TSPAN8 is involved in lipid metabolism in non-alcoholic fatty liver disease in mice / 南方医科大学学报

OBJECTIVE@#To investigate the changes of tetraspanin 8 (TSPAN8) expression levels and its role in lipid metabolism during the development of non-alcoholic fatty liver disease (NAFLD).@*METHODS@#Thirty male C57BL/6J mice were randomly divided into normal diet group and high-fat diet (HFD) group (n=15), and after feeding for 1, 3, and 6 months, the expression levels of TSPAN8 in the liver tissues of the mice were detected with Western blotting. In a HepG2 cell model of NAFLD induced by free fatty acids (FFA), the effect of TSPAN8 overexpression on lipid accumulation was examined using Oil Red O staining and an automated biochemical analyzer, and the mRNA expressions of the key genes involved in lipid metabolism were detected using qRT-PCR.@*RESULTS@#Western blotting showed that compared with that in mice with normal feeding, the expression of TSPAN8 was significantly decreased in the liver tissues of mice with HFD feeding for 3 and 6 months (P < 0.05). In HepG2 cells, treatment with FFA significantly decreased the expression of TSPAN8 at both the mRNA and protein levels (P < 0.01). TSPAN8 overexpression in FFA-treated cells showed significantly lowered intracellular triglyceride levels (P < 0.001) and obviously reduced mRNA expression of fatty acid transport protein 5 (FATP5) (P < 0.01). The expression of FATP5 was significantly increased in FFA-treated cells as compared with the control cells (P < 0.001).@*CONCLUSION@#TSPAN8 is involved in lipid metabolism in NAFLD, and overexpression of TSPAN8 may inhibit cellular lipid deposition by reducing the expression of FATP5.

CD9 Expression in Colorectal Carcinomas and Its Prognostic Significance

BACKGROUND: CD9, a member of the tetraspanin superfamily, is a tumor suppressor in many malignancies. The aim of this study was to evaluate the immunohistochemical expression of CD9 in colorectal carcinomas (CRCs) and determine clinicopathological and prognostic significance of its expression. METHODS: The CD9 expression status of 305 CRCs was evaluated using a semi-quantitative scoring system in tumor cells (T-CD9) and immune cells (I-CD9) by classifying the results as high and low expression. RESULTS: High T-CD9 (T-CD9 [+]) expression was detected in 175 samples (57.6%) and high I-CD9 (I-CD9 [+]) expression was detected in 265 samples (86.9%). Using Kaplan-Meier survival analysis, the T-CD9 (+) group showed a tendency for better disease-free survival (DFS) (p = .057). In left-sided tumors, DFS was significantly longer in the T-CD9 (+) group (p = .021) but no statistical significance was observed with right-sided tumors (p = .453). I-CD9 (+) CRCs significantly correlated with well/moderately differentiation (p = .014). In Kaplan-Meier analysis, the I-CD9 (+) group had a tendency towards worse DFS compared to the I-CD9 (–) group (p = .156). In combined survival analysis of T-CD9 and I-CD9, we found that the longest DFS was among patients in the T-CD9 (+)/I-CD9 (–) group, whereas the T-CD9 (–)/I-CD9 (+) group showed the shortest DFS (p = .054). CONCLUSIONS: High expression of T-CD9 was associated with a favorable DFS, especially in left-sided CRCs. Combined evaluation of T-CD9 and I-CD9 is required to determine the comprehensive prognostic effect of CD9 in CRCs.

Expressions and clinical significance of tetraspanin CO-029 in intrahepatic cholangiocarcinoma / 中华肝胆外科杂志

Objective To investigate the expressions of tetraspanin CO-029 in intrahepatic cholangiocarcinoma (ICC) and to find out their clinical significance.Methods RT-PCR and western blot were used to detect the expressions of CO-029 in ICC and their matched para-tumorous tissues from 20 patients with ICC,as well as in the HCCC-9810 cell lines.The expressions of CO-029 were further detected via tissue microarray (TMA) in the pathological specimens of 40 patients with ICC.Correlations between the expressions of CO-029 and the clinicopathologic features and prognosis were analyzed.Results A high level of CO-029 was detected in the 20 patients with ICC and the HCCC-9810 cell lines via western blot and RTPCR.Moreover,the expression levels of CO-029 in the ICC tissues were higher than the matched para-tumorous tissues (P ＜ 0.05).TMA detection revealed the positive expression rate of CO-029 to be 65％ (26/40).The expression level of CO-029 was much higher in the early recurrence group (Time to recurrence,TTR ＜ 1 year) than the non-recurrence group (TTR≥ 1 year).On analysis,the correlations were significant between the expressions of CO-029 and tumor encapsulation,hilar lymph node metastasis,TNM stage and prognosis (P ＜0.05).Conclusions CO-029 was highly expressed in ICC.It had close correlations with recurrence,metastasis and prognosis of patients with ICC.

