Dataset Information

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

ABSTRACT: Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 ^-/-) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 ^-/- mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 ^fl/fl ;Lyz2 ^cre/+) mice. We found that trabecular bone volume in the Irf8 ^fl/fl ;Lyz2 ^cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 ^fl/fl ;Lyz2 ^cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 ^fl/fl ;Lyz2 ^cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose the existence in vivo of a new lineage of osteoclast precursors among BMCs, which differentiate into osteoclasts without up-regulation of Lyz2 expression.

SUBMITTER: Saito E

PROVIDER: S-EPMC5461233 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

Saito Emi E Suzuki Dai D Kurotaki Daisuke D Mochizuki Ayako A Manome Yoko Y Suzawa Tetsuo T Toyoshima Yoichi Y Ichikawa Takahiro T Funatsu Takahiro T Inoue Tomio T Takami Masamichi M Tamura Tomohiko T Inagaki Katsunori K Kamijo Ryutaro R

Cytotechnology 20160808 3

Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 <sup>-/-</sup>) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 <sup>-/-</sup> mice are severely altered. Theref ...[more]

PMID: 27502007

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Project description:BackgroundInterferon regulatory factor-8 (IRF8) and nuclear factor-activated T cells c1 (NFATc1) are two transcription factors that have an important role in osteoclast differentiation. Thanks to ChIP-seq technology, scientists can now estimate potential genome-wide target genes of IRF8 and NFATc1. However, finding target genes that are consistently up-regulated or down-regulated across different studies is hard because it requires analysis of a large number of high-throughput expression studies from a comparable context.MethodWe have developed a machine learning based method, called, Cohort-based TF target prediction system (cTAP) to overcome this problem. This method assumes that the pathway involving the transcription factors of interest is featured with multiple "functional groups" of marker genes pertaining to the concerned biological process. It uses two notions, Gene-Present Sufficiently (GP) and Gene-Absent Insufficiently (GA), in addition to log2 fold changes of differentially expressed genes for the prediction. Target prediction is made by applying multiple machine-learning models, which learn the patterns of GP and GA from log2 fold changes and four types of Z scores from the normalized cohort's gene expression data. The learned patterns are then associated with the putative transcription factor targets to identify genes that consistently exhibit Up/Down gene regulation patterns within the cohort. We applied this method to 11 publicly available GEO data sets related to osteoclastgenesis.ResultOur experiment identified a small number of Up/Down IRF8 and NFATc1 target genes as relevant to osteoclast differentiation. The machine learning models using GP and GA produced NFATc1 and IRF8 target genes different than simply using a log2 fold change alone. Our literature survey revealed that all predicted target genes have known roles in bone remodeling, specifically related to the immune system and osteoclast formation and functions, suggesting confidence and validity in our method.ConclusioncTAP was motivated by recognizing that biologists tend to use Z score values present in data sets for the analysis. However, using cTAP effectively presupposes assembling a sizable cohort of gene expression data sets within a comparable context. As public gene expression data repositories grow, the need to use cohort-based analysis method like cTAP will become increasingly important.

Dataset Information

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

Publications

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

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