Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1016/j.bone.2017.06.012 http://hdl.handle.net/11449/174783 |
Resumo: | The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions. |
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Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitionsBioengineeringBiomaterialsECMOsteoblastSonic HedgehogThe reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Lab. de Bioensaios e Dinâmica Celular Depto de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista - UNESP, campus BotucatuLaboratory of Bioassays and Signal Transduction Departamento de Bioquímica Instituto de Biologia Universidade Estadual de Campinas (Unicamp), 6109Instituto Nacional de Metrologia Normalização e Qualidade Industrial (INMETRO) Life Sciences Applied Metrology (Dimav)/Bioengineering GroupErasmus MC Cancer Institute Erasmus MC Erasmus University of RotterdamLab. de Bioensaios e Dinâmica Celular Depto de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista - UNESP, campus BotucatuFAPESP: 2014/22689-3CNPq: 301966/2015-0CNPq: 477452/2012-4Universidade Estadual Paulista (Unesp)Universidade Estadual de Campinas (UNICAMP)Life Sciences Applied Metrology (Dimav)/Bioengineering GroupErasmus University of RotterdamMarumoto, Ariane [UNESP]Milani, Renatoda Silva, Rodrigo A. [UNESP]da Costa Fernandes, Célio Junior [UNESP]Granjeiro, José MauroFerreira, Carmen V.Peppelenbosch, Maikel P.Zambuzzi, Willian F. [UNESP]2018-12-11T17:12:50Z2018-12-11T17:12:50Z2017-10-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article55-63application/pdfhttp://dx.doi.org/10.1016/j.bone.2017.06.012Bone, v. 103, p. 55-63.8756-3282http://hdl.handle.net/11449/17478310.1016/j.bone.2017.06.0122-s2.0-850210683262-s2.0-85021068326.pdfScopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengBone1,652info:eu-repo/semantics/openAccess2023-10-24T06:05:46Zoai:repositorio.unesp.br:11449/174783Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-10-24T06:05:46Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions |
title |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions |
spellingShingle |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions Marumoto, Ariane [UNESP] Bioengineering Biomaterials ECM Osteoblast Sonic Hedgehog |
title_short |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions |
title_full |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions |
title_fullStr |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions |
title_full_unstemmed |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions |
title_sort |
Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions |
author |
Marumoto, Ariane [UNESP] |
author_facet |
Marumoto, Ariane [UNESP] Milani, Renato da Silva, Rodrigo A. [UNESP] da Costa Fernandes, Célio Junior [UNESP] Granjeiro, José Mauro Ferreira, Carmen V. Peppelenbosch, Maikel P. Zambuzzi, Willian F. [UNESP] |
author_role |
author |
author2 |
Milani, Renato da Silva, Rodrigo A. [UNESP] da Costa Fernandes, Célio Junior [UNESP] Granjeiro, José Mauro Ferreira, Carmen V. Peppelenbosch, Maikel P. Zambuzzi, Willian F. [UNESP] |
author2_role |
author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) Universidade Estadual de Campinas (UNICAMP) Life Sciences Applied Metrology (Dimav)/Bioengineering Group Erasmus University of Rotterdam |
dc.contributor.author.fl_str_mv |
Marumoto, Ariane [UNESP] Milani, Renato da Silva, Rodrigo A. [UNESP] da Costa Fernandes, Célio Junior [UNESP] Granjeiro, José Mauro Ferreira, Carmen V. Peppelenbosch, Maikel P. Zambuzzi, Willian F. [UNESP] |
dc.subject.por.fl_str_mv |
Bioengineering Biomaterials ECM Osteoblast Sonic Hedgehog |
topic |
Bioengineering Biomaterials ECM Osteoblast Sonic Hedgehog |
description |
The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-10-01 2018-12-11T17:12:50Z 2018-12-11T17:12:50Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1016/j.bone.2017.06.012 Bone, v. 103, p. 55-63. 8756-3282 http://hdl.handle.net/11449/174783 10.1016/j.bone.2017.06.012 2-s2.0-85021068326 2-s2.0-85021068326.pdf |
url |
http://dx.doi.org/10.1016/j.bone.2017.06.012 http://hdl.handle.net/11449/174783 |
identifier_str_mv |
Bone, v. 103, p. 55-63. 8756-3282 10.1016/j.bone.2017.06.012 2-s2.0-85021068326 2-s2.0-85021068326.pdf |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Bone 1,652 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
55-63 application/pdf |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
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1803649506289385472 |