| ObjectiveThe plasma membrane glycoprotein CD147(also known as Basigin or EMMPRIN) is a multifunctional member of the immunoglobulin superfamily. The previous studies have shown that CD147was involved in melanoma cell proliferation, invasiveness, and metastasis, while the mechanism has not been very clear. Calcium-modulating cyclophilin ligand (CAML) is a ubiquitous transmembrane protein localized primarily to the endoplasmic reticulum (ER), which can trigger calcium influx. The aim of the present study was to determine the association between CD147and CAML, then to investigate the interaction region, location and regulation of intracellular calcium concentration ([Ca2+]i) in melanoma cells, in order to confirm the molecular mechanisms of malignant melanoma.Methods1. The yeast two-hybrid system was used to screen the proteins interacting with CD147from a human fetal cDNA library.2. The eukaryotic expression vectors of CAML, full length and mutants of CD147were constructed. 3. The co-immunoprecipitation (co-IP) was used to verify the interaction between CD147and CAML, and confirm the interaction site.4. The location of CD147and CAML in A375cells was detected by cell immunofluorence; meanwhile the plasmids of CAML and mutants of CD147were co-transfected in A375cells to find which region of the CD147was responsible for co-location.5. Small interfering RNAs (siRNAs) were constructed to transfect the A375cells.6. The Fluo-4, AM was used to detected the rest [Ca2+]i in A375cells with CD147silenced compared with the wild type, and the change of [Ca2+]i after the stimulation of tharpsigargin (TG) between the CD147silenced cells and the wild type.7. Both of the PMA and TG were used to elevate the cytoplasmic calcium of A375cells, and the MMP-9in cells expressing the sh-CD147plasmids or expressing the control plasmids were demonstrated by western blotting.Results1. The interaction between CD147and CAML was confirmed by the yeast two-hybrid screen.2. The eukaryotic expression vectors of CAML, full length and mutants of CD147were successfully constructed. 3. The interaction between CD147and CAML was verified by co-IP, and the transmembrane domain as the interaction site was also convinced.4. The CD147and CAML co-localized in ER, but the CD147mutant without transmembrane and intracellular domain couldn’t co-localize with CAML.5. Two fragments of CD147siRNA were successfully designed and utilized, using the nonspecific siRNA as a control.6. The rest [Ca2+]i of A375cells with CD147silenced were lower than the wild type, and the elevating level of cytoplasmic calcium after the TG stimulation was also the same.7. Both of the PMA and TG can promote the production of MMP-9, and the cells transfected with sh-CD147were lower than the cells transfected with the control plasmidsConclusions1. The CD147and CAML interacted with each other, and the interacting region of CD147was in the transmembrane domain.2. The CD147and CAML co-localized in the ER of A375cells, and the transmembrane and intracellular domain was responsible for the co-localization.3. CD147had a positive regulation in cytoplasmic calcium concentration of A375cells. 4. The cytoplasmic calcium may promote the production of MMP-9through the transduction of CD147. |
