Study The Function And Molecular Mechanism Of Erbin In TGF-β1-induced EMT In Renal Tubular Epithelial Cells

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal interstitial fibrosis (RIF), which finally leads to renal failure. As we known, EMT is regulated by different signaling molecules and transforming growth factor β (TGF-β) is considered as the most important one. TGF-β1signals are very complex, contains canonical Smad signaling pathways and non-Smad signaling in certain types of cells, involving p38MAPK, ERK, JNK. AKT/PKB, and RhoA. Erbin, a member of LAP family, is recently reported to inhibit Smads and ERK pathway which are two important types of intracellular signaling involved in TGF-β1-induced EMT. However, the role of Erbin in the regulation of EMT and the underlying mechanisms remain to be fully understood. Along these lines, it is possible that Erbin may participate in process of EMT, even renal fibrosis. To that end, we aimed to evaluate the expression of Erbin in renal interstitial fibrosis and the potential role of Erbin in tubular EMT stimulated by TGF-β1.In this study we demonstrated that the expression of Erbin was up-regulated in the tubular epithelia of5/6-nephrectomized rats. We also showed here that TGF-β1induced Erbin expression in NRK52E cells during their EMT phenotype acquisition. Importantly, elevated expression of Erbin inhibited ERK signaling and partial reversed EMT stimulated by TGF-β1. In the mean time, reducing Erbin expression enhanced ERK phosphorylation, promoted the E-cadherin suppression, induced a-SMA expression and fibronection secretion in response to TGF-β1, which could be rescued if cells were treated with the inhibitor of MEK1/2U0126. However, in the absence of TGF-β1, Erbin failed to affect ERK activation and EMT process. These results suggest that Erbin is a negative feedback molecule induced by TGF-β1and inhibits TGF-β1-induced EMT via ERK signaling pathway. Part1Study the relation between Erbin and renal interstitial fibrosisObjective To investigate the expression change of Erbin in Renal Interstitial Fibrosis (RIF) and TGF-β1-induced epithelial-mesenchymal-transition (EMT) in NRK52E cells.Methods In vivo, the model of renal fibrosis was induced by5/6subtotal nephrectomy in rat. Scr and BUN was detected and Masson staining was used to evaluate the level of renal tissue fibrosis. The location and expression of Erbin in renal tissue were detected by immunohistochemistry and Western blot. In vitro, NRK52E cells were treated by TGF-β1(10ng/ml) for72h, Light microscopy was performed to investigate the morphological changes in cells treated with TGF-β1, immunofluorescence and western blot were used to observe the expression and distribution of E-cadherin and a-SMA. The expression of Erbin mRNA and protein in different stimulated time was detected by RT-PCR or Western blot respectively.Results Compared to sham group with Scr (33.96±7.28) and BUN (8.11± 2.55), rats in5/6nephrectomy model with Scr (140.52±61.11) and BUN (34.23±7.66) appeared renal dysfunction. Masson staining indicated kidney interstitial fibrosis. the expression of Erbin was significantly increased in renal tissue (2.9fold), especially in tubular epithelia. In vitro, Light microscopy showed fibroblast-like morphological changes in cells treated with TGF-β1. Cells disassociated with surrounding cells and were larger and more elongated than control cells. The expression of Erbin and a-SMA were markedly increased (2.3and2.8fold, respectively)(P<0.05) and the expression of E-cadherin was dramatically decreased in NRK52E cells stimulated by TGF-β1, which were consistent with Immunofluorescence results. Meanwhile, our results also indicate TGF-β1-induced Erbin upregulation was time-dependent, and peaked at72h after TGF-β1stimulation. Additionally, TGF-β1treatment resulted in a significant increase in the mRNA level of Erbin, nearly1.8fold compared to control cells.Conclusions Erbin was up-regulated in renal interstitial fibrosis and in cells undergoing EMT induced by TGF-β1. Our data indicate that there is a closely relation between Erbin and renal interstitial fibrosis. Part2Study the role of Erbin in TGF-β1-induced EMT in renal tubular epithelial cellsObjective To investigate the role of Erbin in TGF-β1-induced EMT in renal epithelial cells.Methods Rat proximal tubular epithelial cell line (NRK52E cells) were transfected with Prk5-myc-Erbin or empty vector via Lipofectamine2000complying with the manufacturer's protocol. For evaluating Erbin expression, cells were harvested48h after transfection. Western blot was used to detect the expression of Erbin, E-cadherin and a-SMA in control cells, empty transfected cells and Erbin transfected cells.24h after Erbin plasmid transfected, cells were treated with10ng/ml TGF-β1for72h, and then western blot and Immunofluorescence were used to examine the protein expression of Erbin, E-cadherin and α-SMA in control cells, TGF-β1-treated cells, Erbin transfected cells and Erbin+TGF-β1-treated cells. ELISA was performed to examine the secretion of Fibronectin in above groups. Meanwhile, according to Geenbank, we designed an Erbin specific siRNA to knockdown Erbin expression. For evaluating Erbin expression, cells were harvested72h after transfection, the protein expression of Erbin, E-cadherin and α-SMA were detected by western blot.24h after Erbin specific siRNA transfected, cells were treated with10ng/ml TGF-β1for72h, and then western blot and Immunofluorescence were used to examine the protein expression of Erbin, E-cadherin and α-SMA in