Clusterin/Akt up-regulation is critical for GATA-4 mediated cytoprotection of mesenchymal stem cells against ischemia injury

Background: Clusterin (Clu) is a stress-responding protein with multiple biological functions. Our preliminary microarray studies show that clusterin was prominently upregulated in mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC^GATA-4). We hypothesized that the upregulation of clusterin is involved in overexpression of GATA-4 mediated cytoprotection. Methods: MSCs harvested from bone marrow of rats were transduced with GATA-4. The expression of clusterin in MSCs was further confirmed by real-time PCR and western blotting. Simulation of ischemia was achieved by exposure of MSCs to a hypoxic environment. Lactate dehydrogenase (LDH) released from MSCs was served as a biomarkerof cell injury and MTs uptake was used to estimate cell viability. Mitochondrial function was evaluated by measuring mitochondrial membrane potential (Δm) and caspase 3/7 activity. Results: (1) Clusterin expression was up-regulated in MSC^GATA-4 compared to control MSCs transfected with empty-vector (MSC^Null). MSC^GATA-4 were tolerant to 72 h hypoxia exposure as shown by reduced LDH release and higher MTs uptake. This protection was abrogated by transfecting Clu-siRNA into MSC^GATA-4. (2) Exogenous clusterin significantly decreased LDH release and increased MSC survival in hypoxic environment. Moreover, AMμm was maintained and caspase 3/7 activity was reduced by clusterin in a concentration-dependent manner. (3) p-Akt expression in MSCs was upregulated following pre-treatment with clusterin, with no change in total Akt. Moreover, cytoprotection mediated by clusterin was partially abrogated by Akt inhibitor LY294002. Conclusions: Clusterin/Akt signaling pathway is involved in GATA-4 mediated cytoprotection against hypoxia stress. It is suggested that clusterin may be therapeutically exploited in MSC based therapy for cardiovascular diseases.