Study On Phospho-Smad2/Smad3 And Smad7 Distribution In WB Rat Liver Epithelial Cell Exposed To Exogenous TGF-β₁

PrewordsLiver disease is a disease that treats very harmful to human health, Any kinds of persistent toxic agents can result in different hepatic pathologic process including hepatic damage, liver fibrosis, and even so canceration. It should be noted that liver fibrosis is the critical step of this process. TGF - β is a critical promoting cytokine to this process, However, the effect of TGF -β on hepatic stellate cell ( HSC) signaling pathway depend on activation of membrane receptor and downstream molecular Smad, then activate HSC signaling pathway. A-mong Smad protein family, Smad2 , 3 , 4 and 7 are involved in TGF - β signaling . The transcription regulation depend on transduction of Smad from the cytoplasma to the nuclear, which make the synthesis of extracellular matrix increased and its degradation decreased. HSC plays an important roll in liver fibrosis. So far, it is not clear of TGF - β signaling pathway in the other nonparenchymal cell. This study explore exogenous TGF - β₁ administration to WB rat liver epithelial cell, and check the distribution change of phosphorated - Smad2/3 and Smad7 in the nuclear and the cytoplasma, further accounting for the relationship beteen Smad and TGF - β in nonparenchymal cell. This study also aim to investigate whether TGF - β₁ activate target gene transcription depending on Smad , and its promoting function in the process of liver fibrosis.Materials & MethodsThe subcultured WB rat liver epithelial cell was treated with exogenousTGF -β₁ (5ng/ml) , and collected cells in different treatment time, followed by making suitable samples for electromicroscrope and immuno - colloidal gold staining. The distribution change of P - Smad2/3 and Smad7 in the nuclear and the cytoplasma of WB rat liver epithelial cell were observed under the electromicroscrope. On the other hand, the cells were administrated to different concentration TGF - β₁ ( 0ng/ml, 2. 5ng/ml, 5ng/ml, 7. 5ng/ml and 10ng/ml) , and collected after treatment TGF - β₁ 8 h, then repeated above manipulation. At last the distribution changes of P - Smad2/3 and Smad7 in the nuclear and the cytoplasma were observed utilizing electromicroscorpe.Result1. After treatment with TGF - β₁ (5ng/ml) , It shows that the expression of P - Smad2/3 in different group were increased whether in the nuclear or in the cytoplasma along with extending treatment time, presenting a tendency of trans-duction into the nuclear. After 8h treatment with TGF - β₁, it present a peak expression of P - Smad2/3 in the nuclear, and decrease after administration 24 h. There are some colloidal gold particles of Smad7 in the nuclear and the cytoplasma before TGF — β₁ treatment, that mostly centralizing in the nuclear. After 4h treatment with TGF - β₁, Smad7 particles mostly centralized in the cytoplasma , while they decreased after 8h treatment compare to 4 h in the nuclear and the cytoplasma. After 24h treatment with TGF - β₁, only small amount of particles were observed.2. After treatment with different concentration of TGF - β₁ (0ng/ml, 2. 5ng/ml, 5ng/ml,7. 5ng/ml and 10ng/ml) , It shows that WB cell present some sporadic particles of P - Smad2/3 in the TGF - β₁ (0ng/ ml) group, whereas in the TGF - β₁ (2. 5ng/ ml) group ,more particles than TGF - β₁ (0ng/ ml) were found, especially in the nuclear. After stimulation with TGF - β₁ (5ng/ ml) for 8 h, increasing particles of P - Smad2/3 in the nuclear were observed, no stat-isfic significants, we also find no difference between TGF - β₁ (7. 5ng/ ml) group and TGF - β₁(5ng/ ml) group. When administrated to TGF - β₁ (10ng/ ml) , we also find that the expression of P - Smad2/3 is no significant differencecompare to TGF -β₁ (7. 5ng/ml) group. At the same time, we observed the change of Smad 7 in the WB cell after different concentration TGF - β₁ treatment. The data show that there are some sporadic particles of Smad7 before TGF - β₁ administration , that mostly centralizing in the nuclear. In TGF - β₁ 2. 5ng/ml,5ng/ml ,7.5ng/ml and 10ng/ml group ,the epression of Smad7 shows no significant difference.3. After extension of treatment of TGF - β₁, ratio of dysplastic type nuclear cell increased were found.DiscussionIt were well known that TGF - β is one of very important factors for formation and development of liver fibrosis in the molecular level. Currently TGF - β₁ is arch - cytokine in process of fibrosis, Majority of researchers consider HSC as an important cell for this process, whereas TGF - β carry out its function through Smad signaling pathway and then activate HSC proliferation gene. In this big family protein , there are Smad 2,3,4 and 7 involving in TGF - β signaling pathway.In inactivation normal cell Smad2 and Smad3 exist in the cytoplasma, whereas after exogenous stimulation( for example,TGF - β₁) , TGF - β preceptor I (TβR I ) activate Smad2 and Smad3, followed by phosphorylated Smad2 and Smad 3 combining with C - Smads (Smad4) , then shuttling into the nuclear and regulating target gene