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Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells

Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation fr...

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Autores principales: Lampart, Agata, Sluzalska, Katarzyna Dominika, Czyrek, Aleksandra, Szerszen, Aleksandra, Otlewski, Jacek, Wiedlocha, Antoni, Zakrzewska, Malgorzata
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8833943/
https://www.ncbi.nlm.nih.gov/pubmed/35159330
http://dx.doi.org/10.3390/cells11030522
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author Lampart, Agata
Sluzalska, Katarzyna Dominika
Czyrek, Aleksandra
Szerszen, Aleksandra
Otlewski, Jacek
Wiedlocha, Antoni
Zakrzewska, Malgorzata
author_facet Lampart, Agata
Sluzalska, Katarzyna Dominika
Czyrek, Aleksandra
Szerszen, Aleksandra
Otlewski, Jacek
Wiedlocha, Antoni
Zakrzewska, Malgorzata
author_sort Lampart, Agata
collection PubMed
description Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation.
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spelling pubmed-88339432022-02-12 Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells Lampart, Agata Sluzalska, Katarzyna Dominika Czyrek, Aleksandra Szerszen, Aleksandra Otlewski, Jacek Wiedlocha, Antoni Zakrzewska, Malgorzata Cells Article Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation. MDPI 2022-02-02 /pmc/articles/PMC8833943/ /pubmed/35159330 http://dx.doi.org/10.3390/cells11030522 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lampart, Agata
Sluzalska, Katarzyna Dominika
Czyrek, Aleksandra
Szerszen, Aleksandra
Otlewski, Jacek
Wiedlocha, Antoni
Zakrzewska, Malgorzata
Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells
title Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells
title_full Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells
title_fullStr Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells
title_full_unstemmed Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells
title_short Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells
title_sort nuclear localization sequence of fgf1 is not required for its intracellular anti-apoptotic activity in differentiated cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8833943/
https://www.ncbi.nlm.nih.gov/pubmed/35159330
http://dx.doi.org/10.3390/cells11030522
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