Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Pathway analysis of microRNA expression profile during murine osteoclastogenesis

ABSTRACT: Osteoclasts are multinucleated cells specialized in degrading the mineralized bone matrix. Osteoclast differentiation and function are tightly regulated, to prevent excessive or insufficient bone resorption. Several control mechanisms participate in modulating osteoclastogenesis, and an increasing number of reports describe the role of microRNAs (miRNAs) in this process. Disrupting the expression of specific miRNAs can result in alterations of osteoclast formation and bone homeostasis. We and others have previously characterized 9 miRNAs whose levels change during osteoclast differentiation, and identified some of the target genes that mediate their function. However, little is known about changes in the miRNA expression profile during osteoclastogenesis. In this study, we isolated a murine primary bone marrow population enriched for osteoclast precursors, and used the Agilent microarray platform to analyze the expression of mature miRNAs after 1, 3, and 5 days of RANKL-driven differentiation. 93 miRNAs showed greater than 2 fold-change during these early, middle, and late stages of osteoclastogenesis. Many of these miRNAs were detected for the first time in osteoclasts, and we validated the expression of selected miRNAs by quantitative RT-PCR. We identified clusters of differentially expressed miRNAs, and performed computational analyses to predict functional pathways that may be regulated by these miRNAs. Several miRNAs were predicted to regulate genes involved in cytoskeletal remodeling, a crucial mechanism for the migration of osteoclast precursors, their maturation, and bone resorbing activity. Our results suggest that clusters of miRNAs differentially expressed during the course of osteoclastogenesis converge on the regulation of several key functional pathways. Overall, this study identified miRNAs expressed during early, middle and late osteoclastogenesis, contributing to understanding the molecular mechanisms regulating this complex differentiation process. Mouse primary bone marrow cultures were enriched for osteoclast precursors by depletion of B220/CD45R+ and CD3+ cells (B and T lymphocytes, respectively). Cells were differentiated with M-CSF and RANKL, and miRNA expression was analyzed at days 1, 3, and 5. Four biological replicates for each time point were used.

ORGANISM(S): Mus musculus

SUBMITTER: Anne Delany

PROVIDER: E-GEOD-53017 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Pathway analysis of microRNA expression profile during murine osteoclastogenesis.

Franceschetti Tiziana T Dole Neha S NS Kessler Catherine B CB Lee Sun-Kyeong SK Delany Anne M AM

PloS one 20140915 9

To design novel therapeutics against bone loss, understanding the molecular mechanisms regulating osteoclastogenesis is critical. Osteoclast formation and function are tightly regulated by transcriptional, post-transcriptional and post-translational mechanisms. This stringent regulation is crucial to prevent excessive or insufficient bone resorption and to maintain bone homeostasis. microRNAs (miRNAs) are key post-transcriptional regulators that repress expression of target mRNAs controlling ost ...[more]

PMID: 25222202

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Project description:Osteoclasts are multinucleated cells specialized in degrading the mineralized bone matrix. Osteoclast differentiation and function are tightly regulated, to prevent excessive or insufficient bone resorption. Several control mechanisms participate in modulating osteoclastogenesis, and an increasing number of reports describe the role of microRNAs (miRNAs) in this process. Disrupting the expression of specific miRNAs can result in alterations of osteoclast formation and bone homeostasis. We and others have previously characterized 9 miRNAs whose levels change during osteoclast differentiation, and identified some of the target genes that mediate their function. However, little is known about changes in the miRNA expression profile during osteoclastogenesis. In this study, we isolated a murine primary bone marrow population enriched for osteoclast precursors, and used the Agilent microarray platform to analyze the expression of mature miRNAs after 1, 3, and 5 days of RANKL-driven differentiation. 93 miRNAs showed greater than 2 fold-change during these early, middle, and late stages of osteoclastogenesis. Many of these miRNAs were detected for the first time in osteoclasts, and we validated the expression of selected miRNAs by quantitative RT-PCR. We identified clusters of differentially expressed miRNAs, and performed computational analyses to predict functional pathways that may be regulated by these miRNAs. Several miRNAs were predicted to regulate genes involved in cytoskeletal remodeling, a crucial mechanism for the migration of osteoclast precursors, their maturation, and bone resorbing activity. Our results suggest that clusters of miRNAs differentially expressed during the course of osteoclastogenesis converge on the regulation of several key functional pathways. Overall, this study identified miRNAs expressed during early, middle and late osteoclastogenesis, contributing to understanding the molecular mechanisms regulating this complex differentiation process.

