Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells

Autores
Gutierrez Jofré, Gabriela Mabel; Torlaschi, Camila; Mateos, Melina Valeria; Rotstein, Nora Patricia; Simon, Maria Victoria
Año de publicación
2024
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Introduction Retinal pigment epithelial (RPE) cells play multiple functions in the retina, preserving visual functionality. Autophagy is crucial for RPE degradative functions, and its dysregulation contributes to pathological conditions. The roles of sphingolipids in autophagy regulation are scarcely known. Sphingosine kinase (SphK1), which catalyzes the synthesis of sphingosine-1-phosphate (S1P), has been shown to facilitate endosomal trafficking. Objectives Our purpose was to evaluate whether SphK1 activity modulated autophagic flux in RPE cells. Methods Human RPE cell line (ARPE-19) cultures were treated with 10 μM PF543, a SphK1 inhibitor, with NVP, a ceramide kinase inhi- bitor, with 5 μM S1P or 10 μM C1P, or with 10 μM Bafilomycin 1 (BafA1), for 24 and 48 h. Cell morphology was determined with phalloidin. Cell death was analyzed by MTT assay. Formation of autophagosomes was evaluated by immunocytochemistry, using antibodies for LC3b and p62, specific autophagosome markers. Results Inhibiting SphK1 with PF543 for 24 h promoted morphological changes in ARPE-19 cells and the formation of perinuclear round vesicles, scarce in controls, which increased after 48 h. PF543 also induced endoplasmic reticulum enlargement, but had neither an effect on mitochondria nor on cell viability. Blocking synthesis of ceramide-1-phosphate (C1P), whose functions are similar to those of S1P, did not induce vesicle formation. The vesicles showed intense labeling with LC3b and p62 antibodies, suggesting that they might be “autophagosomes”. Treatment of RPE cells 24 and 48 h with BafA1, which disrupts endocytic flow, led to the accumulation of LC3b- and p62-positive vesicles and alterations in cell morphology remarkably similar to those induced by PF543. The amount of LC3b- and p62-positive vesicles was further increased with the combined addition of PF543 and BafA1, suggesting that PF543 enhanced autophagic flux, and BafA1 prevented vesicle degradation. Supplementation with S1P 1 h after PF543 addition restored cell morphology but did not prevent vesicle formation. Conclusion Our results suggest that inhibition of SPhK1 promoted morphological changes in RPE cells and the formation of LC3b and p62-po- sitive vesicles, possibly autophagosomes. Exogenous S1P preserved morphology but did not prevent autophagosome formation, implying S1P receptor activation did not regulate this formation and suggesting that SphK1 activity was essential for maintaining proper autophagic flux
Fil: Gutierrez Jofré, Gabriela Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Torlaschi, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Simon, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
XXVIth biennial meeting of the International Society of Eye Research
Buenos Aires
Argentina
International Society of Eye Research
Materia
RETINAL PIGMENT EPITHELIAL CELLS
SPHINGOSINE KINASE
AUTOPHAGIC FLUX
SPHINGOLIPIDS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/250493
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/250493
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cellsGutierrez Jofré, Gabriela MabelTorlaschi, CamilaMateos, Melina ValeriaRotstein, Nora PatriciaSimon, Maria VictoriaRETINAL PIGMENT EPITHELIAL CELLSSPHINGOSINE KINASEAUTOPHAGIC FLUXSPHINGOLIPIDShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Introduction Retinal pigment epithelial (RPE) cells play multiple functions in the retina, preserving visual functionality. Autophagy is crucial for RPE degradative functions, and its dysregulation contributes to pathological conditions. The roles of sphingolipids in autophagy regulation are scarcely known. Sphingosine kinase (SphK1), which catalyzes the synthesis of sphingosine-1-phosphate (S1P), has been shown to facilitate endosomal trafficking. Objectives Our purpose was to evaluate whether SphK1 activity modulated autophagic flux in RPE cells. Methods Human RPE cell line (ARPE-19) cultures were treated with 10 μM PF543, a SphK1 inhibitor, with NVP, a ceramide kinase inhi- bitor, with 5 μM S1P or 10 μM C1P, or with 10 μM Bafilomycin 1 (BafA1), for 24 and 48 h. Cell morphology was determined with phalloidin. Cell death was analyzed by MTT assay. Formation of autophagosomes was evaluated by immunocytochemistry, using antibodies for LC3b and p62, specific autophagosome markers. Results Inhibiting SphK1 with PF543 for 24 h promoted morphological changes in ARPE-19 cells and the formation of perinuclear round vesicles, scarce in controls, which increased after 48 h. PF543 also induced endoplasmic reticulum enlargement, but had neither an effect on mitochondria nor on cell viability. Blocking synthesis of ceramide-1-phosphate (C1P), whose functions are similar to those