Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR
Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR
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DOI：
10.1016/j.cellsig.2009.07.016
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发表时间：
2009-12-01
影响因子：
4.8
通讯作者：
Eberhardt, Wolfgang
中科院分区：
文献类型：
--
作者：
Osman, Bashier;Doller, Anke;Eberhardt, Wolfgang
mTOR激酶抑制剂雷帕霉素（西罗莫司）是一种具有有效免疫抑制和抗增殖特性的药物。我们发现雷帕霉素在大鼠系膜细胞（MC）中诱导TGF β /Smad信号级联，分别通过磷酸化Smad- 2、-3和Smad-4的核易位来描述。同时，雷帕霉素增加了受体(R)-和co-Smad蛋白与同源smad结合元件（SBE）的核DNA结合，从而导致纤维化基因表达增加，如结缔组织生长因子（CTGF）和纤溶酶原激活物抑制剂1 （PAI-1）。利用小干扰（si）RNA，我们证明了Smad 2/3被雷帕霉素激活依赖于其内源性受体FK结合蛋白12 （FKBP12）。从机制上说，雷帕霉素诱导Smad是由活性TGF β的增加(1)和中和TGF β抗体的抑制作用启动的。通过激活素受体样激酶(ALK)-5抑制剂和siRNA对TGF β II型受体（TGF β - rii）的抑制，我们进一步证明了两种类型的TGF β受体的功能参与。然而，雷帕霉素并不像放射配体结合实验中发现的那样与TGF β受体结合。除了p38 MAPK的特异性抑制剂SB203580外，活性氧（ROS）清除剂n -乙酰半胱氨酸（NAC）和细胞渗透性超氧化物歧化酶（SOD）模拟物强烈地消除了雷帕霉素对Smad 2和3磷酸化的刺激作用。此外，二氯荧光素（DCF）形成的快速增加表明雷帕霉素主要通过ROS起作用。综上所述，促纤维化TGF β /Smad信号级联的激活伴随着雷帕霉素的免疫抑制和抗增殖作用。(C) 2009爱思唯尔公司版权所有。
The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and anti proliferative properties. We found that rapamycin induces the TGF beta/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGF beta(1) as shown by ELISA and by the inhibitory effects of a neutralizing TGF beta antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGF beta type II receptor (TGF beta-RII) we furthermore demonstrate a functional involvement of both types of TGF beta receptors. However, rapamycin did not compete with TGF beta for TGF beta-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGF beta/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin. (C) 2009 Elsevier Inc. All rights reserved.