Rodrigues Bruna Moretto, Mathias Lucas Solla, Deprá Igor de Carvalho, Cury Sarah Santiloni, de Oliveira Miriane, Olimpio Regiane Marques Castro, De Sibio Maria Teresa, Gonçalves Bianca Mariani, Nogueira Célia Regina
Department of Internal Medicine, Medical School Botucatu, São Paulo State University (UNESP), Botucatu, Brazil.
Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, Brazil.
Front Cell Dev Biol. 2022 Jun 17;10:886136. doi: 10.3389/fcell.2022.886136. eCollection 2022.
Thyroid hormones play a significant role in bone development and maintenance, with triiodothyronine (T3) particularly being an important modulator of osteoblast differentiation, proliferation, and maintenance. However, details of the biological processes (BPs) and molecular pathways affected by T3 in osteoblasts remain unclear. To address this issue, primary cultures of human adipose-derived mesenchymal stem cells were subjected to our previously established osteoinduction protocol, and the resultant osteoblast-like cells were treated with 1 nm or 10 nm T3 for 72 h. RNA sequencing (RNA-Seq) was performed using the Illumina platform, and differentially expressed genes (DEGs) were identified from the raw data using Kallisto and DESeq2. Enrichment analysis of DEGs was performed against the Gene Ontology Consortium database for BP terms using the R package clusterProfiler and protein network analysis by STRING. Approximately 16,300 genes were analyzed by RNA-Seq, with 343 DEGs regulated in the 1 nm T3 group and 467 upregulated in the 10 nm T3 group. Several independent BP terms related to bone metabolism were significantly enriched, with a number of genes shared among them (FGFR2, WNT5A, WNT3, ROR2, VEGFA, FBLN1, S1PR1, PRKCZ, TGFB3, and OSR1 for 1nM T3; and FZD1, SMAD6, NOG, NEO1, and ENG for 10 nm T3). An osteoblast-related search in the literature regarding this set of genes suggests that both T3 doses are unfavorable for osteoblast development, mainly hindering BMP and canonical and non-canonical WNT signaling. Therefore, this study provides new directions toward the elucidation of the mechanisms of T3 action on osteoblast metabolism, with potential future implications for the treatment of endocrine-related bone pathologies.
甲状腺激素在骨骼发育和维持中发挥着重要作用，其中三碘甲状腺原氨酸（T3）尤其是成骨细胞分化、增殖和维持的重要调节因子。然而，T3在成骨细胞中影响的生物学过程（BP）和分子途径的细节仍不清楚。为了解决这个问题，将人脂肪来源的间充质干细胞的原代培养物按照我们先前建立的骨诱导方案进行处理，然后将所得的成骨样细胞用1 nM或10 nM T3处理72小时。使用Illumina平台进行RNA测序（RNA-Seq），并使用Kallisto和DESeq2从原始数据中鉴定差异表达基因（DEG）。使用R包clusterProfiler针对基因本体联盟数据库的BP术语对DEG进行富集分析，并通过STRING进行蛋白质网络分析。通过RNA-Seq分析了约16,300个基因，在1 nM T3组中有343个DEG受到调控，在10 nM T3组中有467个上调。几个与骨代谢相关的独立BP术语显著富集，其中有一些基因是共有的（1 nM T3组中有FGFR2、WNT5A、WNT3、ROR2、VEGFA、FBLN1、S1PR1、PRKCZ、TGFB3和OSR1；10 nM T3组中有FZD1、SMAD6、NOG、NEO1和ENG）。在文献中对这组基因进行的成骨细胞相关搜索表明，两种T3剂量都不利于成骨细胞发育，主要阻碍骨形态发生蛋白（BMP）以及经典和非经典WNT信号传导。因此，本研究为阐明T3对成骨细胞代谢作用的机制提供了新方向，对未来内分泌相关骨病的治疗具有潜在意义。