of S1P, did not induce vesicle formation. The vesicles showed intense labeling with LC3b and p62 antibodies, suggesting that they might be “autophagosomes”. Treatment of RPE cells 24 and 48 h with BafA1, which disrupts endocytic flow, led to the accumulation of LC3b- and p62-positive vesicles and alterations in cell morphology remarkably similar to those induced by PF543. The amount of LC3b- and p62-positive vesicles was further increased with the combined addition of PF543 and BafA1, suggesting that PF543 enhanced autophagic flux, and BafA1 prevented vesicle degradation. Supplementation with S1P 1 h after PF543 addition restored cell morphology but did not prevent vesicle formation. Conclusion Our results suggest that inhibition of SPhK1 promoted morphological changes in RPE cells and the formation of LC3b and p62-po- sitive vesicles, possibly autophagosomes. Exogenous S1P preserved morphology but did not prevent autophagosome formation, implying S1P receptor activation did not regulate this formation and suggesting that SphK1 activity was essential for maintaining proper autophagic fluxFil: Gutierrez Jofré, Gabriela Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Torlaschi, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Simon, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaXXVIth biennial meeting of the International Society of Eye ResearchBuenos AiresArgentinaInternational Society of Eye ResearchInternational Society of Eye Research2024info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectCongresoBookhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/250493Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells; XXVIth biennial meeting of the International Society of Eye Research; Buenos Aires; Argentina; 2024; 617-617CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://iserbiennialmeeting2024.org/abstract-book/Internacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:44:19Zoai:ri.conicet.gov.ar:11336/250493instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:44:20.138CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
title Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
spellingShingle Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
Gutierrez Jofré, Gabriela Mabel
RETINAL PIGMENT EPITHELIAL CELLS
SPHINGOSINE KINASE
AUTOPHAGIC FLUX
SPHINGOLIPIDS
title_short Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
title_full Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
title_fullStr Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
title_full_unstemmed Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
title_sort Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells
dc.creator.none.fl_str_mv Gutierrez Jofré, Gabriela Mabel
Torlaschi, Camila
Mateos, Melina Valeria
Rotstein, Nora Patricia
Simon, Maria Victoria
author Gutierrez Jofré, Gabriela Mabel
author_facet Gutierrez Jofré, Gabriela Mabel
Torlaschi, Camila
Mateos, Melina Valeria
Rotstein, Nora Patricia
Simon, Maria Victoria
author_role author
author2 Torlaschi, Camila
Mateos, Melina Valeria
Rotstein, Nora Patricia
Simon, Maria Victoria
author2_role author
author
author
author
dc.subject.none.fl_str_mv RETINAL PIGMENT EPITHELIAL CELLS
SPHINGOSINE KINASE
AUTOPHAGIC FLUX
SPHINGOLIPIDS
topic RETINAL PIGMENT EPITHELIAL CELLS
SPHINGOSINE KINASE
AUTOPHAGIC FLUX
SPHINGOLIPIDS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Introduction Retinal pigment epithelial (RPE) cells play multiple functions in the retina, preserving visual functionality. Autophagy is crucial for RPE degradative functions, and its dysregulation contributes to pathological conditions. The roles of sphingolipids in autophagy regulation are scarcely known. Sphingosine kinase (SphK1), which catalyzes the synthesis of sphingosine-1-phosphate (S1P), has been shown to facilitate endosomal trafficking. Objectives Our purpose was to evaluate whether SphK1 activity modulated autophagic flux in RPE cells. Methods Human RPE cell line (ARPE-19) cultures were treated with 10 μM PF543, a SphK1 inhibitor, with NVP, a ceramide kinase inhi- bitor, with 5 μM S1P or 10 μM C1P, or with 10 μM Bafilomycin 1 (BafA1), for 24 and 48 h. Cell morphology was determined with phalloidin. Cell death was analyzed by MTT assay. Formation of autophagosomes was evaluated by immunocytochemistry, using antibodies for LC3b and p62, specific autophagosome markers. Results Inhibiting SphK1 with PF543 for 24 h promoted morphological changes in ARPE-19 cells and the formation of perinuclear round vesicles, scarce in controls, which increased after 48 h. PF543 also induced endoplasmic reticulum enlargement, but had neither an effect on mitochondria nor on cell viability. Blocking synthesis of ceramide-1-phosphate (C1P), whose functions are similar to those of S1P, did not induce vesicle formation. The vesicles showed intense labeling with LC3b and p62 antibodies, suggesting that they might be “autophagosomes”. Treatment of RPE cells 24 and 48 h with BafA1, which disrupts