| 1. Background Clonorchis sinensis(C. sinensis), a pathogenic trematode, parasitizes in hepatic duct of human and other mammals, which causes human morbidity and mortality of people worldwide, especially in eastern Asia such as China, Koreaand Vietnam. Currently, there are approximately 35 million people infected with C. sinensis globally, and 15 million of whom are in China. C. sinensisexcretory-secretory products(Cs ESPs) and its mechanical stimulus could induce chronic injury in hepatobiliary system, which results in local expansion of bile ducts, biliary epitheliumhyperplasia, cholangitis, liver fibrosis and even hepatobiliary tumor. Surveys indicated that the morbidity of cirrhosis would rise significantly when people infected with C. sinensis. The persistent activation and proliferation of hepatic stellate cells(HSCs) is the primary source of extracellular matrix(ECM), meanwhile it is the critical link and cytological basis in the development of liver fibrosis.Previous studies revealed that total Cs ESPs enhanced the proliferation of human hepatic stellate cell line LX-2 in vitro, and also induced hepatic fibrosis in rats. C. sinensis secretory phospholipase A2(Css PLA2), an important component of Cs ESPs, could enhanced the proliferation of LX-2 cells alone. The secretory PLA2 s had been onfirmed as an important messenger in signaling pathway, such as inducing a rapid activation of ERK1/2 pathway which is crucial for degranulation and cytokine production. The cell activation and proliferation effect of s PLA2 may not be concerned with its enzyme activity as rate-limiting enzyme of arachidonic acid metabolism, but through binding with relevant receptors on cell membrane. For example, Group IB s PLA2 induced bronchoconstriction and proliferation of fibroblast by means of combining with an 18-k Da protein on cell membrane, and Group IA s PLA2 activated eosinophilia by activating ERK1/2 pathway directly. Our previous studies indicated that the recombinant Css PLA2 could bind to some proteins on LX-2 cells membrane directly, and those proteins could be receptors of Css PLA2 on the LX-2 cell.However, it is not clear about the receptors of Css PLA2, and how Css PLA2-receptors interaction activates and proliferate LX-2 cells is also unknown. In the present study, we used Css PLA2 as a bait to screen a human liver c DNA library using a yeast two-hybrid system, and found human TM7SF3 is the Css PLA2-interacting partner. The TM7SF3-Css PLA2 interaction was confirmed by co-immunoprecipitation and GST Pull-down assay. The localization of TM7SF3 was also tested. Moreover, our study indicated TGFβ/Smad pathway、ERK pathway 、JNK pathway and NF-κB pathway of the proliferation effect of Css PLA2 after blocking the interaction of Css PLA2 and TM7SF3.2. Materials and methods 2.1 MaterialsMouse anti c-Myc, PLA2 antibodies, rabbit anti GST antibodies, goat anti mouse or rabbit secondary antibodies were purchased from Proteintech. The plasmids used in this study were generated by subcloning the indicated c DNA fragments into expression vectors(p CDNA6.0, p EGFP-C1, p GEX-4T-1, p MAL-c2x), and the expression vector of Css PLA2(p MAL-c2x-Css PLA2) was generated and given from Yinjuan Wu in our lab..2.2. Yeast two-hybrid screen PCR generated the Clonorchis sinensis secretory phospholipase A2(Css PLA2) c DNA sequence from plasmid p ET28a-Css PLA2 was cloned into the yeast expression vector p GBKT7 using Ndel/Sal I restriction sites in frame with the Gal4 DNA binding domain. This bait was used to screen the Human Liver Matchmaker? c DNA Library p ACT2. The yeast two-hybrid screens were performed according to the manufacturer’s instructions.2.3. Cell culture and transient transfection 293 T cells were cultured in High Glucose DMEM(GIBCO) with 10% fetal bovine serum(FBS), 100 mg/m L penicillin, and 100 mg/m L streptomycin sulfate at 37 °C under 5% CO2. The Css PLA2 was cloned into an expression vector p EGFP-C1(Clontech), and TM7SF3 gene fragment that interact with Css PLA2 come from the yeast two-hybrid screens was cloned into an expression vector pc DNA6. Lipofectamine 2000 was used in transient transfections according to the manufacturer’s protocol. Recombinant plasmids(p EGFP-Css PLA2,pc DNA6- TM7SF3, p EGFP- Css PLA2 and pc DNA6- TM7SF3) were co-transfected into 293 T cells.2.4. Co-immunopreciptation assay Cell lysis from transfected 293 T cells using RIPA lysis buffer were incubated with anti PLA2 antibodies(Proteintech) and Protein A agarose overnight at 4 °C. After washing five times with RIPA lysis buffer, the beads were eluted with the 2 × SDS sample buffer and boiled for 8 min at 100 °C. The samples were then analyzed by Western blotting using anti c-Myc antibodies.2.5 Expression and purification of N-terminal MBP-tagged Css PLA2 proteins The recombinant plasmids( p MAL-c2x- Css PLA2) were given from Yinjuan Wu of