transcription and expression. At last this signaling system increase matrix expression. In this whole process there is a important step should be noted that is shuttling between the nuclear and the cytoplasma of Smads. In the nuclear, P - Smads is intimate with target gene transcription, and R - Smad can promote development of liver fibrosis. Smad7 can inhibit R - Smad function in the initial stage, but it lose its negative feedback function in the advanced stage of liver fibrosis.Currently, mostly study about expression of P - Smad2/3 and 7 centralize in HSC, whereas there is little knowledge about expression of P - Smad2/3 and 7 in other liver unparenchymal cell after TGF - β treatment except HSC. thisstudy is intensive in check the distribution and expression change of P - Smad2/ 3 and 7 in the nuclear and the cytoplasma of WB rat liver epithelial cell after exogenous TGF - β₁ administration by the method of electromicroscorpe, and provide a direct proof for the issue that TGF - β can induce liver fibrosis.WB rat liver epithelial cell were treated with exogenous TGF - β₁ (5ng/ml) , and the changes of P — Smad 2/3 in the nuclear and the cytoplasma at different dosage and treatment time were investigated . The data show that P - Smad 2/3 can be observed in the nuclear and the cytoplasma before TGF — β₁, treatment. The reason maybe caused by autocrine of cell or other Smads activation pathway independent of TGF - β. We also find that expression of P - Smad2/3 increase in the nuclear and the cytoplasma , and there is an tendency of P - Smad2/3 transduction into the nuclear. After administration 8h with TGF — β₁, P -Smad2/3 get to peak expression in the nuclear. This data is similar to western blotting results documented by Chenghai Liu et al. Exogenous TGF - β₁ can make P - Smad2/Smad3 transduct into the nuclear in activation HSC. It should be noted that our data not only proof this point but also find a direct evidence for illuminating the change of expression and distribution of P - Smad2/3 after TGF- β₁ stimulation.Previous study suggested that the expression of TGF - β is positive correlation to the expression of Smad3, our data show that there is no relationship be-teen TGF -β and P — Smad2/3 epression in WB rat liver epithelial cell, which further suggest that TGF - β signaling pathway in the WB cell is independent of concentration of TGF - β₁. The intensive mechanism need further study.Smad7 carry out a negative feedback function to TGF - p/Smad signaling pathway. When TGF - preceptor I is activated , it also activate Smad7 ( Inhibition Smad) companying with phosphorylating R - Smad. Smad7 can inhibit TGF- β receptor I mediated phosphorylation of Smad2/3 and promote TGF -β receptor I self - degradation of ubiqutin - mediated and further impair TGF -β signaling . Because Smad7 is a special endogenous inhibition protein of TGF -β, we co - culture WB cell and exogenous TGF -β₁ , and observe the distribution changes of Smad7 and cell apoptosis. Before TGF - β₁ was administrated, the original expression of Smad7 in the nuclear and the cytoplasma was found,which meaning Smad7 involved in normal physiological functions, and at the same time this kind of expression of Smad7 dependent on autocrine TGF - β₁ induced. After 4h treatment with TGF - β₁, Smad7 present a transduction into cytoplasma tendency and decrease in the nuclear and the cytoplasma after 8h and 24 h administration. This phenomena suggest that TGF - β₁ can induce upregula-tion of Smad7 along with phosphorylation R - Smad, but this tenuity upregula-tion cann' t antagonize TGF - β induced phosphorylation of R - Smad . In addition, this data also show that the expression of Smad7 is independent of the quantity of TGF -β₁ , which suggest that between TGF - β₁ and its feedback circle molecular Smad7 exist a kind of balance mechanism, and this is dependent of treatment time but independent of TGF - β₁ concentration. On the other hand, we also find that there is increasing ratio of dysplastic type nuclear of these cells under electromicroscrope in normal culture condition after 8h TGF - β₁ treatment. At the same time, we also observed these cell present chromosome edge concentration. These phenomena maybe relate to apoptosis of hepatocyte induced by TGF - β₁ and transformation tendency from liver fibrosis to liver cancer.Our study showed that WB rat liver epithelial cell effect on process of liver fibrosis and involved into TGF - β signaling pathway, however, whether WB cell play assistant role in HSC induced liver fibrosis need further study.In conclusion, more and more data show TGF - β signaling pathway close relate with liver fibrosis.Conclution1. P - Smad 2/3 present a transduction into the nuclear tendency by exogenous TGF - β₁ treatment, After administration TGF - β₁ for 8h, P -Smad2/3 get to peak expression in the nuclear, the expression of p -Smad2/3 in the nuclear decrease in 24h.2. Smad7 present a transduction into the cytoplasma tendency by exogenous TGF - β₁ treatment, and decrease in the nuclear and the cytoplasma after 8h and 24h TGF - β₁ treatment.