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Differential Setup of PI3K/AKT and JAK/STAT pathways in fibrotic vs regenerative healing in mammals
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Abstract(s)
A regeneração epimórfica em mamíferos é um fenómeno raro e pouco compreendido, mas há muito tempo que captura a imaginação humana, com a promessa de uma completa recuperação na sequência de um ferimento grave. Por esta razão, os cientistas estão há mais de um século a tentar compreender porque alguns animas têm um potencial desta característica tão superior aos humanos, para que talvez sejamos capazes de o recriar em nós.
Os vertebrados inferiores, como salamandras, sapos e peixes teleósteos, são capazes de regenerar vários tecidos e órgãos, incluindo o coração, mandíbula, apêndices, etc., recuperando completamente a funcionalidade. Compreender como um processo regenerativo é estabelecido em resposta a um ferimento, nestes organismos, tem-nos ajudado a compreender algumas limitações do nosso próprio sistema, mas uma compreensão completa requer o nosso entendimento de como este fenómeno ocorre em espécies mais próximas de nós, com as quais partilhamos, por exemplo, uma resposta imunitária adaptativa complexa.
Recentemente, algumas espécies de cervídeos e murídeos foram associados a uma impressionante capacidade de regenerar as suas hastes e tecidos associados, e os seus pavilhões auriculares e pele do dorso, respetivamente. Além disso, foi demonstrado que a regeneração nestes modelos tem uma forte relação com a resposta imunitária, e é dependente da sinalização MAPK/ERK, algo que também já foi demonstrado em processos regenerativos de vertebrados inferiores.
A sinalização MAPK/ERK é conhecida por controlar vários processos celulares, principalmente proliferação, portanto não é de espantar que esteja envolvida em eventos regenerativos. A sinalização MAPK/ERK também é conhecida por interagir com outras vias de sinalização intracelulares, principalmente com a sinalização PI3K/AKT, contribuindo para a integração de outros sinais, como disponibilidade nutricional e presença de fatores de stress, ajudando a célula a decidir o seu destino.
Apesar de ser indispensável para a regeneração, e ser capaz de induzir algumas características de regeneração num processo fibrótico, a sobre ativação da sinalização MAPK/ERK em mamíferos não é suficiente para o transformar completamente num evento regenerativo. Isto é indicativo de que provavelmente haverá outras vias de sinalização diferencialmente ativas entre os processos, e um melhor entendimento de como a proliferação é regulada nestes processos, em particular na regeneração em mamíferos, é necessário. Em particular, como é que células, quando confrontadas com tal stress, tomam a decisão de proliferar, em vez de escolherem outro destino, como morte celular programada, e como é que o sistema imunitário influência este processo.
Com esse fim, de melhor entender essa regulação, neste trabalho procedemos ao isolamento de populações de células proliferativas que se formam durante o processo de resposta a uma perfuração circular do pavilhão auricular de duas espécies de mamífero, uma apresenta uma resposta regenerativa, e outra uma fibrótica, e analisámos os seus perfis transcricionais em vários pontos da sua resposta inicial a esse ferimento (hemóstase, inflamação e proliferação). Ao fazermos isso, observámos que, apesar de alguma, ainda que escassa, semelhança, estas duas populações proliferativas representam respostas amplamente distintas.
Em particular, o perfil transcricional de A. cahirinus mostrou uma forte associação com processos envolvidos no metabolismo energético e proliferação, enquanto o perfil transcricional das populações proliferativas de M. musculus associou-se mais significativamente com processos relacionados com dinâmicas citoplasmáticas, provavelmente associadas a migração, e diferenciação de vários tecidos.
Enquanto a regulação da proliferação de M. musculus se associou principalmente à via de sinalização de MAPK/ERK, o perfil transcritómico de A. cahirinus associou-se a mais vias de sinalização, nomeadamente à via PI3K/AKT, WNT e NF-KB, além de MAPK/ERK.
A comparação dos perfis transcricionais também sugeriu um envolvimento distinto de fatores de transcrição na regulação da proliferação. Também encontrámos uma associação do perfil transcricional de A. cahirinus com uma resposta apoptótica, associação essa que não foi observada em M. musculus.
Apesar de grupos distintos de vias de sinalização estarem associados com a resposta de cada uma das espécies estudadas, ambas as respostas são conhecidas por envolver igualmente algumas das mesmas vias de sinalização, mas com resultados distintos. Por causa disso, decidimos analisar como é que estas vias de sinalização estão montadas, e as dinâmicas da sua ativação em ambas as espécies.
