Hamzavi Jafar, Ehnert Sabrina, Godoy Patricio, Ciuclan Loredana, Weng Honglei, Mertens Peter R, Heuchel Rainer, Dooley Steven
Molecular Alcohol Research in Gastroenterology, Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Germany.
J Cell Mol Med. 2008 Oct;12(5B):2130-44. doi: 10.1111/j.1582-4934.2008.00262.x. Epub 2008 Feb 4.
Transforming growth factor-beta (TGF-beta) signalling is induced in liver as a consequence of damage and contributes to wound healing with transient activation, whereas it mediates fibrogenesis with long-term up-regulation in chronic disease. Smad-dependent TGF-beta effects are blunted by antagonistic Smad7, which is transcriptionally activated as an immediate early response upon initiation of TGF-beta signalling in most cell types, thereby providing negative feedback regulation. Smad7 can be induced by other cytokines, e.g. IFN-gamma, leading to a crosstalk of these signalling pathways. Here we report on a novel mouse strain, denoted S7DeltaE1, with a deletion of exon I from the endogenous smad7 gene. The mice were viable and exhibited normal adult liver architecture. To obtain insight into Smad7-depend-ent protective effects, chronic liver damage was induced in mice by carbon tetrachloride (CCI4) administration. Subsequent treatment, elevated serum liver enzymes indicated enhanced liver damage in mice lacking functional Smad7. CCI4-dependent Smad2 phosphorylation was pronounced in S7DeltaE1 mice and accompanied by increased numbers of alpha-smooth muscle actin positive 'activated' HSCs. There was evidence for matrix accumulation, with elevated collagen deposition as assessed morphometrically in Sirius red stained tissue and confirmed with higher levels of hydroxyproline in S7DeltaE1 mice. In addition, the number of CD43 positive infiltrating lymphocytes as well as of apoptotic hepatocytes was increased. Studies with primary hepatocytes from S7DeltaE1 and wild-type mice indicate that in the absence of functional Smad7 protein, hepatocytes are more sensitive for TGF-beta effects resulting in enhanced cell death. Furthermore, S7DeltaE1 hepatocytes display increased oxidative stress and cell damage in response to CCI4, as measured by reactive oxygen species production, glutathione depletion, lactate dehydrogenase release and lipid peroxidation. Using an ALK-5 inhibitor all investigated CCI4 effects on hepatocytes were blunted, confirming participation of TGF-beta signalling. We conclude that Smad7 mediates a protective effect from adverse TGF-beta signalling in damaged liver, re-iterating its negative regulatory loop on signalling.
转化生长因子-β（TGF-β）信号通路在肝脏受损时被激活，在损伤修复过程中短暂激活以促进伤口愈合，而在慢性疾病中则通过长期上调介导肝纤维化。拮抗蛋白Smad7可抑制Smad依赖的TGF-β效应，在大多数细胞类型中，TGF-β信号通路启动后，Smad7作为即时早期反应被转录激活，从而提供负反馈调节。Smad7可被其他细胞因子如IFN-γ诱导，导致这些信号通路之间发生串扰。在此，我们报道了一种新型小鼠品系，命名为S7DeltaE1，其内源smad7基因的外显子I缺失。这些小鼠存活且成年肝脏结构正常。为深入了解Smad7依赖性保护作用，通过给予四氯化碳（CCl4）诱导小鼠慢性肝损伤。后续治疗发现，缺乏功能性Smad7的小鼠血清肝酶升高，表明肝损伤加剧。在S7DeltaE1小鼠中，CCl4依赖性的Smad2磷酸化明显，同时α-平滑肌肌动蛋白阳性的“活化”肝星状细胞数量增加。有证据表明存在基质积聚，天狼星红染色组织形态计量分析显示胶原沉积增加，S7DeltaE1小鼠中羟脯氨酸水平升高也证实了这一点。此外，CD43阳性浸润淋巴细胞以及凋亡肝细胞的数量增加。对S7DeltaE1和野生型小鼠原代肝细胞的研究表明，在缺乏功能性Smad7蛋白的情况下，肝细胞对TGF-β效应更敏感，导致细胞死亡增加。此外，通过活性氧生成、谷胱甘肽消耗、乳酸脱氢酶释放和脂质过氧化检测发现，S7DeltaE1肝细胞对CCl4的反应表现为氧化应激增加和细胞损伤。使用ALK-5抑制剂后，所有研究的CCl4对肝细胞的影响均被减弱，证实了TGF-β信号通路的参与。我们得出结论，Smad7在受损肝脏中介导了对不良TGF-β信号的保护作用，再次强调了其在信号通路中的负调节作用。