endocytic flow, led to the accumulation of LC3b- and p62-positive vesicles and alterations in cell morphology remarkably similar to those induced by PF543. The amount of LC3b- and p62-positive vesicles was further increased with the combined addition of PF543 and BafA1, suggesting that PF543 enhanced autophagic flux, and BafA1 prevented vesicle degradation. Supplementation with S1P 1 h after PF543 addition restored cell morphology but did not prevent vesicle formation. Conclusion Our results suggest that inhibition of SPhK1 promoted morphological changes in RPE cells and the formation of LC3b and p62-po- sitive vesicles, possibly autophagosomes. Exogenous S1P preserved morphology but did not prevent autophagosome formation, implying S1P receptor activation did not regulate this formation and suggesting that SphK1 activity was essential for maintaining proper autophagic flux
Fil: Gutierrez Jofré, Gabriela Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Torlaschi, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Simon, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
XXVIth biennial meeting of the International Society of Eye Research
Buenos Aires
Argentina
International Society of Eye Research
description Introduction Retinal pigment epithelial (RPE) cells play multiple functions in the retina, preserving visual functionality. Autophagy is crucial for RPE degradative functions, and its dysregulation contributes to pathological conditions. The roles of sphingolipids in autophagy regulation are scarcely known. Sphingosine kinase (SphK1), which catalyzes the synthesis of sphingosine-1-phosphate (S1P), has been shown to facilitate endosomal trafficking. Objectives Our purpose was to evaluate whether SphK1 activity modulated autophagic flux in RPE cells. Methods Human RPE cell line (ARPE-19) cultures were treated with 10 μM PF543, a SphK1 inhibitor, with NVP, a ceramide kinase inhi- bitor, with 5 μM S1P or 10 μM C1P, or with 10 μM Bafilomycin 1 (BafA1), for 24 and 48 h. Cell morphology was determined with phalloidin. Cell death was analyzed by MTT assay. Formation of autophagosomes was evaluated by immunocytochemistry, using antibodies for LC3b and p62, specific autophagosome markers. Results Inhibiting SphK1 with PF543 for 24 h promoted morphological changes in ARPE-19 cells and the formation of perinuclear round vesicles, scarce in controls, which increased after 48 h. PF543 also induced endoplasmic reticulum enlargement, but had neither an effect on mitochondria nor on cell viability. Blocking synthesis of ceramide-1-phosphate (C1P), whose functions are similar to those of S1P, did not induce vesicle formation. The vesicles showed intense labeling with LC3b and p62 antibodies, suggesting that they might be “autophagosomes”. Treatment of RPE cells 24 and 48 h with BafA1, which disrupts endocytic flow, led to the accumulation of LC3b- and p62-positive vesicles and alterations in cell morphology remarkably similar to those induced by PF543. The amount of LC3b- and p62-positive vesicles was further increased with the combined addition of PF543 and BafA1, suggesting that PF543 enhanced autophagic flux, and BafA1 prevented vesicle degradation. Supplementation with S1P 1 h after PF543 addition restored cell morphology but did not prevent vesicle formation. Conclusion Our results suggest that inhibition of SPhK1 promoted morphological changes in RPE cells and the formation of LC3b and p62-po- sitive vesicles, possibly autophagosomes. Exogenous S1P preserved morphology but did not prevent autophagosome formation, implying S1P receptor activation did not regulate this formation and suggesting that SphK1 activity was essential for maintaining proper autophagic flux
publishDate 2024
dc.date.none.fl_str_mv 2024
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Congreso
Book
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/250493
Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells; XXVIth biennial meeting of the International Society of Eye Research; Buenos Aires; Argentina; 2024; 617-617
CONICET Digital
CONICET
url http://hdl.handle.net/11336/250493
identifier_str_mv Inhibiting sphingosine kinase 1 modulated autophagic flux in retinal pigment epithelial cells; XXVIth biennial meeting of the International Society of Eye Research; Buenos Aires; Argentina; 2024; 617-617
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://iserbiennialmeeting2024.org/abstract-book/
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
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eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.coverage.none.fl_str_mv Internacional
dc.publisher.none.fl_str_mv International Society of Eye Research
publisher.none.fl_str_mv International Society of Eye Research
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
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instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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