Nós observámos que, aparentemente, a proporção de mediadores e as interações que estabelecem, em três vias de sinalização intracelulares, nomeadamente MAPK/ERK, PI3K/AKT e JAK/STAT, conhecidas por interagirem entre si na regulação de vários processos celulares, em particular na proliferação, não são idênticas entre as espécies. Por exemplo, M. musculus aparenta ter uma preferência por ativar uma das isoformas de ERK1/2, enquanto o mesmo não se observa em A. cahirinus. Adicionalmente, as dinâmicas temporais de ativação sugerem que cada isoforma tem a sua própria dinâmica, com ERK2 aparentemente associado a uma resposta precoce, e ERK1 possivelmente envolvido no suporte da resposta proliferativa.
Quanto à via de sinalização PI3K/AKT, nós observámos uma abundância relativa consistentemente superior de AKT1 ativado em M. musculus, onde a sua ativação parece aumentar em particular nas primeiras 12h depois do ferimento. Além disso, a resposta desta via de sinalização aparenta ser bimodal, com um tipo de resposta precoce e outro mais tardio, possivelmente associado com uma indução de apoptose em A. cahirinus brevemente após lesão, algo que aparenta não acontecer em M. musculus. Esta indução talvez contribua para suster a proliferação, algo que tem sido associado com uma constante produção de fatores mitogénicos por parte de células pré-apoptóticas, que podem permanecer neste estado por largos períodos.
A via de sinalização JAK/STAT, por outro lado, exibe uma clara e sustentada proporção de JAK2 ativado mais elevada em A. cahirinus. No entanto, esta atividade superior não se traduziu em consideráveis diferenças nos níveis de ativação de STAT3, o que sugere que JAK2 poderá ter outros papéis no processo de resposta a ferimento nestas duas espécies. Isto poderá constituir um método que as células proliferativas, neste contexto, usam para integrar sinais do sistema imunitário na regulação da sua proliferação, visto que JAK2 pode transativar recetores que atuam acima da sinalização MAPK/ERK e PI3K/AKT, como os recetores ERBB.
No seu todo, o nosso trabalho sugere a existência de duas estratégias muito distintas de resposta a um ferimento, entre processos regenerativos e fibróticos em mamíferos, e sugere que isto não só se deve a uma resposta distinta aos mesmos estímulos, mas também que esta resposta distinta poderá dever-se a composições alternativas destas vias de sinalização intracelulares, que vão elas próprias gerar os resultados distintos destes dois tipos de resposta.
Mais exploração destas vias de sinalização, e da resposta dos seus participantes, aos estímulos presentes durante a resposta a ferimentos irão ajudar-nos a perceber o conjunto ideal de fatores a utilizar para transformar processos fibróticos em regenerativos em animais com potencial regenerativo inferior.
Epimorphic regeneration in mammals is a rare and poorly understood phenomenon, but for long it has captured human imagination, with the promise of complete recovery from serious injury. For this reason, scientists have spent over a century attempting to understand why in some animal species this characteristic has a much higher potential than in Humans, so that we might recreate it in ourselves. Lower vertebrates, like salamanders, anurans and teleost fish, are capable of regenerating several tissues and organs to full functionality, including heart, jaw, fins, limbs, etc. Understanding how regeneration is established in response to wounding, in these organisms, has helped us comprehend some limitations in our system, but full comprehension requires the understanding of how these phenomena occur in species closer to us, with which we share, for example, a complex adaptative immune response to injury. Recently, a few species of cervids and murids have been shown to have the remarkable ability to regenerate antlers and associated tissues, and ear pinnas and dorsal skin, respectively. Furthermore, it was shown that regeneration in these models has a strong relationship with the immune response, and is dependent on MAPK/ERK signaling, something that had also been shown in the regenerative processes of lower vertebrates. MAPK/ERK signaling is known for controlling several cellular processes, chiefly amongst them proliferation, so it is no wonder that it also does so during regeneration. MAPK/ERK is also known to interact with other intracellular pathways to perform this regulation, chiefly amongst them the PI3K/AKT pathway, which contributes to an integration of other cues, like nutrient availability and presence of stress factors, helping the cell decide its fate. Despite the indispensability to regeneration, MAPK/ERK signaling overactivation in mammals is only capable of inducing some characteristics of regeneration in a fibrotic wound healing process, but not to fully transform