Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells
Activation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cy...
| Autores: | , , , , , , , , |
|---|---|
| Tipo de documento: | artigo |
| Data de publicação: | 2022 |
| País: | España |
| Recursos: | Universidad Complutense de Madrid (UCM) |
| Repositório: | Docta Complutense |
| Idioma: | inglês |
| OAI Identifier: | oai:docta.ucm.es:20.500.14352/116128 |
| Acesso em linha: | https://hdl.handle.net/20.500.14352/116128 |
| Access Level: | Acceso aberto |
| Palavra-chave: | 612.017 Integrin RIAM PHAGOCYTOSIS Neutrophil αMβ2 VASP Ciencias Biomédicas 2412 Inmunología 32 Ciencias Médicas |
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Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cellsTorres Gómez, ÁlvaroFiyouzi, TaraGuerra Espinosa, ClaudiaCardeñes, BeatrizClares, IreneToribio, VíctorReche Gallardo, Pedro AntonioCabañas Gutiérrez, CarlosLafuente Duarte, María Esther612.017IntegrinRIAMPHAGOCYTOSISNeutrophilαMβ2VASPCiencias Biomédicas2412 Inmunología32 Ciencias MédicasActivation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (β2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMβ2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMβ2 and αXβ2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.FrontiersUniversidad Complutense de Madrid20222022-09-2720222022-09-27journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/20.500.14352/116128reponame:Docta Complutenseinstname:Universidad Complutense de Madrid (UCM)InglésengMinisterio de Economía y Competitividad http://dx.doi.org/10.13039/501100003329 PLAN ESTATAL DE INVESTIGACION CIENTIFICA Y TÉCNICA Y DE INNOVACIÓN 2013-2016 SAF2016-77096-RMinisterio de Economía y Competitividad http://dx.doi.org/10.13039/501100003329 l Plan Estatal de Investigación Científica, Técnica y de Innovación 2021 -2023 PID2021-123199OB-I00open accesshttp://purl.org/coar/access_right/c_abf2Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:docta.ucm.es:20.500.14352/1161282026-06-02T12:44:21Z |
| dc.title.none.fl_str_mv |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells |
| title |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells |
| spellingShingle |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells Torres Gómez, Álvaro 612.017 Integrin RIAM PHAGOCYTOSIS Neutrophil αMβ2 VASP Ciencias Biomédicas 2412 Inmunología 32 Ciencias Médicas |
| title_short |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells |
| title_full |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells |
| title_fullStr |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells |
| title_full_unstemmed |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells |
| title_sort |
Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells |
| dc.creator.none.fl_str_mv |
Torres Gómez, Álvaro Fiyouzi, Tara Guerra Espinosa, Claudia Cardeñes, Beatriz Clares, Irene Toribio, Víctor Reche Gallardo, Pedro Antonio Cabañas Gutiérrez, Carlos Lafuente Duarte, María Esther |
| author |
Torres Gómez, Álvaro |
| author_facet |
Torres Gómez, Álvaro Fiyouzi, Tara Guerra Espinosa, Claudia Cardeñes, Beatriz Clares, Irene Toribio, Víctor Reche Gallardo, Pedro Antonio Cabañas Gutiérrez, Carlos Lafuente Duarte, María Esther |
| author_role |
author |
| author2 |
Fiyouzi, Tara Guerra Espinosa, Claudia Cardeñes, Beatriz Clares, Irene Toribio, Víctor Reche Gallardo, Pedro Antonio Cabañas Gutiérrez, Carlos Lafuente Duarte, María Esther |
| author2_role |
author author author author author author author author |
| dc.contributor.none.fl_str_mv |
Universidad Complutense de Madrid |
| dc.subject.none.fl_str_mv |
612.017 Integrin RIAM PHAGOCYTOSIS Neutrophil αMβ2 VASP Ciencias Biomédicas 2412 Inmunología 32 Ciencias Médicas |
| topic |
612.017 Integrin RIAM PHAGOCYTOSIS Neutrophil αMβ2 VASP Ciencias Biomédicas 2412 Inmunología 32 Ciencias Médicas |
| description |
Activation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (β2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMβ2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMβ2 and αXβ2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype. |
| publishDate |
2022 |
| dc.date.none.fl_str_mv |
2022 2022-09-27 2022 2022-09-27 |
| dc.type.none.fl_str_mv |
journal article http://purl.org/coar/resource_type/c_6501 VoR http://purl.org/coar/version/c_970fb48d4fbd8a85 |
| dc.type.openaire.fl_str_mv |
info:eu-repo/semantics/article |
| format |
article |
| dc.identifier.none.fl_str_mv |
https://hdl.handle.net/20.500.14352/116128 |
| url |
https://hdl.handle.net/20.500.14352/116128 |
| dc.language.none.fl_str_mv |
Inglés eng |
| language_invalid_str_mv |
Inglés |
| language |
eng |
| dc.relation.none.fl_str_mv |
Ministerio de Economía y Competitividad http://dx.doi.org/10.13039/501100003329 PLAN ESTATAL DE INVESTIGACION CIENTIFICA Y TÉCNICA Y DE INNOVACIÓN 2013-2016 SAF2016-77096-R Ministerio de Economía y Competitividad http://dx.doi.org/10.13039/501100003329 l Plan Estatal de Investigación Científica, Técnica y de Innovación 2021 -2023 PID2021-123199OB-I00 |
| dc.rights.none.fl_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ |
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info:eu-repo/semantics/openAccess |
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open access http://purl.org/coar/access_right/c_abf2 Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ |
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openAccess |
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application/pdf |
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Frontiers |
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Frontiers |
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reponame:Docta Complutense instname:Universidad Complutense de Madrid (UCM) |
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Universidad Complutense de Madrid (UCM) |
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Docta Complutense |
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Docta Complutense |
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