May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Adenosine Analog NECA Inactivates the Glycogen Synthase Kinase-3 (GSK-3) and Increases HIF-1 in HREC and HEK293 Cells
Author Affiliations & Notes
  • R.C. Miller
    Pharmacology and Therapeutics, University of Florida, Gainesville, FL, United States
  • S. Baker
    Pharmacology and Therapeutics, University of Florida, Gainesville, FL, United States
  • P.E. Spoerri
    Pharmacology and Therapeutics, University of Florida, Gainesville, FL, United States
  • M.B. Grant
    Pharmacology and Therapeutics, University of Florida, Gainesville, FL, United States
  • Footnotes
    Commercial Relationships  R.C. Miller, None; S. Baker, None; P.E. Spoerri, None; M.B. Grant, None.
  • Footnotes
    Support  The Juvenile Diabetes Research Foundation International; NIH grants EY012601 and EY007739
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2891. doi:
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      R.C. Miller, S. Baker, P.E. Spoerri, M.B. Grant; Adenosine Analog NECA Inactivates the Glycogen Synthase Kinase-3 (GSK-3) and Increases HIF-1 in HREC and HEK293 Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2891.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Ischemia induced increase in the nucleoside adenosine and vascular endothelial growth factor (VEGF) are implicated in vascular pathologies such as diabetic retinopathy and retinopathy of prematurity. Previously we have shown that stimulation of human retinal endothelial cells (HRECs) with adenosine or its analogue 5’N-ethylcarboxamide-adenosine (NECA) upregulates VEGF by activating the A2B receptor, one of the four G-protein coupled receptors (A1, A2A, A2B and A3). We sought to elucidate the cellular mechanism of the upregulation by studying whether adenosine affects the transcription enhancer HIF-1α, a regulator of VEGF expression. HIF-1α protein is stabilized by hypoxia or stimulation with insulin or IGF-I in certain cell types. The latter involves the PI3K/Akt pathway. Akt inactivates the glycogen synthase kinase 3 (GSK-3) that has been proposed to destabilize HIF-1α. We studied whether stimulation of HRECs and a second human cell line, HEK 293, with NECA inactivates GSK-3 and increases the HIF-1α protein. Methods: HRECs and HEK293 cells were stimulated with NECA in the presence and absence of selective adenosine receptor (AdoR) antagonists. Cytosolic and nuclear proteins were separated by fractionation. Phosphorylated GSK-3 (pGSK-3), HIF-1α and phosphorylated Akt (pAkt) proteins were measured by Western blotting. Results: NECA stimulation increased nuclear pGSK3, HIF-1α, and pAkt in the nucleus in a concentration-dependent manner in HEK-293 cells. Similarly, in HRECs, pGSK-3 and HIF-1α increased in the nucleus within 30 minutes when the cells were treated with NECA. The effects were attenuated by the AdoR antagonist, 3-isobutyl-8-pyrrolidinoxanthine (IPDX). Conclusion: NECA treatment resulted in inactivation of nuclear GSK-3 and in accumulation of HIF-1α in HRECs and HEK 293 cells. The results suggest that adenosine may stabilize HIF-1α by the PI3K/Akt/GSK-3 signaling pathway. Future studies will determine whether GSK-3 interacts directly with HIF-1α. These studies support a critical role of adenosine in HIF-1α accumulation and subsequent VEGF expression implicated in the development of proliferative retinopathies.

Keywords: neovascularization • retinal culture • signal transduction 
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