it into a regenerative one. This indicates that other differentially regulated pathways are also likely involved, and a better understanding of how proliferation is regulated in the wound healing events of vertebrates, in particular in mammalian regeneration, is required. Particularly, how the cells, when faced with such stress, make the decision to proliferate, rather than another fate, like programmed cell death, and how the immune system exacts its influence over the process. To that end, in this work we isolated proliferative cell populations formed during the wound healing response to full-thickness ear pinna punching of two mammalian species, one that mounts a regenerative response, and the other a fibrotic one, and analyzed their transcriptional profiles at several timepoints during the initial phases of wound healing (homeostasis, inflammation and proliferation). By doing this, we observed that despite some scarce similarities, the two proliferative populations mostly represented two wildly distinct responses to wounding. In particular, the transcriptomic profile of A. cahirinus had a strong association with processes involved in energy metabolism and proliferation, while M. musculus proliferative cell population transcriptional profiles associated more significantly with processes associated with cytoplasmic dynamics, likely related with migration, and differentiation of several tissues. While M. musculus proliferative regulation primarily associates with MAPK/ERK signaling, A. cahirinus transcriptomic profile associated with more signaling pathways, primarily PI3K/AKT, WNT and NF-KB, besides MAPK/ERK. Comparison of transcriptomic profiles also suggested differential involvement of transcription factors in the regulation of proliferation. We also found an association of the transcriptomic profile of A. cahirinus with an apoptotic response, and this was not observed for M. musculus. Because, despite distinctive groups of signaling pathways being associated with the response of each species, both responses are known to equally involve some of the same pathways, but have distinct results, we decided to look at how these pathways are set up, and the dynamics of their activation in both species. We found that it appears that the proportion of mediators and the interactions they establish, in three intracellular pathways, MAPK/ERK, PI3K/AKT and JAK/STAT, known for interacting in the regulation of several cellular processes, particularly proliferation, are not the same between species. For example, M. musculus appears to have a preference for activating a particular isoform of ERK, while the same preference is not found in A. cahirinus. Furthermore, the temporal dynamics of activation suggest that each isoform has its own dynamic activation, with ERK2 appearing to be an early responder, and ERK1 involved in sustaining proliferation. As for the PI3K/AKT pathway, we found a consistently higher abundance of activated AKT1 in M. musculus, where its activation appears to be particularly increased in the first 12h. Furthermore, the response of this pathway also appears to be bimodal, with an early type of response and a later one, possibly associated with an early induction of apoptosis in A. cahirinus that does not occur in M. musculus. This induction might contribute to sustain proliferation, which has been associated with a constant production of mitogens by proapoptotic cells, which can remain in this state for a long time. In the JAK/STAT pathway, on the other hand, we saw a clear and sustained higher proportion of activated JAK2 in A. cahirinus. However, this higher activation did not translate to greatly differing activation levels of STAT3, which suggests to us that JAK2 might have other roles in the wound healing process of these two species. This might constitute a method for proliferative cells, in this context, to integrate immune signals in the regulation of their proliferation, seeing as JAK2 is known to transactivate receptors that operate upstream of the MAPK/ERK and PI3K/AKT pathways, like the ERBB receptors. Overall, our work points to two very distinct strategies of response to wound healing between regenerative and fibrotic events in mammals, and suggest that this is due not just to a differential response to the same stimuli, but that this differential response actually comes from inherent differences to the setups of several intracellular pathways, which will themselves have different activities between these two types of response. Further exploration of these pathways and the response of their effectors to the stimuli present in response to wounding will help us understand the set of ideal factors to be able to transform fibrotic processes into regenerative ones in organisms with lower regenerative potentials.
