肾病（ESRD）的特征是炎症细胞间质浸润，并伴有肾小管萎缩、上皮-间充质转分化（EMT）和间质纤维化。人近端肾小管上皮细胞（HK2细胞）在活化的外周血单个核细胞条件培养基（aPBMC-CM）作用下发生EMT，表现为获得成纤维细胞样形态、纤连蛋白-EDA（EDA）表达增加、连接蛋白E-钙黏蛋白定位丧失以及细胞角蛋白19（K19）表达丧失。调控HK2细胞对aPBMC-CM反应性EMT的信号通路尚未完全明确。本研究表明，aPBMC-CM在15分钟至48小时内可诱导HK2细胞中RhoA、Rac1和Cdc42活性快速激活。此外，用表达组成型活性RhoA、Rac1和Cdc42的腺病毒感染显著增加了EDA的表达，并下调了E-钙黏蛋白和K19的表达。显性负性RhoA表达抑制了aPBMC-CM诱导的EDA上调，但未恢复E-钙黏蛋白和K19的表达。组成型活性RhoA激活了Rho激酶及其下游效应器，而组成型活性Rac1和Cdc42激活了上皮细胞中的P21活化蛋白激酶。在进一步实验中，HK2细胞用毒素B、外切酶C3、Y-27632和HA1077处理。这些抑制Rho/Rho激酶通路以及Rac1/Cdc42/P21活化蛋白激酶通路的策略，阻断了HK2细胞对aPBMC-CM的转分化。总之，这些结果表明，aPBMC-CM激活了RhoA、Rac1和Cdc42及其下游效应器，导致HK2细胞发生转分化。因此，小RhoGTP酶的激活是EMT机制中的关键步骤，可能是肾小管间质纤维化的一个促成因素。
