Transcriptional Repression of Mad-Max Complex by Human Umbilical Cord Blood Stem Cells Downregulates Extracellular Signal-Regulated Kinase in Glioblastoma

Abstract

Previously, we have shown that human umbilical cord blood stem cell (hUCBSC) treatment downregulate cyclin D1 in glioma cells. To study the cell cycle progression and investigate the upstream molecules regulating cyclin D1 expression, we analyzed the involvement of extracellular signal-regulated kinase (ERK) and its functionality after treatment with hUCBSC. We observed downregulation of pERK after hUCBSC treatment at both transcriptional and translational levels. Increased translocation of ERK from cytoplasm to the nucleus was observed in glioma cells, whereas hUCBSC cocultures with glioma cells showed suppressed nuclear translocation. This finding suggests that hUCBSC regulates ERK by suppressing its phosphorylation at phospho-Thr²⁰²/Tyr²⁰⁴ retarding pERK nuclear translocation. ERK promoter analysis has shown c-Myc binding sites, indicative of possible transcriptional interactions that regulate cyclin D1 and ERK expression levels. Treatment of U251 and 5310 glioma cells with U0126, a MEK/ERK inhibitor receded pERK and c-Myc levels. In another experiment, U251 and 5310 cells treated with 10074-G5, c-Myc/Max inhibitor displayed reduction in pERK and c-Myc levels suggestive of a positive feedback loop between ERK/c-Myc/Max molecules. In the present study, we show that glioma cells exhibit abundant c-Myc expression and increased c-Myc/Max activity. In contrast, the glioma cells cocultured with hUCBSC demonstrated high Mad1 expression that competitively binds to Max to repress the c-Myc/Max mediated gene transcription. Our studies thus elucidate the potential role of hUCBSC in controlling glioma cell cycle progression and invasion by limiting Max binding to c-Myc, thus regulating the expression of glioma cell cycle and invasion associated molecules such as ERK, integrins via increased levels of Mad1 expression.