Ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) is one of the deubiquitinating enzymes in the ubiquitin-proteasome system. It has been shown that UCH-L1 could markedly decrease neointima formation through suppressing vascular smooth muscle cell (VSMC) proliferation in the balloon-injured rat carotid. However, whether UCH-L1 plays roles in VSMC migration remains to be determined. In this study, the primary VSMCs were isolated from aortic media of rats and TNF-α to was used to induce VSMC migration. Using a modified Boyden chamber and wound healing assay, it was found that TNF-α can dose and time-dependently induce VSMC migration with a maximal effect at 10 ng/mL. Moreover, UCH-L1 expression increased gradually with the prolonged induction time at 10 ng/mL of TNF-α. UCH-L1 content in VSMC was then modulated by recombinant adenoviruses expressing UCH-L1 or RNA interference to evaluate its roles in cell migration. The results showed that over-expression of UCH-L1 attenuated VSMC migration, while knockdown of it enhanced cell migration significantly no matter whether TNF-α treatment or not. Finally, the effect of UCH-L1 on NF-κB activation was demonstrated by NF-κB nuclear translocation and DNA binding activity, and the levels of IL-6 and IL-8 in cell culture media were examined by ELISA. It was showed that UCH-L1 over-expression inhibited NF-κB activation and decrease IL-6 and IL-8 levels, while knockdown of it enhanced NF-κB activation and increase IL-6 and IL-8 levels during TNF-α treatment. These data suggest that UCH-L1 can inhibit TNF-αinduced VSMCs migration, and this kind of effect may partially due to its suppression role in NF-κB activation.

The aim of the present study was to examine the protective effects and mechanisms of S100 calcium-binding protein A4 (S100A4) on endothelial cell apoptosis induced by oxidative stress injury. Endothelial cells were cultured and divided into control and oxidative stress injury groups, with the latter state induced by H2O2. Endothelial cells in every group were incubated with or without 50 or 100 µM S100A4. The cell viability and amounts of malondialdehyde, nitric oxide and lactate dehydrogenase in the culture medium were measured. The apoptotic index was detected by TUNEL staining. Western blot and immunoprecipitation analyses were used to detect the expression levels and the association between S100A4 and P53. H2O2 treatment led to oxidative stress injury in the cultured vascular endothelial cells, a decrease in the cell viability and an increase in the rate of apoptosis of vascular endothelial cells compared with the negative control group. Exogenous S100A4 serves a significant function against oxidative stress injury (P<0.05), increasing the viability and attenuating the apoptotic rate of endothelial cells. Western blotting results suggested that the protein levels of S100A4 and P53 increased subsequent to oxidative stress injury and that exogenous S100A4 increased the expression of P53 in the cytoplasm and decreased the expression of P53 in nucleus. The immunoprecipitation assay results revealed a protein-protein interaction between S100A4 and P53. These results suggested that rat recombinant S100A4 serves an anti-apoptotic function in oxidative stress injury. This effect of S100A4 is mediated, at least in part, via the inhibition of the translocation of P53 to the nucleus.

The EF-hand calcium-binding protein S100B presents a wide range of biological activities and functions. This binding protein is involved in various human diseases, including cancer, brain trauma and ischemia, neuro-degenerative disease (Alzheimer's disease), and psychiatric disorders. In this study, we prepared human S100B protein and its monoclonal antibodies. Human S100B protein was expressed in Escherichia coli, successfully purified by diethylaminoethyl cellulose anion-exchange chromatography, and then identified by western blot analysis. Monoclonal antibodies (mAbs) were produced by the standard hybridoma method and validated by enzyme-linked immunosorbent assay and western blot analysis. The prepared human S100B protein and its mAbs demonstrated potential biological activities. The KD of one mAb is approximately 4.72x10-8 mol/l, and its cross reactivity is low with human S100A4, mouse S100A4, and human S100A1. Recombinant Soluble S100B can promote the migration and invasion of HeLa cells. The expression of S100B protein in tumor tissues can be detected effectively by using the prepared monoclonal antibodies. Increasing concentration of the anti-human S100B mAbs showed a reduced expression of the S100B protein. Subsequently, the expression of p53 increased significantly (P<0.05) in A375 cells. A significant increase in apoptosis in A375 cells was observed with increasing S100B mAb concentration. Results showed that our prepared S100B mAbs were suitable for detecting S100B expression in human tissues, furnishing promising tools for further functional investigation and clinical applications.

Preparations utilizing monoclonal antibodies against S100A4 provide useful tools for functional studies to investigate the clinical applications of the human S100A4 protein. In the present study, human S100A4 protein was expressed in Escherichia coli (E. coli) BL21 (DE3), successfully purified by diethylaminoethyl cellulose anion-exchange chromatography and identified by western blot analysis. Soluble S100A4 bioactivity was confirmed by Transwell migration and invasion assays in the human HeLa cell line. Monoclonal antibodies (mAbs) were generated utilizing the standard hybridoma method and were validated by enzyme-linked immunosorbent assay and western blot analysis. The antibody was then used to examine human gastric carcinoma specimens by immunohistochemistry. Recombinant S100A4 was functionally expressed in E. coli and promoted the migration and invasion of HeLa cells. Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained. One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli. The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.