Tetraspanin CD9 modulates ADAM17-mediated shedding of LR11 in leukocytes

LR11, also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding on cleavage with a disintegrin and metalloproteinase 17 (ADAM17). A shedding mechanism is presumed to have a key role in the functions of LR11, but the evidence for this has not yet been demonstrated. Tetraspanin CD9 has been recently shown to regulate the ADAM17-mediated shedding of tumor necrosis factor-alpha and intercellular adhesion molecule-1 on the cell surface. Here, we investigated the role of CD9 on the shedding of LR11 in leukocytes. LR11 was not expressed in THP-1 monocytes, but it was expressed and released in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1). Confocal microscopy showed colocalization of LR11 and CD9 proteins on the cell surface of PMA/THP-1. Ectopic neo-expression of CD9 in CCRF-SB cells, which are LR11-positive and CD9-negative, reduced the amount of sLR11 released from the cells. In contrast, incubation of LR11-transfected THP-1 cells with neutralizing anti-CD9 monoclonal antibodies increased the amount of sLR11 released from the cells. Likewise, the PMA-stimulated release of sLR11 increased in THP-1 cells transfected with CD9-targeted shRNAs, which was negated by treatment with the metalloproteinase inhibitor GM6001. These results suggest that the tetraspanin CD9 modulates the ADAM17-mediated shedding of LR11 in various leukemia cell lines and that the association between LR11 and CD9 on the cell surface has an important role in the ADAM17-mediated shedding mechanism.

Tetraspanin CD9 induces MMP-2 expression by activating p38 MAPK, JNK and c-Jun pathways in human melanoma cells

Expression of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), which correlates with tumor invasion and metastasis, has been known to be regulated by several intracellular signaling pathways. Since the CD9 membrane protein has been implicated in signal transduction and malignant progression of cancer cells, we examined the functional involvement of CD9 in the regulation of MMP-2 and MMP-9 expression by using stable CD9 transfectant clones of MelJuso human melanoma cells. The CD9 cDNA-transfected cells with elevated CD9 expression displayed increased MMP-2 and decreased MMP-9 expression when compared with the mock transfectant cells. Among several signal pathway inhibitors tested, SB203580 and SP600125, which inhibit p38 MAPK and JNK respectively, completely blocked the CD9-stimulated MMP-2 expression. Phosphorylation levels of p38 MAPK and c-Jun in MelJuso cells were also significantly increased by CD9 transfection. In addition, the down-regulation of p38 MAPK and JNK by siRNA transfection resulted in a decrease in MMP-2 expression by MelJuso cells. Promoter analysis and gel shift assay showed that the CD9-induced MMP-2 expression is mediated by a functional AP-1 site through interactions with AP-1 transcription factors including c-Jun. These results suggest that CD9 induces MMP-2 expression by activating c- Jun through p38 MAPK and JNK signaling pathways in human melanoma cells.

Cloning, Expression and Identification of Membrane Protein Tetraspanin 2-A of Schistosoma japonicum / 中国寄生虫学与寄生虫病杂志

Objective To clone and express a membrane protein(Tetraspanin 2) gene of Schistosoma japonicum (SjTsp2). Methods A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a(+). The recombinant plasmid was transformed into E. coli BL21(D3) and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting. Results Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant (1 ∶ 32 000 in maximum) was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting. Conclusion SjTsp2 has been expressed and shows certain antigenicity.

Effect of up-regulation of Tspan-5 expression on the metastatic ability of colorectal cancer cells / 解放军医学杂志

Objective To investigate the effect of up-regulation of tetraspanin-5 (Tspan-5) expression on the metastatic ability of colorectal cancer (CRC) cells. Methods The coding sequence of Tspan-5 mRNA was amplified from total RNA of LoVo cells by RT-PCR and cloned into pEGFP-C1 to construct Tspan-5/pEGFP-C1,a eukaryotic expression vector. The expression of Tspan-5 in LST-R1 cells was up-regulated by gene tranfection. Western blotting was used to detect the expression level of Tspan-5/EGFP fusion protein,and laser scanning confocal microscopy (LSCM) was used to observe the distribution of Tspan-5/EGFP in LST-R1 cells. The changes in adhesion,migration and invasion ability of LST-R1 cells were determined by adhesion,migration and invasion experiment in vitro,respectively. Liver metastasis model of nude mice was used to test the changes in liver metastasis ability of LST-R1 cells in vivo. Results The Tspan-5/EGFP fusion protein could be detected by Western blotting in Tspan-5/pEGFP-C1 transfectants. LSCM showed that Tspan-5/EGFP protein located in cell membrane. Adhesion and migration assays showed that up-regulation of Tspan-5 expression level significantly promoted the adhesion and migration ability of LST-R1 cells on a series of extracellular matrix (ECM) components,including collagen IV,FN,LN and VN (P