TGF-β1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation
TGF-β1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation
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DOI：
10.1152/ajprenal.00406.2005
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发表时间：
2006-05-01
影响因子：
4.2
通讯作者：
Watson, RWG
中科院分区：
文献类型：
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作者：
Docherty, NG;O'Sullivan, OE;Watson, RWG
肾小管上皮细胞凋亡和上皮-间质转分化（EMT）发生在应激的肾小管上皮细胞，并有助于肾纤维化。转化生长因子（TGF）-β（1）促进这些反应，我们在体外研究了这些过程是否相互依赖。直接（半胱天冬酶抑制）和间接[表皮生长因子（EGF）受体刺激]策略被用来阻断TGF-β（1）刺激期间的细胞凋亡，并评估对EMT的后续影响。将HK-2细胞暴露于TGF-β（1），同时或不与ZVAD-FMK（泛半胱天冬酶抑制剂）预孵育，或同时用EGF加或减与LY-294002（PI 3-激酶抑制剂）预孵育。然后通过流式细胞术、结晶紫测定和蛋白质印迹法评估细胞的凋亡和增殖。通过显微镜、免疫荧光、实时RT-PCR、Western blotting、派-1报告基因测定和胶原凝胶收缩试验评估EMT标志物。TGF-β（1）引起细胞凋亡并引发星形孢菌素诱导的细胞凋亡。这被ZVAD-FMK阻止。然而，ZVAD-FMK不能预防TGF-β 1治疗后的EMT。EGF通过增加细胞增殖和E-cadherin丢失抑制细胞凋亡并促进TGF-β 1诱导EMT。此外，EGF显着增强TGF-β（1）诱导的I型胶原凝胶收缩。EGF在EMT期间增加Akt磷酸化，并且使用LY-294002证实了这一促生存作用，LY-294002减少EGF诱导的Akt磷酸化并逆转其抗凋亡和促增殖作用。TGF-β（1）诱导EMT独立于其促凋亡作用。TGF-β（1）和EGF共同导致EMT。EGF以PI 3-K Akt依赖的方式增加EMT期间的增殖和对凋亡的抵抗。在体内，EGF受体活化可能有助于转分化的促纤维化细胞类型的选择性存活。
Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)-beta(1) promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [ epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF-beta(1) stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF-beta(1) with or without preincubation with ZVAD-FMK (pan-caspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF-beta(1) caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF-beta(1) treatment. EGF inhibited apoptosis and facilitated TGF-beta(1) induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF-beta(1)-induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF-beta(1) induces EMT independently of its proapoptotic effects. TGF-beta(1) and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type.