Avaliação da ação do hormônio tireoidiano na expressão dos RNAs codificantes em células osteoblásticas derivadas do tecido adiposo humano.
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Data
2018-02-26
Autores
Rodrigues, Bruna Moretto
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Universidade Estadual Paulista (Unesp)
Resumo
O sistema esquelético é um sistema complexo com intenso metabolismo composto de células, proteínas e minerais. Os osteoblastos, são células fundamentais para o tecido, desempenhando duas funções principais: formação óssea e regulação da reabsorção por meio da modulação da osteoclastogênese. Sendo assim, essas células desempenham funções primordiais para o desenvolvimento e manutenção óssea. Diversas moléculas sistêmicas atuam no tecido, sendo os hormônios tireoidianos um destes. Eles são fundamentais para o metabolismo ósseo já que alterações hormonais culminam em desordens ósseas. Osteoblastos possuem receptores nucleares para T3 e apesar de pouco compreendido, ele afeta diversos aspectos do desenvolvimento da célula, assim como vias modulatórias da remodelação óssea. Diversas linhagens celulares têm sido utilizadas para estudos de osteoblastos, sendo as células-tronco mesenquimais derivadas do tecido adiposo humano (hA-CTMs) um modelo promissor para osteoindução. Desta forma, o objetivo deste trabalho foi verificar a influência do T3 suprafisiológico (T3S) na expressão gênica diferencial em osteoblastos diferenciados a partir de hA-CTMs. As células obtidas de 3 doadores foram submetidas a osteoindução por 16 dias com coquetel de diferenciação (dexametasona, ácido ascórbico e β-glicerofosfato) e caracterizadas pela presença de osteocalcina, fosfatase alcalina e matriz mineralizada. O tratamento com T3 (10-8M) foi realizado por 72h e o RNA foi extraído para preparação das bibliotecas de mRNAs e sequenciamento na plataforma Illumina. A análise bioinformática englobou os softwares: FastQC para controle de qualidade, Kallisto para alinhamento (Hg19), Deseq2 no R para análise de expressão gênica diferencial (DE), análise de enriquecimento para Gene Ontology (GO) e REVIGO para construções de gráficos. A diferenciação osteoblástica foi confirmada pela presença de seus marcadores, assim como foi identificada a expressão gênica de outros marcadores, como fibronectina1 (FN1), osteonectina (SPARC) e colágeno tipo I (COL1A1/2). A qualidade dos RNAs, bibliotecas de mRNAs e reads obtidas do sequenciamento foram confirmadas, proporcionando a identificação de 659 genes DE. A maioria deles (55,5%) foram UP regulados pela condição T3S identificando 30 termos GO enriquecidos. Dentre eles, o T3S modulou diversos termos ósseos: desenvolvimento ósseo, morfogênese da célula, diferenciação osteoblástica, regulação negativa da proliferação celular e ossificação; Termos de vias mecânicas (adesão célula-substrato, adesões focais, resposta ao estresse de cisalhamento líquido); Vias bioquímicas (prostaglandinas, calmodulina, TGF-β/BMPs); Ritmo Circadiano; E diversos termos relacionados com ações moleculares. Além disso, o T3S aumentou a expressão dos genes SMAD6/7 e NOG, exercendo ação negativa sobre as vias BMPs e SMADs, favorecendo a regulação das vias TGF-β e MAPK. As informações obtidas neste estudo comprovam a ação do T3 em osteoblastos obtidos de hA-CTMs, evidenciando seu papel modulatório em diversos níveis biológicos. Além disso, as vias bioquímicas identificadas, não estão relacionadas apenas com osteoblastos, mas com reabsorção óssea osteoclástica, apontando a complexidade da ação do T3 no metabolismo ósseo. Por fim, foi proposto uma esquematização de vias de sinalização reguladas pela condição T3S, que pode nortear investigações mais específicas desta ação hormonal em osteoblastos e no tecido ósseo.