control cells, TGF-β1-treated cells, Erbin siRNA transfected cells and Erbin siRNA+TGF-β1treated cells. ELISA was also performed to examine the secretion of Fibronectin in above groups.Results Compared to control and empty plasmid transfected cells, Erbin plasmid transfection significantly increased Erbin expression. However, Erbin overexpression did not impact the basal protein level of E-cadherin and a-SMA in untreated NRK52E cells. After TGF-β1treatment, the expression of Erbin and a-SMA were markedly increased and the expression of E-cadherin was dramatically decreased in NRK52E cells. Interestingly, Erbin overexpression inhibited TGF-β1-induced E-cadherin decrease and a-SMA increase. Immunofluorescence results showed, after TGF-β1treatment, E-cadherin at the cell-cell junctions changed its liner pattern into a zipper-like pattern, and most cells lost contact with neighboring cells. Compared with paralleled-transfected cells, NRK52E cells with Erbin overexpression were resistant to TGF-β1treatment, partially retained E-cadherin at the cell-cell adherent junction. ELISA results showed TGF-β1induced fibronectin secretion compared to control cells. In agreement with the results of western blot, Erbin overexpression cells were resistant to TGF-β1-induced fibronectin secretion. Meanwhile, compared to scramble siRNA, Erbin expression was efficiently attenuated in cells transfected with Erbin target siRNA. We demonstrated previously that Erbin overexpression did not impact EMT process without TGF-β1stimulation. Depletion of Erbin didn't have any effect on the protein level of E-cadherin and a-SMA either. However, Erbin deficient profoundly enhanced E-cadherin decrease and α-SMA increase in response to TGF-β1. Immunofluorescence results showed further disruption of E-cadherin in the cell-cell junction in Erbin deficient cells after TGF-β1treatment compared with cells transfected with scramble siRNA. Moreover, Erbin deficient cells with TGF-β1showed a significant increase in fibronectin accumulation compared to TGF-β1treatment cells.Conclusion Erbin overexpression blocked the EMT of NRK52E cells induced by TGF-β1, in contrast, Erbin deficient profoundly enhanced EMT in response to TGF-β1. Thereby, these experiments support that Erbin plays a protective role in TGF-β1-induced EMT. Part3Study the molecular mechanism of Erbin in TGF-β1-induced EMT in renal tubular epithelial cellsObjective To investigate the regulation role of Erbin in TGF-β/Smad and TGF-β/ERK signal pathway, finally demonstrate the specific molecular mechanism of Erbin in TGF-β1-induced EMT in renal epithelial cells.Methods After lOng/ml TGF-β1treated for indicated time (0,15min,30min,60min,120min and240min), Western blot was performed to examine the expression of p-Smad2and p-ERK in response to TGF-β1stimulation. Rat proximal tubular epithelial cell line (NRK52E cells) were transfected with Prk5-myc-Erbin or empty vector via Lipofectamine2000complying with the manufacturer's protocol, then the effect of Erbin on the expression of p-Smad2, p-ERK and nuclear translocation of Smad2/3complex were detected by western blot. Meanwhile, Erbin specific siRNA was transfected into NRK52E cells to knockdown Erbin expression via Lipofectamine2000. Western blot was selected to investigate the role of Erbin deficient in TGF-β/ERK. signal pathway. To further evaluate whether ERK activation is necessary for the effect of Erbin on EMT in NRK52E cells, we pretreated NRK52E cells with U0126. a pharmacological inhibitor of MEK1/2. Western blot was performed to detect the expression of p-ERK, t-ERK, Erbin, E-cadherin and a-SMA in control cells, TGF-β1treated cells, TGF-β1+U0126treated cells, Erbin siRNA+TGF-β1-treated cells, Erbin siRNA+TGF-β1cells+U0126treated. Immunofluorescence and ELISA were used to evaluate the distribution of E-cadherin and secretion of fibronetin respectively.Results Smad2was phosphorylated as early as15min after TGF-β1incubation, peaked at30min and remained active at240min in NRK52E cells. TGF-β1stimulated NRK52E cells did significantly enhance transposition of Smad2/3complex from cytoplasm to nucleus as compared with their un-stimulated counterparts. However, elevated expression of Erbin did not change the extent of p-Smad2expression or nuclear accumulation of Smad2/3. We next evaluated whether Erbin had a role in regulating ERK signaling in NRK52E cells. Our results showed TGF-β1induced ERK phosphorylation from30min and peaked at60min. ERK phosphorylation induced by TGF-β1was blocked by Erbin overexpression and enhanced by Erbin silencing. Meanwhile, administering U0126(1μM) to NRK52E cells inhibits ERK activation stimulated by TGF-β1but not basal phosphorylated ERK. Interestingly, the increased activation of ERK by Erbin silencing was almost abolished by U0126(Figure7A). In addition, our data also indicated the effect of Erbin depletion on TGF-β1-induced EMT can be blocked by U0126. E-cadherin decrease. a-SMA expression and fibronectin secretion increase induced by Erbin depletion are reversed to the level present in control cells.Conclusion ERK signaling is essential for Erbin-mediated regulation of EMT and Erbin inhibits TGF-β1-induced EMT via ERK suppression. But Erbin is failed to affect canonical Smads signaling activation stimulated by TGF-β1in NRK52E cells. This article provides evidence for a protective role of Erbin in renal fibrosis by counteracting TGF-β1-induced EMT via an ERK dependent pathway. These observations may help identifying a potential target of therapeutic interventions of related renal disorders.