Effects Of Targeting β-arrestin2 On Extracellular Matrix Synthesis In Hepatic Stellate Cells

Hepatic fibrosis is a pathologic process that leads to deposition of an excess of extracellular matrix (ECM), and hepatic stellate cell (HSC) is a major fibrogenic cell type that contributes to collagen accumulation by producing ECM. Transforming growth factor β1 (TGF-β1) is a strongly promoter of activation of HSC and synthesis of ECM such as Collagen I and Collagen Ⅲ. The TGF-β receptor type Ⅲ (TβRⅢ) acts as co-receptor to regulate the TGF-β downstream signaling pathways. β-arrestin2 is adaptor protein and signal transduction protein belong to arrestin family, which is well known for negatively regulating G-protein-coupled receptors (GPCRs) signaling and participating in receptor desensitization and internalization. Many researches found that β-arrestin2 plays an important role in fibrotic diseases. Based on the previous researches, the present study was designed to investigate the role of β-arrestin2 in ECM production of hepatic fibrosis and the possibility mechanism. The expressions of β-arrestin2, TβRⅢ and collagen proteins were detected in porcine serum (PS)-induced liver fibrosis rats in vivo. Additionally, the expression of β-arrestin2, TβRⅢ and collagen expression and related signaling pathways were detected in TGF-β1 stimulated HSC in vitro. Furthermore, the small interfering RNA (siRNA) of TβRⅢ and β-arrestin2 was transfected in HSC respectively, detected the expression of β-arrestin2, TβRⅢ, collagen and the activation of signaling pathways, laser confocal microscopy and co-immunoprecipitation were measured to investigate the relationship between β-arrestin2, TβRIII in HSC.OBJECTIVE PS-induced liver fibrosis model and TGF-β1 stimulated HSC were used to detect the expressions of collagen, β-arrestin2, TβRIII and related signaling molecules. The siRNA technique was applied to explore the role of β-arrestin2 in collagen production of HSC. The interaction of β-arrestin2 and TβRIII were detected by laser confocal microscopy and co-immunoprecipitation to further illustrate the possible mechanism.METHODS PS-induced liver fibrosis was established in male Wistar rats. At five time points, the dynamic expressions of β-arrestin2, TGF-β1, TβRII, TβRIII, Collagen I and Collagen III in rat liver tissues were measured by Western blot. In vitro, the dynamic expressions of β-arrestin2, TβRIII, collagen and related signaling molecules in TGF-β1 stimulated HSC were assessed by Western blot. Furthermore, siRNA technique was used to explore the role of β-arrestin2 in TGF-β-induced ECM synthesis. The interaction of β-arrestin2 and TβRIII was assessed by laser confocal microscopy and co-immunoprecipitation.RESULTS1. Time course analysis of Collagen I, Collagen III, β-arrestin2, TGF-β1, TβRII and TβRⅢ expression in PS-induced liver fibrotic ratsWestern blot analysis showed that β-arrestin2 expression increased in the process of liver fibrosis, Collagen I and Collagen III expression were also gradually increased. The expression of β-arrestin2 was positively correlated with the collagen levels. TGF-β1 was increased, while the expression of TβRIII was decreased, and there was no significant change in the expression of TβRII. The expression of β-arrestin2 was negatively correlated with TβRIII expression.2. The dynamic expressions of β-arrestin2, TβRⅢ, collagen, and Smad2, Smad3, Akt signaling pathway in TGF-β1 stimulated HSCHSC was stimulated with TGF-β1 at different time points (0.25h,0.5h, 1h,4h,8h, 12h). The results of Western blot showed that expression of β-arrestin2 increased, but TβRⅢ expression was decreased. Collagen I, Collagen Ⅲ production was also gradually increased. The activation of Smad2, Smad3 and Akt reached peak at 0.5h, 0.25h,4h respectively.3. Expressions of TβRIH and collagen in HSC transfected with TβRⅢ siRNAIn order to investigate the role of TβRⅢ in collagen synthesis of HSC, TβRⅢ siRNA was used to silence the mRNA expression of TβRⅢ in HSC. The Western blot results showed that silencing of TβRⅢ gene expression significantly inhibited TβRⅢ, while the Collagen I, Collagen Ⅲ expressions were increased.4. Activation of Smad2, Smad3 and Akt in HSC transfected with TβRⅢ siRNATo further investigate the effect of TβRⅢ on TGF-β1-induced signaling pathway, we examined p-Smad2, Smad2, p-Smad3, Smad3, p-Akt and Akt expression. The results showed that decreased expression of TβRⅢ by transfecting siRNA significantly, promoted TGF-β1-induced Smad2, Smad3 and Akt activation.5. Expressions of β-arrestin2, collagen in HSC transfected with β-arrestin2 siRNATo investigate the role of β-arrestin2 in collagen synthesis of HSC, β-arrestin2 siRNA was used to silence the mRNA expression of β-arrestin2 in HSC. The Western blot results showed that silencing of β-arrestin2 gene expression significantly inhibited β-arrestin2 and the Collagen Ⅰ, Collagen Ⅲ expression in TGF-β1 stimulated HSC.6. Activation of Smad2, Smad3 and Akt in HSC transfected with β-arrestin2 siRNAWestern blot results found that the decreased expression of β-arrestin2 can also effect Smad2, Smad3 and Akt activation. The low expression of β-arrestin2 by transfecting siRNA significantly inhibited TGF-β1-induced high expressions of p-Smad2, p-Smad3 and p-Akt.7. The interaction of β-arrestin2 and TβRⅢ in TGF-β1 stimulated HSCThe results of laser confocal microscopy and co-immunoprecipitation showed that β-arrestin2 had interaction with TβRⅢ in HSC, and after stimulaion with TGF-β1, the interaction became stronger. After transfected β-arrestin2 siRNA in HSC, we found that TβRIII expression increased, but the interaction of β-arrestin2 and TβRIII had no significant change with TGF-β1 stimulation. These results indicated that decreased β-arrestin2 expression in HSC maybe through downregulating the interaction with TβRIII, thus suppressed TGF-β1 signaling pathways.CONCLUSIONS1. In the progression of liver fibrosis, the expressions of P-arrestin2, Collagen I and Collagen Ⅲ gradually increased. And TGF-β1 was increased, TβRII expression had no significant change among the different time points, while the expression of TβRIII was decreased. The results of correlation analysis found that β-arrestin2 was positively correlated with the collagen expression and negatively correlated with TβRIII expression in liver fibrosis.2. In TGF-β1 stimulated HSC, the expressions of β-arrestin2, Collagen I, Collagen Ⅲ were increased, but TβRIII expression was decreased. β-arrestin2 and TβRIII existed interaction in HSC, and after stimulaion with TGF-β1, the interaction became stronger.3. Silenced endogenous TβRⅢ by siRNA promoted the TGF-β1 stimulated collagen synthesis in HSC. And low TβRⅢ promoted the TGF-β1-induced Smad 2, Smad 3 and Akt activation.4. Silenced β-arrestin2 expression by siRNA suppressed its interaction with TβRⅢ, downregulated Smad 2/3 and Akt activation, thus inhibited the collagen synthesis in HSC stimulated by TGF-β1. These results suggested that selective targeting of P-arrestin2 might present as a novel strategy for inhibited the process of liver fibrosis.