Direct GSK-3 beta Inhibition Enhances Mesenchymal Stromal Cell Migration by Increasing Expression of Beta-PIX and CXCR4

Abstract

Mesenchymal stromal cells (MSCs) are emerging as candidate cells for the treatment of neurological diseases because of their neural replacement, neuroprotective, and neurotrophic effects. However, the majority of MSCs transplanted by various routes fail to reach the site of injury, and they have demonstrated only minimal therapeutic benefit in clinical trials. Therefore, enhancing the migration of MSCs to target sites is essential for this therapeutic strategy to be effective. In this study, we assessed whether inhibition of glycogen synthase kinase-3 beta (GSK-3 beta) increases the migration capacity of MSCs during ex vivo expansion. Human bone marrow MSCs (hBM-MSCs) were cultured with various GSK-3 beta inhibitors (LiCl, SB-415286, and AR-A014418). Using a migration assay kit, we found that the motility of hBM-MSCs was significantly enhanced by GSK-3 beta inhibition. Western blot analysis revealed increased levels of migration-related signaling proteins such as phospho-GSK-3 beta, beta-catenin, phospho-c-Raf, phospho-extracellular signal-regulated kinase (ERK), phospho-beta-PAK-interacting exchange factor (PIX), and CXC chemokine receptor 4 (CXCR4). In addition, real-time polymerase chain reaction demonstrated increased expression of matrix metalloproteinase-2 (MMP-2), membrane-type MMP-1 (MT1-MMP), and beta-PIX. In the reverse approach, treatment with beta-PIX shRNA or CXCR4 inhibitor (AMD 3100) reduced hBM-MSC migration. These findings suggest that inhibition of GSK-3 beta during ex vivo expansion of hBM-MSCs may enhance their migration capacity by increasing expression of beta-catenin, phospho-c-Raf, phospho-ERK, and beta-PIX and the subsequent up-regulation of CXCR4. Enhancing the migration capacity of hBM-MSCs by treating these cells with GSK-3 beta inhibitors may increase their therapeutic potential.