Project description:Purpose: Among the diverse cytokines involved in osteoclast differentiation, IL-3 has been shown to inhibit RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. In the present study, we demonstrate that IL-3 activation of STAT5 inhibits RANKL-induced osteoclastogenesis through the induction of Id genes. Methods: To investigate the effect of STAT5 on osteoclast differentiation and IL-3-mediated inhibition of osteoclast differentiation, bone marrow derived macrophages isolated from STAT5 wild-type (Stat5fl/fl) or STAT5 cKO (STAT5;MX1-Cre) were differentiated to osteoclast in the presence of M-CSF and RANKL with or without IL-3; and bone marrow derived macrophges from STAT5 wild-type and STAT5 cKO were overexpressed with PMX-FIG (control) or STAT5A1*6 (constitutively active form of STAT5A) and differentiated to osteoclast. To analyze bone phenotype, femurs and tibiae of 16 week-old STAT5 wild-type and STAT5 cKO were subjected to micro CT analysis and histomorphometry, respectively. Results: Overexpression of STAT5 inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate either expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting that STAT5 plays an important role in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated expression of the Id1 and Id2 genes, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Moreover, micro-computed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in STAT5 conditional knockout mice than in wild-type mice in a RANKL injection model. Conclusion: Taken together, our results suggest that STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis through Id gene expression. Examination of 4 different combination of osteoclast differentiation condition of bone marrow derived macrophages.

Project description:Paget’s disease of bone (PDB) is a chronic focal skeletal disorder that affects 2-3% of the population over the age of 60. PDB is inherited as an autosomal dominant trait with genetic heterogeneity. SQSTM1/p62 UBA domain mutation (p62P392L) is widely identified in PDB and has been shown to increase osteoclastogenesis. Further, environmental factors such as paramyxovirus are implicated in PDB and MVNP has been shown to induce Pagetic phenotype in osteoclasts. However, the molecular mechanisms underlying p62P392L and MVNP stimulation of osteoclast differentiation in PDB are unclear. We therefore determined MVNP regulated gene expression profiling during osteoclast differentiation. We identified 8.4% of genes were upregulated (> 4-fold) in MVNP transduced cells. MVNP increased integrin β3 (63 fold), NFAT activating protein (6.5 fold), OSCAR (5.5 fold), TRAF5 (8.5 fold) mRNA expression compared to empty vector (EV) transduced cells. MVNP also elevated gene expression of cytokines/growth factors such as IL-17 (18 fold), IL-1F7 (10 fold), IL-17R (4.5 fold) and IL-11 (7 fold). Interestingly, MVNP transduced cells demonstrated high level expression of signal regulatory protein beta 1 (SIRPβ1) (353 fold). SIRPβ1 has been shown to interact with DAP 12, an ITAM containing adaptor protein which plays an important role in osteoclast differentiation. Real-time PCR analysis of total RNA isolated from normal human peripheral blood monocytes transduced with MVNP confirmed upregulation of SIRPβ1 mRNA expression in the absence of RANKL stimulation. In contrast, RANKL stimulation did not alter SIRPβ1 expression in these cells. Furthermore, bone marrow mononuclear cells derived from patients with PDB showed high levels of SIRPβ1 mRNA expression compared to normal subjects. Thus, MVNP regulated gene expression profiling during osteoclast differentiation provides new insights into molecular mechanisms and therapeutic targets to control elevated osteoclast activity in PDB. Total RNA isolated from normal human bone marrow mononuclear cells transduced with EV, MVNP retroviral expression vectors and stimulated with M-CSF and RANKL for 48 h were subjected to Agilent microarray (~26,000 genes) analysis. One replicate per treatment was hybridized.

Dataset Information

Pathway analysis of microRNA expression profile during murine osteoclastogenesis

Publications

Pathway analysis of microRNA expression profile during murine osteoclastogenesis.

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