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Qiuhua Zhang, Jena J. Steinle; DNA-PK Phosphorylation of IGFBP-3 Is Required to Prevent Apoptosis in Retinal Endothelial Cells Cultured in High Glucose. Invest. Ophthalmol. Vis. Sci. 2013;54(4):3052-3057. doi: 10.1167/iovs.12-11533.
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The goal of this study was to determine whether Compound 49b stimulates insulin-like growth factor binding protein-3 (IGFBP-3) activation in retinal endothelial cells (REC) through DNA-dependent protein kinase (DNA-PK).
REC were grown in a normal glucose (5 mM) or high glucose medium (25 mM). Some cells were transfected with protein kinase A (PKA) siRNA, following treatment with 50 nM Compound 49b, a novel β-adrenergic receptor agonist. Cell proteins were extracted and analyzed for DNA-PK expression by Western blotting. Additional cells were treated with or without NU7441 (a specific DNA-PK inhibitor) prior to Compound 49b treatment. Cell lysates were processed for IGFBP-3 ELISA analyses and Western blotting to measure casein kinase 2 (CK2). Immunoprecipitation for total and phospho–IGFBP-3, cell proliferation and cell death measurements were done after transfection with the S156A IGFBP-3 mutation (key phosphorylation site involved in DNA-PK) plasmid DNA.
Compound 49b required DNA-PK to activate IGFBP-3 in REC. IGFBP-3 activation was significantly reduced following treatment with either the DNA-PK inhibitor or following transfection with the IGFBP-3 S156A mutant plasmid (P < 0.05). Significant increases in cell death and decreases in cell proliferation were also observed in cells transfected with the IGFBP-3 S156A mutant plasmid (P < 0.05). Casein kinase levels were not altered after treatment with NU7741 or Compound 49b.
Our findings suggest Compound 49b induces DNA-PK levels through PKA activity. DNA-PK is required for Compound 49b–induced IGFBP-3 expression, leading to inhibition of REC cell death.
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