Tetraspanin Family Member, CD82, Regulates Expression of EZH2 via Inactivation of p38 MAPK Signaling in Leukemia Cells

PURPOSE We recently found that the tetraspanin family member, CD82, which is aberrantly expressed in chemotherapy-resistant CD34(+)/CD38- acute myelogenous leukemia (AML) cells, negatively regulates matrix metalloproteinase 9, and plays an important role in enabling CD34(+)/CD38(-) AML cells to adhere to the bone marrow microenvironment. This study explored novel functions of CD82 that contribute to AML progression. MATERIALS AND METHODS We employed microarray analysis comparing the gene expression profiles between CD34(+)/CD38(-) AML cells transduced with CD82 shRNA and CD34(+)/CD38(-) AML cells transduced with control shRNA. Real-time RT-PCR and western blot analysis were performed to examine the effect of CD82 knockdown on the expression of the polycomb group member, enhancer of zeste homolog 2 (EZH2), in leukemia cells. A chromatin immunoprecipitation assay was performed to examine the effect of CD82 expression on the amount of EZH2 bound to the promoter regions of tumor suppressor genes in leukemia cells. We also utilized methylation-specific PCR to examine whether CD82 expression influences the methylation status of the tumor suppressor gene promoter regions in leukemia cells. RESULTS Microarray analysis revealed that levels of EZH2 decreased after shRNA-mediated depletion of CD82 in CD34(+)/CD38(-) AML cells. Moreover, the antibody-mediated blockade of CD82 in leukemia cells lowered EZH2 expression via activation of p38 MAPK signaling, decreased the amount of EZH2 bound to the promoter regions of the tumor suppressor genes, and inhibited histone H3 lysine 27 trimethylation in these promoter regions, resulting in upregulation of the tumor suppressors at both the mRNA and protein levels.