Abstract 10808: Clusterin Up-regulation and Akt Pathway Activation Are Key to GATA-4 Mediated Cytoprotection of Mesenchymal Stem Cells Against Ischemia Injury

Clusterin (Clu) is a stress-responding protein with multiple biological functions, including the inhibition of apoptosis and inflammation. Our preliminary microarray studies show that clusterin was prominently upregulated in mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC GATA-4 ). Here, we directly investigate whether the upregulation of clusterin is involved in GATA-4 mediated cytoprotection. Methods: MSCs harvested from bone marrow of SD rats were transduced with GATA-4. The expression of clusterin in MSCs was documented by real-time PCR and western blotting. Simulation of ischemia was achieved by MSCs exposure to a hypoxic environment. Lactate dehydrogenase (LDH) released from MSCs was served as a biomarker of cell injury and MTT uptake was used to estimate cell viability. Mitochondrial function was assessed using mitochondrial membrane potential (ΔΨm) and caspase 3/7 activity. Results: (1) Clusterin expression was up-regulated in MSC GATA-4 compared to control MSCs which transfected with empty-vector (MSC Null ) ( Fig. A1 - A2 ). MSC GATA-4 were tolerant to 72 h hypoxia exposure; expressed as less LDH release and higher MTT uptake ( Fig. A3 ). This protection was abrogated by transfecting with Clu-siRNA ( Fig. A4 - A5 ). (2) Exogenous clusterin significantly decreased LDH release and increased MSC survival in hypoxic environment. Moreover, ΔΨm was maintained and caspase 3/7 activity was reduced by exogenous clusterin in a concentration-dependent manner (Fig. B ). (3) p-Akt expression in MSCs was upregulated following pre-treated with clusterin, although there was no significant change in total Akt ( Fig. C1 - C2 ). Moreover, cytoprotection mediated by clusterin (2μM/ml) was partially abrogated by Akt inhibitor LY294002 (5μMml) ( Fig. C3 ). Conclusions: Up-regulation of clusterin and activation of Akt signaling pathway is involved in GATA-4 mediated cytoprotection consequent to hypoxia stress.