The skeletal system is complex and have an intense metabolism compound with cells, proteins and minerals. The osteoblasts are fundamental cells to the tissue, executing two mainly functions: bone formation and regulation of reabsorption through osteoclastogenesis modulation. Therefore, these cells perform primordial functions to the bone development and maintenance. Several systemic molecules act in the tissue and the thyroid hormones are one of them. They are fundamental to the bone metabolism, once hormone alterations result in bone disorders. Osteoblasts have T3 nuclear receptors, and although not well elucidated it affects many aspects of the cell development so as pathways that modulates the bone remodeling. Several cells lineages have been used in osteoblasts studies, and human adipose-derived Mesenchymal stem-cells (hA-MSCs) are a promising model to osteoinduction. Thus, the aim of this study was to investigate the supraphysiological T3 (T3S) influence in the gene differential expression in osteoblasts differentiated from hA-MSCs. The cells obtained from three donators were submitted to osteoinduction for 16 days with a differentiation cocktail (dexamethasone, ascorbic acid, β-glycerophosphate) and characterized by the presence of osteocalcin, alkaline phosphate and mineralized matrix. The cells were treated with T3 (10-8M) for 72h and the RNA was extracted to mRNA library prepare and sequenced by Illumina platform. The bioinformatic analysis included the software: FastQC to quality control, Kallisto to alignment (Hg19), Deseq2 in R to differential gene expression (DE) analysis, enrichment analysis to Gene Ontology (GO) and REVIGO to graphs construction. The osteoblastic differentiation was confirmed by the presence of their markers as well as was identified the gene expression of other markers, such as: fibronectin 1 (FN1), osteonectin (SPARC) and collagen type 1 (COL1A1/2). The RNAs, mRNA library and sequencing reads quality were confirmed, providing the identification of 659 genes DE. The majority (55,5%) were upregulated by T3S condition with 30 GO terms identified. Between them, the T3S modulated several bone terms: bone development, cell morphogenesis, osteoblastic differentiation, negative regulation of cell proliferation and ossification; Mechanical pathway terms (cell-substrate adhesion, focal adhesion and response to fluid shear stress); Biochemical pathways (prostaglandins, calmodulins, TGF- β/BMPs); Circadian rhythm; And diverse terms related to molecular action. Also, the T3S increased the expression of the genes SMAD6/7 and NOG, exercising negative action on BMPs and SMADs pathways and promoting the regulation of TGF-β and MAPK pathways. The information obtained in this study confirm the T3 action in osteoblasts from hA-MSCs, demonstrating their modulatory role in several biologic levels. Furthermore, the biochemical pathways identified, are not related only to osteoblasts, but with bone osteoclastic reabsorption, highlighting to the complexity of T3 action in the bone metabolism. Finally, it was proposed a diagram of the signaling pathways regulated by T3S, which can be a guiding study to more specific investigations of this hormonal action in osteoblasts and in the bone tissue.
The skeletal system is complex and have an intense metabolism compound with cells, proteins and minerals. The osteoblasts are fundamental cells to the tissue, executing two mainly functions: bone formation and regulation of reabsorption through osteoclastogenesis modulation. Therefore, these cells perform primordial functions to the bone development and maintenance. Several systemic molecules act in the tissue and the thyroid hormones are one of them. They are fundamental to the bone metabolism, once hormone alterations result in bone disorders. Osteoblasts have T3 nuclear receptors, and although not well elucidated it affects many aspects of the cell development so as pathways that modulates the bone remodeling. Several cells lineages have been used in osteoblasts studies, and human adipose-derived Mesenchymal stem-cells (hA-MSCs) are a promising model to osteoinduction. Thus, the aim of this study was to investigate the supraphysiological T3 (T3S) influence in the gene differential expression in osteoblasts differentiated from hA-MSCs. The cells obtained from three donators were submitted to osteoinduction for 16 days with a differentiation cocktail (dexamethasone, ascorbic acid, β-glycerophosphate) and characterized by the presence of osteocalcin, alkaline phosphate and mineralized matrix. The cells were treated with T3 (10-8M) for 72h and the RNA was extracted to mRNA library prepare and sequenced by Illumina platform. The bioinformatic analysis included the software: FastQC to quality control, Kallisto to alignment (Hg19), Deseq2 in R to differential gene expression (DE) analysis, enrichment analysis to Gene Ontology (GO) and REVIGO to graphs construction. The osteoblastic differentiation was confirmed by the presence of their markers as well as was identified the gene expression of other markers, such as: fibronectin 1 (FN1), osteonectin (SPARC) and collagen type 1 (COL1A1/2). The RNAs, mRNA library and sequencing reads quality were confirmed, providing the identification of 659 genes DE. The majority (55,5%) were upregulated by T3S condition with 30 GO terms identified. Between them, the T3S modulated several bone terms: bone development, cell morphogenesis, osteoblastic differentiation, negative regulation of cell proliferation and ossification; Mechanical pathway terms (cell-substrate adhesion, focal adhesion and response to fluid shear stress); Biochemical pathways (prostaglandins, calmodulins, TGF- β/BMPs); Circadian rhythm; And diverse terms related to molecular action. Also, the T3S increased the expression of the genes SMAD6/7 and NOG, exercising negative action on BMPs and SMADs pathways and promoting the regulation of TGF-β and MAPK pathways. The information obtained in this study confirm the T3 action in osteoblasts from hA-MSCs, demonstrating their modulatory role in several biologic levels. Furthermore, the biochemical pathways identified, are not related only to osteoblasts, but with bone osteoclastic reabsorption, highlighting to the complexity of T3 action in the bone metabolism. Finally, it was proposed a diagram of the signaling pathways regulated by T3S, which can be a guiding study to more specific investigations of this hormonal action in osteoblasts and in the bone tissue.
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Palavras-chave
Metabolismo ósseo, Osteoblastos, Triiodotironina, T3S, RNASeq, Bone Metabolism, Triiodothyronine, Osteoblasts