DNA-PK phosphorylation of IGFBP-3 is required to Prevent Apoptosis in Retinal Endothelial cells cultured in High Glucose.

DNA-PK phosphorylation of IGFBP-3 is required to Prevent Apoptosis in Retinal Endothelial cells cultured in High Glucose.

Invest Ophthalmol Vis Sci. 2013 Apr 4;

Authors: Zhang Q, Steinle JJ

Abstract
PURPOSE: 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). METHODS: REC were grown in 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 ?-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 and cell death measurements were done after transfection with the S156A IGFBP-3 mutation (key phosphorylation site involved in DNA-PK) plasmid DNA. RESULTS: 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 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. CONCLUSIONS: Our finding suggested Compound 49b induces DNA-PK levels through PKA activity. DNA-PK is required for Compound 49b-induced activation of IGFBP-3, leading to inhibition of REC cell death.

PMID: 23557743 [PubMed - as supplied by publisher]

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