title: Proteasome-dependent regulation of signal transduction in retinal pigment epithelial cells.
abstract: As in many other types of cells, retinal pigment epithelial (RPE) cells have an active ubiquitin-proteasome pathway (UPP). However, the function of the UPP in RPE remains to be elucidated. The objective of this study is to determine the role of the UPP in controlling the levels and activities of transcription factors hypoxia-inducible factor (HIF) and NF-kappaB. We inhibited the UPP with proteasome-specific inhibitors and determined the activation of HIF and NF-kappaB as well as the expression and secretion of pro-angiogenic factors. HIF-1alpha was not detectable in ARPE-19 cells under normal culture conditions. However, when proteasome activity was inhibited, HIF-1alpha accumulated in RPE in a time-dependent manner. Consistent with accumulation of HIF-1alpha in the cells, levels of mRNA for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in RPE were up to 7-fold higher upon inhibition of the proteasome. Proteasome inhibition was also associated with a 2-fold increase in levels of mRNA for angiopoietin-1 (Ang-1). ARPE-19 cells secrete significant levels of VEGF under normal culture conditions. Inhibition of proteasome activity increased the secretion of VEGF by 2-fold. In contrast to the increase in HIF activity, NF-kappaB activation was reduced by proteasome inhibition. In addition, the expression and secretion of monocyte chemoattractant protein-1 (MCP-1) by RPE were substantially attenuated by the inhibition of proteasome activity. These data demonstrate that the UPP plays an important role in modulating the activities of HIF and NF-kappaB in the RPE. Consequences of an impairment of the UPP include accumulation of HIF-1alpha and diminished NF-kappaB activation, which lead to enhanced expression and secretion of pro-angiogenic factors and attenuated expression of MCP-1. Taken together, these data predict that the impairment of the UPP could lead to the development of AMD-related phenotypes.
authors.affiliation: Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111, USA.
authors.forename: Alexandre F
authors.initials: AF
authors.lastname: Fernandes
authors.forename: Weimin
authors.initials: W
authors.lastname: Guo
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhang
authors.forename: Matthew
authors.initials: M
authors.lastname: Gallagher
authors.forename: Mircea
authors.initials: M
authors.lastname: Ivan
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
authors.forename: Paulo
authors.initials: P
authors.lastname: Pereira
authors.forename: Fu
authors.initials: F
authors.lastname: Shang
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D025801:Ubiquitin / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D043925:Angiogenesis Inducing Agents; C111835:CCL2 protein, human; D018932:Chemokine CCL2; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D025801:Ubiquitin; D042461:Vascular Endothelial Growth Factor A; D046988:Proteasome Endopeptidase Complex