Epimorphic regeneration in mammals is a rare and poorly understood phenomenon, but for long it has captured human imagination, with the promise of complete recovery from serious injury. For this reason, scientists have spent over a century attempting to understand why in some animal species this characteristic has a much higher potential than in Humans, so that we might recreate it in ourselves. Lower vertebrates, like salamanders, anurans and teleost fish, are capable of regenerating several tissues and organs to full functionality, including heart, jaw, fins, limbs, etc. Understanding how regeneration is established in response to wounding, in these organisms, has helped us comprehend some limitations in our system, but full comprehension requires the understanding of how these phenomena occur in species closer to us, with which we share, for example, a complex adaptative immune response to injury. Recently, a few species of cervids and murids have been shown to have the remarkable ability to regenerate antlers and associated tissues, and ear pinnas and dorsal skin, respectively. Furthermore, it was shown that regeneration in these models has a strong relationship with the immune response, and is dependent on MAPK/ERK signaling, something that had also been shown in the regenerative processes of lower vertebrates. MAPK/ERK signaling is known for controlling several cellular processes, chiefly amongst them proliferation, so it is no wonder that it also does so during regeneration. MAPK/ERK is also known to interact with other intracellular pathways to perform this regulation, chiefly amongst them the PI3K/AKT pathway, which contributes to an integration of other cues, like nutrient availability and presence of stress factors, helping the cell decide its fate. Despite the indispensability to regeneration, MAPK/ERK signaling overactivation in mammals is only capable of inducing some characteristics of regeneration in a fibrotic wound healing process, but not to fully transform it into a regenerative one. This indicates that other differentially regulated pathways are also likely involved, and a better understanding of how proliferation is regulated in the wound healing events of vertebrates, in particular in mammalian regeneration, is required. Particularly, how the cells, when faced with such stress, make the decision to proliferate, rather than another fate, like programmed cell death, and how the immune system exacts its influence over the process. To that end, in this work we isolated proliferative cell populations formed during the wound healing response to full-thickness ear pinna punching of two mammalian species, one that mounts a regenerative response, and the other a fibrotic one, and analyzed their transcriptional profiles at several timepoints during the initial phases of wound healing (homeostasis, inflammation and proliferation). By doing this, we observed that despite some scarce similarities, the two proliferative populations mostly represented two wildly distinct responses to wounding. In particular, the transcriptomic profile of A. cahirinus had a strong association with processes involved in energy metabolism and proliferation, while M. musculus proliferative cell population transcriptional profiles associated more significantly with processes associated with cytoplasmic dynamics, likely related with migration, and differentiation of several tissues. While M. musculus proliferative regulation primarily associates with MAPK/ERK signaling, A. cahirinus transcriptomic profile associated with more signaling pathways, primarily PI3K/AKT, WNT and NF-KB, besides MAPK/ERK. Comparison of transcriptomic profiles also suggested differential involvement of transcription factors in the regulation of proliferation. We also found an association of the transcriptomic profile of A. cahirinus with an apoptotic response, and this was not observed for M. musculus. Because, despite distinctive groups of signaling pathways being associated with the response of each species, both responses are known to equally involve some of the same pathways, but have distinct results, we decided to look at how these pathways are set up, and the dynamics of their activation in both species. We found that it appears that the proportion of mediators and the interactions they establish, in three intracellular pathways, MAPK/ERK, PI3K/AKT and JAK/STAT, known for interacting in the regulation of several cellular processes, particularly proliferation, are not the same between species. For example, M. musculus appears to have a preference for activating a particular isoform of ERK, while the same preference is not found in A. cahirinus. Furthermore, the temporal dynamics of activation suggest that each isoform has its own dynamic activation, with ERK2 appearing to be an early responder, and ERK1 involved in sustaining proliferation. As for the PI3K/AKT pathway, we found a consistently higher abundance of activated AKT1 in M. musculus, where its activation appears to be particularly increased in the first 12h. Furthermore, the response of this pathway also appears to be bimodal, with an early type of response and a later one, possibly associated with an early induction of apoptosis in A. cahirinus that does not occur in M. musculus. This induction might contribute to sustain proliferation, which has been associated with a constant production of mitogens by proapoptotic cells, which can remain in this state for a long time. In the JAK/STAT pathway, on the other hand, we saw a clear and sustained higher proportion of activated JAK2 in A. cahirinus. However, this higher activation did not translate to greatly differing activation levels of STAT3, which suggests to us that JAK2 might have other roles in the wound healing process of these two species. This might constitute a method for proliferative cells, in this context, to integrate immune signals in the regulation of their proliferation, seeing as JAK2 is known to transactivate receptors that operate upstream of the MAPK/ERK and PI3K/AKT pathways, like the ERBB receptors. Overall, our work points to two very distinct strategies of response to wound healing between regenerative and fibrotic events in mammals, and suggest that this is due not just to a differential response to the same stimuli, but that this differential response actually comes from inherent differences to the setups of several intracellular pathways, which will themselves have different activities between these two types of response. Further exploration of these pathways and the response of their effectors to the stimuli present in response to wounding will help us understand the set of ideal factors to be able to transform fibrotic processes into regenerative ones in organisms with lower regenerative potentials.
Description
Keywords
A. cahirinus Proliferação JAK/STAT PI3K/AKT Transcritómica