DNA-PK phosphorylation of IGFBP-3 is required to prevent apoptosis in retinal endothelial cells cultured in high glucose.

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 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 S(156)A 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 S(156)A 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 S(156)A mutant plasmid (P < 0.05). Casein kinase levels were not altered after treatment with NU7741 or Compound 49b.
CONCLUSIONS: 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.