Search

Background: 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. Methods: 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. Results: 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. Conclusions: 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rodrigues, Mathias, Deprá, Cury, de Oliveira, Olimpio, De Sibio, Gonçalves and Nogueira.)

Adiponectin and leptin, important for metabolic regulation, are synthesized and secreted by adipose tissue and are influenced by triiodothyronine (T3) that activates the MAPK/ERK and integrin αVβ3 pathways, modulating gene expression. Adipocytes were treated with T3 (10 nM), for 1 h, in the absence or presence of PD98059 (PD) and tetraiodothyroacetic acid (Tetrac), which are pathways inhibitors. The cells were incubated with Adipo Red/Oil Red O reagents, and intracellular lipid accumulation [glycerol and triacylglycerol (TAG)], MTT, 8-hydroxideoxyguanosine (8-OH-dG), and mRNA and protein expression were assessed. T3 increased leptin mRNA and protein expression, and, in contrast, there was a decrease in the Tetrac + T3 group. Adiponectin mRNA expression was not altered by T3, though it had increased its protein expression, which was terminated by inhibitors PD + T3 and Tetrac + T3. However, T3 did not alter PPARγ protein expression, lipid accumulation, TAG, glycerol, and DNA damage, but PD + T3 and Tetrac + T3 reduced these parameters. T3 activated the MAPK/ERK pathway on adipocytes to modulate the adiponectin protein expression and integrin αvβ3 to alter the leptin gene expression., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A considerable increase in endocrine abnormalities has been reported over the last few decades worldwide. A growing exposure to endocrine-disrupting chemicals (EDCs) can be one of the causes of endocrine disorders in populations, and these disorders are not only restricted to the metabolic hormone system but can also cause abnormal functions. Thyroid hormone (TH) disruption is defined as an abnormal change in TH production, transport, function, or metabolism, which results in some degree of impairment in body homeostasis. Many EDCs, including organotin compounds (OTCs), are environmental contaminants that are commonly found in antifouling paints used on ships and in several other industrial procedures. OTCs are obesogenic and can disrupt TH metabolism; however, abnormalities in thyroid function resulting from OTC exposure are less well understood. OTCs, one of the most prevalent EDCs that are encountered on a daily basis, modulate the thyroid axis. In most toxicology studies, it has been reported that OTCs might contribute to hypothyroidism.

Objective: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway., Materials and Methods: The cell line was treated with T3 at a physiological dose (10-9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%., Results: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA., Conclusion: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.

Adipose tissue (AT), an endocrine organ that modulates several physiological functions by synthesizing and releasing adipokines such as adiponectin, is a metabolic target of triiodothyronine (T3). T3 and adiponectin play important roles in controlling normal metabolic functions such as stimulation of fatty acid oxidation and increase in thermogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway is important for the differentiation of preadipocytes into adipocytes and can be activated by T3 for the transcription of specific genes, such as adiponectin. We examined the role of PI3K in adiponectin modulation by T3 action in murine adipocytes (3T3-L1). The 3T3-L1 adipocytes were treated with 1000 nM T3 for 1 h in the presence or absence of 50 μM LY294002 (LY), a PI3K inhibitor. Then, we assessed the expression of adiponectin and the phosphorylated serine/threonine kinase Akt (pAkt), a PI3K signaling protein, in the adipocytes. Adiponectin and pAKT levels were higher in the T3-adipocyte cells, whereas in the LY group adiponectin was elevated and pAKT was decreased compared to the control (C). PI3K pathway inhibition for 1 h and posterior treatment with T3, in LY + T3, reduced the adiponectin level and increased pAKT levels compared to those in LY. T3 stimulated adiponectin levels by PI3K pathway activation and T3 can compensate alteration in the PI3K pathway, because with inhibition of the pathway it is able to maintain the basal levels of adiponectin and pAKT., (© 2019 AOCS.)

Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.