Sun Yi-Min, Wu Yu, Li Gan-Xun, Liang Hui-Fang, Yong Tu-Ying, Li Zifu, Zhang Bixiang, Chen Xiao-Ping, Jin Guan-Nan, Ding Ze-Yang
Hepatic Surgery Center, Hubei Province for The Clinical Medicine Research Center of Hepatic Surgery and Hubei Key Laboratory of Hepatic-Biliary-Pancreatic Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
Present address: Department of Gastrointestinal Surgery, Affiliated First Hospital, Yangtze University, Jingzhou, Hubei 434000, China.
Aging (Albany NY). 2024 Apr 5;16(7):6588-6612. doi: 10.18632/aging.205725.
Liver progenitor cells (LPCs) are a subpopulation of cells that contribute to liver regeneration, fibrosis and liver cancer initiation under different circumstances.
By performing adenoviral-mediated transfection, CCK-8 analyses, F-actin staining, transwell analyses, luciferase reporter analyses and Western blotting, we observed that TGF-β promoted cytostasis and partial epithelial-mesenchymal transition (EMT) in LPCs. In addition, we confirmed that TGF-β activated the Smad and MAPK pathways, including the Erk, JNK and p38 MAPK signaling pathways, and revealed that TGFβ-Smad signaling induced growth inhibition and partial EMT, whereas TGFβ-MAPK signaling had the opposite effects on LPCs. We further found that the activity of Smad and MAPK signaling downstream of TGF-β was mutually restricted in LPCs. Mechanistically, we found that TGF-β activated Smad signaling through serine phosphorylation of both the C-terminal and linker regions of Smad2 and 3 in LPCs. Additionally, TGFβ-MAPK signaling inhibited the phosphorylation of Smad3 but not Smad2 at the C-terminus, and it reinforced the linker phosphorylation of Smad3 at T179 and S213. We then found that overexpression of mutated Smad3 at linker phosphorylation sites intensifies TGF-β-induced cytostasis and EMT, mimicking the effects of MAPK inhibition in LPCs, whereas mutation of Smad3 at the C-terminus caused LPCs to blunt TGF-β-induced cytostasis and partial EMT.
These results suggested that TGF-β downstream of Smad3 and MAPK signaling were mutually antagonistic in regulating the viability and partial EMT of LPCs. This antagonism may help LPCs overcome the cytostatic effect of TGF-β under fibrotic conditions and maintain partial EMT and progenitor phenotypes.
肝祖细胞（LPCs）是一类细胞亚群，在不同情况下对肝脏再生、纤维化及肝癌起始发挥作用。
通过腺病毒介导的转染、CCK-8分析、F-肌动蛋白染色、Transwell分析、荧光素酶报告基因分析及蛋白质免疫印迹法，我们观察到转化生长因子-β（TGF-β）促进LPCs的细胞生长停滞及部分上皮-间质转化（EMT）。此外，我们证实TGF-β激活了Smad和丝裂原活化蛋白激酶（MAPK）信号通路，包括细胞外信号调节激酶（Erk）、c-Jun氨基末端激酶（JNK）和p38 MAPK信号通路，并揭示TGFβ-Smad信号诱导生长抑制和部分EMT，而TGFβ-MAPK信号对LPCs具有相反作用。我们进一步发现，TGF-β下游的Smad和MAPK信号活性在LPCs中相互限制。机制上，我们发现TGF-β通过LPCs中Smad2和Smad3的C末端及连接区的丝氨酸磷酸化激活Smad信号。此外，TGFβ-MAPK信号抑制Smad3的C末端磷酸化，但不抑制Smad2的C末端磷酸化，并且增强Smad3在T179和S213处的连接区磷酸化。然后我们发现，连接区磷酸化位点的突变型Smad3过表达增强了TGF-β诱导的细胞生长停滞和EMT，模拟了LPCs中MAPK抑制的作用，而Smad3的C末端突变使LPCs对TGF-β诱导的细胞生长停滞和部分EMT反应减弱。
这些结果表明，Smad3和MAPK信号下游的TGF-β在调节LPCs的活力和部分EMT方面相互拮抗。这种拮抗作用可能有助于LPCs在纤维化条件下克服TGF-β的细胞生长停滞作用，并维持部分EMT和祖细胞表型。
