title: Clusterin/Akt Up-Regulation Is Critical for GATA-4 Mediated Cytoprotection of Mesenchymal Stem Cells against Ischemia Injury.
abstract: 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 (MSCGATA-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 biomarker of cell injury and MTs uptake was used to estimate cell viability. Mitochondrial function was evaluated by measuring mitochondrial membrane potential (ΔΨm) and caspase 3/7 activity.
  
  
  RESULTS
  (1) Clusterin expression was up-regulated in MSCGATA-4 compared to control MSCs transfected with empty-vector (MSCNull). MSCGATA-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 MSCGATA-4. (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 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Bin
authors.initials: B
authors.lastname: Yu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Yueting
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Huan
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Min
authors.initials: M
authors.lastname: Gong
authors.affiliation: Department of Pharmacology & Cell Biophysics, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Millard
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Yi-Gang
authors.initials: YG
authors.lastname: Wang
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ashraf
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Meifeng
authors.initials: M
authors.lastname: Xu
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D015687:Cell Hypoxia; D051152:Clusterin / Q000096:biosynthesis*; D050991:GATA4 Transcription Factor / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: C498500:Clu protein, rat; D051152:Clusterin; D050991:GATA4 Transcription Factor; C084365:Gata4 protein, rat; D007770:L-Lactate Dehydrogenase; D051058:Oncogene Protein v-akt; C505410:Casp3 protein, rat; D053148:Caspase 3; D053179:Caspase 7