our lab to overexpress N-terminal MBP-tagged Css PLA2 proteins. Then the ecombinant plasmids were transformed to E. coli BL21(DE3). Overnight cultures carrying the recombinant plasmid p MAL-c2x- Css PLA2 were inoculated in 2L of Luria–Bertani broth medium containing 50 μg/ml of ampicillin. Cells were grown at 37 °C until the A600 had reached 0.6. The expression of protein was induced by adding IPTG at a final concentration of 0.5 m M, followed by incubation at 37 °C for 4h with vigorous shaking at 250 rpm. Css PLA2 protein was purified by a single step of Amylose Resin chromatography under native conditions, following the procedure of the manufacturer. The purity of MBP-Css PLA2 was analyzed by 10 % SDS-PAGE followed by Coomassie blue staining. The final concentration of purified Css PLA2 was determined by the method of BCA. The purified recombinant protein and plasmids were stored at-80 °C for later use.2.6 Recombinant plasmid construction, expression of N-terminal GST-tagged TM7SF3 proteins The TM7SF3 gene fragment that interacted with Css PLA2 come from the yeast two-hybrid screens was cloned into an expression vector p GEX-4T-1 to overexpress and purify soluble TM7SF3 protein in Escherichia coli. The recombinant plasmids were transformed to E. coli BL21(DE3) to produce soluble forms of N-terminal GST-tagged TM7SF3 proteins. All sequences of the recombinant genes were confirmed by sequencing at Invitrogen.Overnight cultures carrying the recombinant plasmid were inoculated in 2L of Luria–Bertani broth medium containing 50 μg/ml of ampicillin. Cells were grown at 37 °C until the A600 had reached 0.6. The expression of protein was induced by adding IPTG at a final concentration of 0.1 m M, followed by incubation at 37 °C for 5h with vigorous shaking at 250 rpm. TM7SF3 protein was purified by a single step of Glutathione Sepharose 4B chromatography under native conditions, following the procedure of the manufacturer. The purity of GST-TM7SF3 was analyzed by 10 % SDS-PAGE followed by Coomassie blue staining. The final concentration of purified TM7SF3 was determined by the method of BCA. The purified recombinant protein nd plasmids were stored at-80 °C for later use.2.7GST Pull-down assay The recombinant proteins MBP-Css PLA2 1Xmg and GST-TM7SF3 0.5mg were incubated with Amylose resin 1mg for 2h at room temperature, and protein GST、MBP and MBP-Css PLA2 were incubated with Amylose resin at the same condition as negative control. After the incubation, the mixture of two proteins was purified by a single step of Amylose resin chromatography under native conditions, following the procedure of the manufacturer. The samples were then analyzed by Western blotting using anti GST antibodies.2.8 Immunohistochemical localization of TM7SF3 in LX-2 cellsThe LX-2 cells were fixed with 4% paraformaldehyde(GIBCO) for 30 min and 0.25% Tritonx-100(GIBCO) for 10min(30ul each well) at room temperature. After washing procedure, the fixed cells were incubated with 3% H2O2 for 30 min and blocked with 5%BSA for 30 min at temperature. After the blocking, the cells were incubated with rabbit anti-TM7SF3 antibodies(1:200 dilutions in 5%BSA) at 4°C overnight, and the cells incubated without anti-TM7SF3 antibodies were negative control. After washing procedure, the cells were incubated with goat anti-rabbit secondary antibodies(1:1000 dilutions in 5%BSA) for 20 min at room temperature. Then the cells were stained with DAB for 10 min and hematoxylin for 1min at room temperature. After washing procedure, the cells were observed under the microscope.2.9. CCK-8 assayThe cell survival rate was examined using Cell Counting Kit-8(Dojindo Molecular Technologies). Cells were plated in 96-well plates at 6000 cells/well. After 24 h of culture, the LX2 cells were incubated with the protein MBP-Css PLA2(40μg/ml) or the protein Css PLA2(40μg/ml) and anti-TM7SF3 antibodies(1:200 dilutions) or PBS at the indicated concentrations. After incubation for 24 h, 10 μL CCK-8(Takara) was dded to each well, and cells were further incubated for 2h. Absorbance was read at 450 nm using an enzyme micro-plate reader. The OD value was calculated using Gen5 CHS 1.09 software.2.10. Quantitative real-time PCRTotal RNA was isolated from LX-2 cells incubated with PBS, Css PLA2 OR Css PLA2 and anti-TM7SF3 antibodies using a TRIzol kit, and c DNA was synthesized using a c DNA synthesis kit. Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix.The PCR amplification program was 95 °C for 30 s, followed by 44 cycles of 95 °C for 5 s and 60 °C for 20 s. The melting curve was performed using a program of 95 °C for 30 s and 65 °C for 15 s. The Bio Rad i Q5 software(version 2.1) was used to analyze the data according to the 2-ΔΔCt method(Livak and Schmittgen 2001). SPSS(version 16.0) software was used in the present study for statistical analysis.2.12. Statistical analysisWe determined the significance of differences using Pearson’s correlation test and Student’s t-test(two-tailed). P< 0.05 was considered to be significant. Statistical significance is displayed as *p< 0.05, **p <0.01. SPSS(version 16.0) software was used in the present study for statistical analysis.3. Results3.1 Identification of Css PLA2 interacting protein using the yeast two-hybrid systemConsidering the importance of Css PLA2 in liver fibrosis induced by Clonorchis sinensis, we performed a yeast two-hybrid screen of a human liver c DNA library to dentify novel Css PLA2 interacting partners. Then through gene sequencing and BLAST analysis from NCBI database, 5 interacting partners of Css PLA2 were identified finally. However, among these proteins, metallothionein-2、aminoacylase-1 isoform b and cytochrome c oxidase subunit I are located in mitochondria, actually they can’t combine with Css PLA2 directly, meanwhile hemopexin is a kind of secreted proteins in the blood which always flow around the body. Finally according to our purpose to look for the proteins located on the cell membrane, the transmembrane 7 times super family 3(TM7SF3), the only transmembrane protein after the Yeast two-hybrid screen, was chosen to be the possible binding partner of Css PLA2 to be further studied. The interaction between Css PLA2 and TM7SF3 was validated in an independent yeast two-hybrid assay. To further analyze their interaction, we transiently over-expressed GFP-tagged Css PLA2 and myc-tagged TM7SF3 in 293 T cells for 24 h. By co-immunoprecipitation(co-IP) analysis, we showed that TM7SF3 pulled down Css PLA2. Also, we over-expressed MBP-tagged Css PLA2 and GST-tagged TM7SF3 in E.coli. By GST-Pull down analysis, we showed that TM7SF3 pulled down Css PLA2. These results confirm the proteins interaction between Css PLA2 and human TM7SF3.3.2 Expression, purification and characterization of Css PLA2The DNA fragment of Css PLA2 without signal peptide sequence was cloned into p MAL-c2 x expression vector, and the recombinant fusion protein was expressed and then purified by affinity chromatography using Amylose Resin under native conditions. The purified protein was analyzed with SDS-PAGE and confirmed by Western blot.3.3 Expression, purification and characterization of TM7SF3The DNA fragment of TM7SF3 the yeast two-hybrid system preyed was cloned into p GEX-4T-1 expression vector, and the recombinant fusion protein was expressed and then purified by affinity chromatography using Glutathione Sepharose 4B under ative conditions. The purified protein was analyzed with SDS-PAGE(Fig. 4A) and confirmed by Western blot(Fig. 4B).3 Expression, purification and characterization of TM7SF3.3.4 Localization of TM7SF3 in LX2 cellsTo determine whether TM7SF3 existed in LX-2 cells and its subcellular localization, we performed an immunohistochemical localization of TM7SF3 in LX-2 cells. The result showed that TM7SF3 existed in LX2 cells, and according to the specific coloration it mainly located in cytomembrane.3.5 Blocking of TMTSF3 decreased Css PLA2-mediated cell proliferation in LX2 cellsTo investigate whether TM7SF3 regulate Css PLA2-mediated cell proliferation in LX2 cells, the LX2 cells were incubated with Css PLA2, anti TM7SF3 antibodies and Css PLA2, and PBS, respectively. Then we performed CCK-8 assay and q-RT PCR. Compared with PBS control group, cell proliferation of Css PLA2 group significantly increased, however, the increasing cell proliferation induced by Css PLA2 was inhibited effectively after blocking of TM7SF3. Recent studies showed that activation of pathways of TGFβ、ERK、JNK and NF-κB can upregulate cell proliferation of LX-2 cells, therefore we investigated whether Css PLA2 had an effect on those signal pathways in LX-2 cells. Compared with control group, expression level of involved genes of ERK, JNK, NF-κB and TGFβ/Smadpathways in Css PLA2 group increased significantly, while the blocking of TM7SF3 inhibited this effect of Css PLA2. In other words, these changes mean the interaction of Css PLA2 and human TM7SF3 may influence LX-2 cells through ERK, JNK, NF-κB and TGFβ/Smadpathways.4. ConclusionsUsing a yeast two-hybrid system and screening a human liver c DNA library, then we found 5 interacting proteins of Css PLA2 including TM7SF3, which was one of the strongest binding partners of Css PLA2 in Y2 HGold, and the interacting effect was confirmed by co-immunoprecipitation and GST-Pull down assay further. We also confirmed the localization of the TM7SF3, in LX-2 cells, a human hepatic stellate cell line. Moreover, our study indicated some relevant pathways of the proliferation effect of Css PLA2, including TGFβ/Smad pathway、ERK pathway 、JNK pathway and NF-κB pathway, and these signal pathways are generally considered to be concerned with development of liver fibrosis. |
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