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UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts

BACKGROUND: Pterygium is a condition characterized by epithelial overgrowth of the cornea, inflammatory cell infiltration and an abnormal extracellular matrix accumulation. Chronic UV exposure is considered as a pathogenic factor of this disease. Proteasome is an intracellular multi-subunit protease...

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Autores principales: Aletras, Alexios J., Trilivas, Ioannis, Christopoulou, Maria-Elpida, Drakouli, Sotiria, Georgakopoulos, Constantine D., Pharmakakis, Nikolaos
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299496/
https://www.ncbi.nlm.nih.gov/pubmed/30563490
http://dx.doi.org/10.1186/s12886-018-0987-8
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author Aletras, Alexios J.
Trilivas, Ioannis
Christopoulou, Maria-Elpida
Drakouli, Sotiria
Georgakopoulos, Constantine D.
Pharmakakis, Nikolaos
author_facet Aletras, Alexios J.
Trilivas, Ioannis
Christopoulou, Maria-Elpida
Drakouli, Sotiria
Georgakopoulos, Constantine D.
Pharmakakis, Nikolaos
author_sort Aletras, Alexios J.
collection PubMed
description BACKGROUND: Pterygium is a condition characterized by epithelial overgrowth of the cornea, inflammatory cell infiltration and an abnormal extracellular matrix accumulation. Chronic UV exposure is considered as a pathogenic factor of this disease. Proteasome is an intracellular multi-subunit protease complex that degrades intracellular proteins. Among proteasome subunits the β5 (PSMB5), bearing chymotrypsin-like activity. It is considered as the main proteasome subunit and its expression is mediated by Nrf2-ARE pathway in many cell types. This study investigates the expression of PSMB5 in pterygium and the effect of UVB irradiation on its expression and activity in pterygium fibroblasts. METHODS: Normal conjunctival and pterygium specimens were obtained from the bulbar conjunctiva of patients undergoing cataract surgery and from patients with pterygium undergoing surgical removal of primary tissue, respectively. Fibroblasts were isolated upon treatment of specimens with clostridium collagenase. The expression of PSMB5 and Nrf2 in tissues and cells was ascertained by RT-PCR analysis and western blotting. Cell survival was measured by the MTT method and the proteasome chymotrypsin-like activity was determined by fluorometry. RESULTS: RT-PCR analysis showed that the expression of PSMB5 was significantly lower in pterygium than in normal conjunctiva. The expression of PSMB5 was mediated by the Nrf2/ARE pathway as indicated by using the Nrf2 activator Oltipraz. The expression of PSMB5 and Nrf2 by pterygium fibroblasts was suppressed in a dose dependent manner following UVB radiation of 0–50 mJ/cm(2) doses. The expression of PSMB5, but not of Nrf2, remained at almost the control levels, when UVB exposure was performed after pre-incubation of cells with the src kinases inhibitor PP2. UVB irradiation had very low deleterious effect on fibroblasts survival, while it did not affect the proteasome chymotrypsin-like activity. CONCLUSION: In pterygium fibroblasts, UVB exposure leads to down-regulation of Nrf2/ARE-mediated PSMB5 gene expression, in which src kinases may be implicated. This effect may be partially responsible for the lower expression of PSMB5 detected in pterygium as compared to normal conjunctiva.
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spelling pubmed-62994962018-12-20 UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts Aletras, Alexios J. Trilivas, Ioannis Christopoulou, Maria-Elpida Drakouli, Sotiria Georgakopoulos, Constantine D. Pharmakakis, Nikolaos BMC Ophthalmol Research Article BACKGROUND: Pterygium is a condition characterized by epithelial overgrowth of the cornea, inflammatory cell infiltration and an abnormal extracellular matrix accumulation. Chronic UV exposure is considered as a pathogenic factor of this disease. Proteasome is an intracellular multi-subunit protease complex that degrades intracellular proteins. Among proteasome subunits the β5 (PSMB5), bearing chymotrypsin-like activity. It is considered as the main proteasome subunit and its expression is mediated by Nrf2-ARE pathway in many cell types. This study investigates the expression of PSMB5 in pterygium and the effect of UVB irradiation on its expression and activity in pterygium fibroblasts. METHODS: Normal conjunctival and pterygium specimens were obtained from the bulbar conjunctiva of patients undergoing cataract surgery and from patients with pterygium undergoing surgical removal of primary tissue, respectively. Fibroblasts were isolated upon treatment of specimens with clostridium collagenase. The expression of PSMB5 and Nrf2 in tissues and cells was ascertained by RT-PCR analysis and western blotting. Cell survival was measured by the MTT method and the proteasome chymotrypsin-like activity was determined by fluorometry. RESULTS: RT-PCR analysis showed that the expression of PSMB5 was significantly lower in pterygium than in normal conjunctiva. The expression of PSMB5 was mediated by the Nrf2/ARE pathway as indicated by using the Nrf2 activator Oltipraz. The expression of PSMB5 and Nrf2 by pterygium fibroblasts was suppressed in a dose dependent manner following UVB radiation of 0–50 mJ/cm(2) doses. The expression of PSMB5, but not of Nrf2, remained at almost the control levels, when UVB exposure was performed after pre-incubation of cells with the src kinases inhibitor PP2. UVB irradiation had very low deleterious effect on fibroblasts survival, while it did not affect the proteasome chymotrypsin-like activity. CONCLUSION: In pterygium fibroblasts, UVB exposure leads to down-regulation of Nrf2/ARE-mediated PSMB5 gene expression, in which src kinases may be implicated. This effect may be partially responsible for the lower expression of PSMB5 detected in pterygium as compared to normal conjunctiva. BioMed Central 2018-12-18 /pmc/articles/PMC6299496/ /pubmed/30563490 http://dx.doi.org/10.1186/s12886-018-0987-8 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Aletras, Alexios J.
Trilivas, Ioannis
Christopoulou, Maria-Elpida
Drakouli, Sotiria
Georgakopoulos, Constantine D.
Pharmakakis, Nikolaos
UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
title UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
title_full UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
title_fullStr UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
title_full_unstemmed UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
title_short UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
title_sort uvb-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299496/
https://www.ncbi.nlm.nih.gov/pubmed/30563490
http://dx.doi.org/10.1186/s12886-018-0987-8
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