HMGA2 调节人晶状体上皮-间充质转化中的 TGFβ/Smad、TGFβ/ERK 和 Notch 信号通路。

HMGA2 Modulates the TGFβ/Smad, TGFβ/ERK and Notch Signaling Pathways in Human Lens Epithelial-Mesenchymal Transition.

机构信息

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.

Department of Ophthalmology, Ophthalmology & Optometry Center, Peking University People's Hospital, Beijing Key Laboratory of Diagnosis and Therapy of Retinal and Choroid Diseases, Beijing 100044, China.

出版信息

Curr Mol Med. 2018;18(2):71-82. doi: 10.2174/1566524018666180705104844.

DOI:10.2174/1566524018666180705104844

PMID:29974827

Abstract

BACKGROUND AND OBJECTIVE

Multiple signaling pathways coordinately promote epithelial-mesenchymal transition (EMT) in lens epithelial cells (LECs), where transforming growth factor beta (TGFβ)-mediated signaling plays a central role. But the mechanism of crosstalk among these pathways remains obscure. The objective of this study is to investigate the regulatory effect of the high mobility group protein A2 (HMGA2) on the signaling pathways in lens fibrosis.

METHODS

The human anterior capsulorhexis specimens were collected. The human SRA01/04 LEC line was cultured and treated with recombinant human TGFβ2 (5ng/ml). For inhibition of signaling pathways, a selective inhibitor SB431542, U0126 or DAPT was added to LECs respectively. The specific small interfering RNA (siRNA) were transfected to LECs for gene silence. The mRNAs expressions were measured by realtime PCR and the proteins expressions were determined by western blot and immunofluorescent staining.

RESULTS

HMGA2 and EMT markers α-smooth muscle actin (SMA), fibronectin (FN) and collagen type I (Col I) were overexpressed in human ASC specimens and TGFβ2 stimulated EMT in LECs. While blockage of EMT by a selective inhibitor of TGFβ/Smad, TGFβ/extracellular signal-regulated kinase (ERK) or Notch signaling pathway could significantly inhibited HMGA2 protein expression. And silence of HMGA2 by siRNA could significantly inhibit TGFβ2 induced expression of EMT markers including FN, Col I, collagen type IV (Col IV), key transcription factors Snail and Slug, and remarkably upregulate the epithelial markers E-cadherin and tight junction protein (ZO-1). In addition, silence of HMGA2 gene could abrogate TGFβ2 induced phosphorylation of Smad2, Smad3 as well as ERK1/2. Blockage of HMGA2 could also inhibit the upregulation of Jagged1, Notch2, and Notch3 induced by TGFβ2.

CONCLUSION

This study indicated that HMGA2 functions as a shared effector in TGFβ2- induced lens fibrosis, modulating the signaling network necessary for EMT in a positive feedback loop.

摘要

背景与目的

多种信号通路协同促进晶状体上皮细胞（LEC）中的上皮间质转化（EMT），其中转化生长因子β（TGFβ）介导的信号转导起着核心作用。但是这些途径之间的串扰机制尚不清楚。本研究旨在探讨高迁移率族蛋白 A2（HMGA2）对晶状体纤维化信号通路的调节作用。

方法

收集人前囊膜撕裂标本。培养人 SRA01/04 LEC 细胞系，并给予重组人 TGFβ2（5ng/ml）处理。为了抑制信号通路，分别向 LEC 中加入选择性抑制剂 SB431542、U0126 或 DAPT。转染特异性小干扰 RNA（siRNA）对 LEC 进行基因沉默。通过实时 PCR 测量 mRNA 表达，通过 Western blot 和免疫荧光染色测定蛋白表达。

结果

HMGA2 和 EMT 标志物 α-平滑肌肌动蛋白（SMA）、纤维连接蛋白（FN）和 I 型胶原（Col I）在人 ASC 标本中过度表达，TGFβ2 刺激 LEC 中的 EMT。而通过 TGFβ/Smad、TGFβ/细胞外信号调节激酶（ERK）或 Notch 信号通路的选择性抑制剂阻断 EMT，可显著抑制 HMGA2 蛋白表达。通过 siRNA 沉默 HMGA2 可显著抑制 TGFβ2 诱导的 EMT 标志物包括 FN、Col I、IV 型胶原（Col IV）、关键转录因子 Snail 和 Slug 的表达，并显著上调上皮标志物 E-钙粘蛋白和紧密连接蛋白（ZO-1）。此外，沉默 HMGA2 基因可阻断 TGFβ2 诱导的 Smad2、Smad3 及 ERK1/2 的磷酸化。抑制 HMGA2 还可以抑制 TGFβ2 诱导的 Jagged1、Notch2 和 Notch3 的上调。