title: DNA-PK phosphorylation of IGFBP-3 is required to prevent apoptosis in retinal endothelial cells cultured in high glucose.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, USA.
authors.forename: Qiuhua
authors.initials: Q
authors.lastname: Zhang
authors.forename: Jena J
authors.initials: JJ
authors.lastname: Steinle
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D047388:Casein Kinases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D042783:Endothelial Cells / Q000473:pathology*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation; D034741:RNA, Small Interfering; D012160:Retina / Q000473:pathology*; D014162:Transfection
chemical_list: C499693:8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one; D000318:Adrenergic beta-Agonists; D002867:Chromones; D018972:Insulin-Like Growth Factor Binding Protein 3; D009025:Morpholines; D034741:RNA, Small Interfering; D047388:Casein Kinases; D051747:DNA-Activated Protein Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose
references.citation: J Endocrinol. 2001 Nov;171(2):273-84
references.pmid: 11691647
references.citation: Circ Res. 2009 Oct 23;105(9):897-905
references.pmid: 19762684
references.citation: Endocrinology. 2003 May;144(5):1984-93
references.pmid: 12697706
references.citation: Biogerontology. 2003;4(1):1-8
references.pmid: 12652183
references.citation: Mol Cell Endocrinol. 2012 Sep 25;361(1-2):172-8
references.pmid: 22548717
references.citation: Invest Ophthalmol Vis Sci. 2003 Jan;44(1):365-9
references.pmid: 12506097
references.citation: Cancer Res. 2006 Mar 15;66(6):3024-33
references.pmid: 16540651
references.citation: Biochem Biophys Res Commun. 1993 Nov 15;196(3):1267-73
references.pmid: 7504473
references.citation: Invest Ophthalmol Vis Sci. 2012 May 17;53(6):3004-13
references.pmid: 22467575
references.citation: Biochem Biophys Res Commun. 2003 Oct 10;310(1):163-8
references.pmid: 14511665
references.citation: Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10595-600
references.pmid: 17567755
references.citation: Mol Endocrinol. 2009 Oct;23(10):1624-33
references.pmid: 19556345
references.citation: FASEB J. 2004 Nov;18(14):1719-21
references.pmid: 15371331
references.citation: Diabetes Res Clin Pract. 2009 Nov;86(2):146-51
references.pmid: 19783316
references.citation: J Endocrinol. 1999 Mar;160(3):379-87
references.pmid: 10076184
references.citation: Blood. 2011 Sep 1;118(9):2622-31
references.pmid: 21551235
references.citation: Cancer Lett. 2002 Feb 25;176(2):149-58
references.pmid: 11804742
references.citation: Endocrinology. 2012 Oct;153(10):4799-807
references.pmid: 22910030
references.citation: PLoS One. 2012;7(7):e39398
references.pmid: 22792172
references.citation: J Clin Endocrinol Metab. 1994 May;78(5):1119-27
references.pmid: 7513716
references.citation: Am J Physiol Cell Physiol. 2009 May;296(5):C954-76
references.pmid: 19279229
references.citation: Diabetes Technol Ther. 2000 Spring;2(1):69-80
references.pmid: 11467325
references.citation: J Biol Chem. 1999 Oct 15;274(42):30215-21
references.pmid: 10514513
references.citation: J Med Chem. 2005 Dec 1;48(24):7829-46
references.pmid: 16302822
references.citation: J Mol Endocrinol. 2005 Feb;34(1):163-75
references.pmid: 15691886
references.citation: Cancer Res. 2006 Nov 15;66(22):10878-84
references.pmid: 17108124
references.citation: Clin Cancer Res. 2002 Dec;8(12):3796-802
references.pmid: 12473592
references.citation: Endocrinology. 2000 Feb;141(2):564-70
references.pmid: 10650937
references.citation: Oncol Res. 2003;13(6-10):359-71
references.pmid: 12725526
references.citation: J Cell Physiol. 2009 Jul;220(1):182-8
references.pmid: 19259947
references.citation: Apoptosis. 2013 Mar;18(3):361-8
references.pmid: 23291901
references.citation: J Cell Biochem. 2002;86(3):583-9
references.pmid: 12210764
references.citation: Clin Biochem. 2010 Jun;43(9):725-31
references.pmid: 20307522