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Myonectin inhibits the differentiation of osteoblasts and osteoclasts in mouse cells.

ABSTRACT: Myonectin is a myokine, which is involved in the pathophysiology of diabetes and obesity, and various myokines are involved in the interactions between skeletal muscle and bone. However, roles of myonectin in bone have still remained unknown. We therefore examined the effects of myonectin on mouse osteoblast and osteoclast differentiation in vitro. Myonectin significantly suppressed the mRNA levels of osteogenic genes and alkaline phosphatase (ALP) activity in mouse osteoblasts. As for osteoclasts, myonectin significantly suppressed osteoclast formation as well as the mRNA levels of osteoclast-related genes enhanced by receptor activator nuclear factor ?B ligand (RANKL) from mouse monocytic RAW264.7 cells. Moreover, myonectin significantly suppressed osteoclast formation from mouse bone marrow cells in the presence of macrophage-colony stimulating factor and RANKL. On the other hand, myonectin significantly suppressed RANKL-induced oxygen consumption rate and peroxisome proliferator-activated receptor ? coactivator-1? mRNA levels in RAW264.7 cells, although myonectin did not affect these mitochondrial biogenesis parameters in mouse osteoblasts. In conclusion, the present study demonstrated that myonectin suppresses the differentiation and ALP activity in mouse osteoblasts. Moreover, myonectin suppressed osteoclast differentiation from mouse bone marrow and RAW264.7 cells partly through an inhibition of mitochondrial biogenesis.

SUBMITTER: Kawaguchi M

PROVIDER: S-EPMC7266783 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Myonectin inhibits the differentiation of osteoblasts and osteoclasts in mouse cells.

Kawaguchi Miku M Kawao Naoyuki N Takafuji Yoshimasa Y Ishida Masayoshi M Kaji Hiroshi H

Heliyon 20200515 5

Myonectin is a myokine, which is involved in the pathophysiology of diabetes and obesity, and various myokines are involved in the interactions between skeletal muscle and bone. However, roles of myonectin in bone have still remained unknown. We therefore examined the effects of myonectin on mouse osteoblast and osteoclast differentiation <i>in vitro</i>. Myonectin significantly suppressed the mRNA levels of osteogenic genes and alkaline phosphatase (ALP) activity in mouse osteoblasts. As for os ...[more]

PMID: 32514479

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Project description:BackgroundReal-time quantitative RT-PCR (qPCR) is a powerful technique capable of accurately quantitating mRNA expression levels over a large dynamic range. This makes qPCR the most widely used method for studying quantitative gene expression. An important aspect of qPCR is selecting appropriate controls or normalization factors to account for any differences in starting cDNA quantities between samples during expression studies. Here, we report on the selection of a concise set of housekeeper genes for the accurate normalization of quantitative gene expression data in differentiating osteoblasts, osteoclasts and macrophages. We implemented the use of geNorm, an algorithm that determines the suitability of genes to function as housekeepers by assessing expression stabilities. We evaluated the expression stabilities of 18S, ACTB, B2M, GAPDH, HMBS and HPRT1 genes.FindingsOur analyses revealed that 18S and GAPDH were regulated during osteoblast differentiation and are not suitable for use as reference genes. The most stably expressed genes in osteoblasts were ACTB, HMBS and HPRT1 and their geometric average constitutes a suitable normalization factor upon which gene expression data can be normalized. In macrophages, 18S and GAPDH were the most variable genes while HMBS and B2M were the most stably expressed genes. The geometric average of HMBS and B2M expression levels forms a suitable normalization factor to account for potential differences in starting cDNA quantities during gene expression analysis in macrophages. The expression stabilities of the six candidate reference genes in osteoclasts were, on average, more variable than that observed in macrophages but slightly less variable than those seen in osteoblasts. The two most stably expressed genes in osteoclasts were HMBS and B2M and the genes displaying the greatest levels of variability were 18S and GAPDH. Notably, 18S and GAPDH were the two most variably expressed control genes in all three cell types. The geometric average of HMBS, B2M and ACTB creates an appropriate normalization factor for gene expression studies in osteoclasts.ConclusionWe have identified concise sets of genes suitable to use as normalization factors for quantitative real-time RT-PCR gene expression studies in osteoblasts, osteoclasts and macrophages.

Dataset Information

Myonectin inhibits the differentiation of osteoblasts and osteoclasts in mouse cells.

Publications

Myonectin inhibits the differentiation of osteoblasts and osteoclasts in mouse cells.

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