| Objective:Caveolaes are invaginations of the plasma membrane that were firstly described in 1953 in the Capillary Endothelium by G.E. Palade. Caveolae mediates endocytosis, pinocytosis, cholesterol homeostasis and signal transduction. There are three identified caveolin proteins, Caveolin-1(α,β), Caveolin-2(α,β,γ) and Caveolin-3, which serve as markers for identification of caveolae and are critically involved in caveolae function. Caveolin-1 expresses in most cell types. Caveolin-2 co-localizes with Caveolin-1 and is found in the same cells as Caveolin-1. Caveolin-3 is a muscle specific caveolin and is found in muscle cells.Many results show that Caveolin-1 is a nodal point of many signal molecules. Caveolin-1 plays important roles in cell proliferation,transfor- mation,apoptosis and metastasis. There are some modified sites on Cave- olin-1, for instance, phosphorylation of Tyr14, palmitoylation of Cys133, Cys143 and Cys156. There are some other domains such as transmembrane domain and Caveolin scaffolding domain(CSD) which play different actions in cell biological behaviours. For example, Caveolin-1, through its CSD, is able to bind to upstream and downstream components of cell signaling pathways and then change activities of some proteins. It is now known that Caveolin-1 expression is sufficient and necessary to drive the formation of morphologically identifiable caveolae. Caveolae affects downstream molecules through modulating the internalization of signal molecules and the duration of signal.CD147 belongs to the highly glycosylated immunoglobulin super- family transmembrane protein, enriched on the surface of many malignant tumor cells. As a result of heterogeneous N-glycosylation, CD147 exists in both a highly glycosylated form, HG-CD147 and lowly glycosylated form, LG-CD147. CD147 mediates the interaction of tumor cells and matrix, and is responsible for inducing the expression of fibroblasts and tumor cells themselves MMPs, accelerating angiogenesis which promote tumor metastasis.Our previous study showedCaveolin-1 expression levels were higher in Hca-F cells than that in Hca-P and Hepa1-6 cells which have character of high lymphatic metastatic potential, low lymphatic metastatic potential and no lymphatic metastasis potential respectively. And the expression of caveolin-1 was undetectable in Hepa1-6 cells. These results indicate that caveolin-1 may play a role in Hepatocacinoma cell lines lymphatic metastasis. When caveolin-1 expression was down-regulated specifically in Hca-F cells using RNAi technology, Western blot analysis showed that the Caveolin-1 levels were significantly reduced in Hca-F/RNAi cells. HG- CD147 expression was obviously decreased while LG-CD147 expression showed a marked increase.The main aim of this study was to construct Cav-1 mutants expression vectors, to express Cav-1(wt) and mutants in Hepa1-6 cells and observe their location and their effect on CD147 glycosylation.Methods:Cav-1 mutants of phosphorylation site mutant (Y14F), palmitoylation site mutant (C133A, C143A, C156A), 133-178 residues at C-terminal cyto- solic domain deletion (△133-178) and 102-134 residues of transmembrane domain deletion(△TMD) were constructed. The above mutants were subcloned into pMD18-T simple vector, and then transformed into E.coli. DH5a. The positive clones were selected by PCR, restriction enzyme ana- lysis and DNA sequencing. The positive plasmid DNA was subcloned into expression vector pcDNA3.1/Myc-His(-)B and the recombinant plasmids were identified by restriction enzymes, PCR and DNA sequencing.Recombinant plasmids,pcDNA3.1/Cav-1(Y14F), pcDNA3.1/Cav-1 (C 133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178), pcDNA3.1/Cav-1(△TMD) were transfected into Hepa1-6 cells by liposome, respectively. Untransfected cells and trasfected pc DNA3.1/Cav-1(wt) or empty vectors cells were used as controls.The Cav-1 expression and CD147 glycosylation in Cav-1 mutants were revealed by immunocytochemistry and Western blot analysis.Results:1. The recombinant plasmids of pcDNA3.1/Cav-1(Y14F), pcDNA3. 1/Cav-1(C133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178), pcDNA3.1/Cav-1(△TMD) were constructed successfully. The temporarily expressed Cav-1 mutants proteins were found in the cytosol of Hepa1-6 cells, but the expression level of Cav-1(Y14F) was lower.2. The expression of LG-CD147 protein was undetectable. But this changes of CD147 glycosylation in Hepa1-6/Cav-1(wt) cells were not also found among Hepa1-6 cells transfected pcDNA3.1/Cav-1(C133A), pc DNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178).3. The expression of Cav-1(△TMD) protein reached top level at 36h after transfection and then the protein level of Cav-1(△TMD) decreased and disappeared at 96h. The reduction of Cav-1(△TMD) protein was inhibited by treatment Heap1-6/ Cav-1(△TMD) with 0.2μM of proteosome inhibitor MG132.Conclusions:1. 6 recombinant Cav-1 mutant plasmids of pcDNA3.1/Cav-1(Y14F), pcDNA3.1/Cav-1(C133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178), pcDNA3.1/Cav-1(△TMD) expressed in Hepa1-6 cells and the proteins of those Cav-1 mutants mainly localized in the cytosol of Hepa1-6 cells.2. The palmitoylation rather than phosphorylation of Cav-1 may contri- bute to CD147 glycosylation in Hepa1-6 cells.3. Transmembrane domain of Cav-1 protects Cav-1 protein from prote- olysis by ubiquitin-proteosome pathway. |
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