BMP-7 fails to attenuate TGF-β1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells
BMP-7 fails to attenuate TGF-β1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells
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DOI：
10.1093/ndt/gfn662
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发表时间：
2009-05-01
影响因子：
6.1
通讯作者：
Farrell, Francis X.
中科院分区：
文献类型：
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作者：
Dudas, Paul L.;Argentieri, Rochelle L.;Farrell, Francis X.
背景在慢性肾脏疾病的啮齿动物模型中，骨形态发生蛋白-7（BMP-7）已被证明可以阻止疾病进展并促进恢复。随后利用永生化啮齿动物肾细胞系的研究表明，BMP-7通过拮抗TGF-β 1刺激的上皮细胞向间充质细胞转化（EMT）而具有肾脏保护作用。本研究试图确定BMP-7是否阻止TGF-β 1诱导的原代（RPTEC）和永生化（HK-2）人近端小管上皮细胞的EMT。通过实时荧光定量PCR分析TGF-β 1处理后RPTEC和HK-2细胞中e-cadherin、vimentin、CTGF和TGF-β 1转录本的表达，以及免疫细胞化学分析ZO-1和α-平滑肌肌动蛋白（α-SMA）蛋白的表达来确定EMT。在RPTEC和HK-2细胞中，TGF-β 1显著降低e-cadherin表达，显著增加波形蛋白、CTGF和TGF-β 1表达。TGF-β 1还减少ZO-1免疫反应性，并增加融合细胞单层中的α-SMA表达。TGF-β 1与抗TGF-β 1中和抗体的共孵育显著降低了细胞因子的作用，这表明这些细胞中的EMT是可逆转的。在RPTEC或HK-2细胞中，BMP-7与TGF-β 1在较宽的浓度范围（0.01-100 μ g/ml）内的共同给药不能减弱EMT，如通过改变的e-钙粘蛋白、波形蛋白、CTGF和TGF-β 1表达没有抑制以及ZO-1免疫反应性没有恢复所证明的。此外，当BMP-7单独应用于近端小管细胞时，它还降低了e-钙粘蛋白的表达，并增加了波形蛋白、CTGF和TGF-β 1的表达。此外，BMP-7未能诱导NRK-49 F大鼠肾成纤维细胞中的间充质-上皮转化（MET）。然而，BMP-7确实阻止了TCMK-1小鼠肾小管上皮细胞中TGF-β 1介导的e-cadherin下调。通过检测BMP-7诱导的SMADs 1/5/8的磷酸化、BMP-7对BMPR-IA的调节、BMP-7介导的IL-6转录表达的减少以及BMP-7介导的分泌的IL-6和IL-8蛋白的减少来常规地证实BMP-7活性。在本研究中，尽管证实了BMP-7调节受体表达和诱导下游信号传导事件，但我们无法证明BMP-7抑制原代或永生化人近端小管细胞中的EMT。此外，我们不能证明BMP-7刺激MET在大鼠肾成纤维细胞。然而，在小鼠肾小管上皮细胞中，在升高的BMP-7浓度下观察到保护作用。
Background. In rodent models of chronic renal disease bone morphogenetic protein-7 (BMP-7) has been shown to halt disease progression and promote recovery. Subsequent studies utilizing immortalized rodent renal cell lines showed that BMP-7 was renoprotective by antagonizing TGF-beta 1-stimulated epithelial-to-mesenchymal transition (EMT). The present study sought to determine if BMP-7 prevents TGF-beta 1-induced EMT in primary (RPTEC) and immortalized (HK-2) human proximal tubule epithelial cells.Methods. EMT was determined by quantitative real-time PCR analysis of e-cadherin, vimentin, CTGF and TGF-beta 1 transcript expression and immunocytochemical analysis of ZO-1 and alpha-smooth muscle actin (alpha-SMA) protein expression following TGF-beta 1 treatment in RPTEC and HK-2 cells.Results. In RPTEC and HK-2 cells, TGF-beta 1 significantly reduced e-cadherin expression and significantly increased vimentin, CTGF and TGF-beta 1 expression. TGF-beta 1 also diminished ZO-1 immunoreactivity and increased alpha-SMA expression in confluent cell monolayers. Co-incubation of TGF-beta 1 with an anti-TGF-beta 1 neutralizing antibody substantially reduced the cytokine's effects, which indicated EMT in these cells was inhibitable. Co-administration of BMP-7 over a broad concentration range (0.01-100 mu g/ml) with TGF-beta 1 failed to attenuate EMT in RPTEC or HK-2 cells, as demonstrated by no inhibition of altered e-cadherin, vimentin, CTGF and TGF-beta 1 expression and no restoration of ZO-1 immunoreactivity. Furthermore, when BMP-7 was applied to proximal tubule cells alone, it also decreased e-cadherin expression and increased vimentin, CTGF and TGF-beta 1 expression. Additionally, BMP-7 failed to induce the mesenchymal-to-epithelial transition (MET) in NRK-49F rat renal fibroblasts. BMP-7 did however prevent TGF-beta 1-mediated e-cadherin downregulation in TCMK-1 mouse renal tubular epithelial cells. BMP-7 activity was routinely confirmed by examining BMP-7-induced phosphorylation of SMADs 1/5/8, BMP-7 regulation of BMPR-IA, BMP-7-mediated reduction of IL-6 transcript expression and BMP-7-mediated reduction of secreted IL-6 and IL-8 proteins.Conclusions. In the present study, despite confirming BMP-7 regulation of receptor expression and induction of downstream signalling events, we were unable to demonstrate BMP-7 inhibition of EMT in either primary or immortalized human proximal tubule cells. Moreover, we were unable to demonstrate BMP-7-stimulated MET in rat renal fibroblasts. A protective effect was however observed at an elevated BMP-7 concentration in mouse renal tubular epithelial cells.