Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Microarray studies on genes stimulated by RANKL in bone marrow macrophages in a cyclooxygenase-2-dependent manner

ABSTRACT: Overall goal was to look for the differentially expressed genes between receptor activator of nuclear factor kappa-B ligand (RANKL)-stimulated wild type (WT) bone marrow macrophages (BMMs) and the BMMs in which the expression or the enzyme activity of cyclooxygenase-2 (Cox2), the major enzyme for prostaglandin E2 (PGE2) synthesis, is absent. For this purpose, two separate microarrays (experiment 1 and 2) were done. In both the experiments, Serum Amyloid A3 (Saa3) was one of the most highly differentially expressed gene. For experiment 1, BMMs were isolated from the bone marrow of Cox2 knockout (KO) and WT mice. Out of the three sample groups, two were treated with 30 ng/ml each of macrophage-colony stimulating factor (M-CSF) and RANKL, while the third group was treated with M-CSF only, for 1 day. Two of the sample groups had n=3 biological replicates (i.e. samples), while the third one had n=4 biological replicates. The differential gene expression comparisons among the sample groups were done as (A) WT+MCSF+RANKL_d1 versus Cox2KO+MCSF+RANKL_d1 and (B) WT+MCSF+RANKL_d1 versus WT+MCSF_d1. For experiment 2 also, BMMs were obtained from Cox2 KO and WT mice. All the four sample groups were treated with M-CSF and RANKL for 3 days. All the four sample groups had n=4 biological replicates. One of the WT sample group was also treated with an inhibitor of Cox2 activity,NS398 (0.1 Âµm) and one of the KO sample group was also treated with the product of Cox2 enzyme, PGE2 (1 Âµm). The differential gene expression comparisons among the sample groups were done as (A) WT+MCSF+RANKL_d3 versus Cox2KO+MCSF+RANKL_d3; (B) WT+MCSF+RANKL_d3 versus WT+MCSF+RANKL+NS398_d3 and (C) Cox2KO+MCSF+RANKL+PGE2_d3 versus Cox2KO+MCSF+RANKL_d3.

ORGANISM(S): Mus musculus

SUBMITTER: Carol Pilbeam

PROVIDER: E-GEOD-72047 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Serum Amyloid A3 Secreted by Preosteoclasts Inhibits Parathyroid Hormone-stimulated cAMP Signaling in Murine Osteoblasts.

Choudhary Shilpa S Goetjen Alexandra A Estus Thomas T Jacome-Galarza Christian E CE Aguila Hector L HL Lorenzo Joseph J Pilbeam Carol C

The Journal of biological chemistry 20151223 8

Continuous parathyroid hormone (PTH) blocks its own osteogenic actions in marrow stromal cell cultures by inducing Cox2 and receptor activator of nuclear factor κB ligand (RANKL) in the osteoblastic lineage cells, which then cause the hematopoietic lineage cells to secrete an inhibitor of PTH-stimulated osteoblast differentiation. To identify this inhibitor, we used bone marrow macrophages (BMMs) and primary osteoblasts (POBs) from WT and Cox2 knock-out (KO) mice. Conditioned medium (CM) from RA ...[more]

PMID: 26703472

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Project description:Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts, and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule is the enigmatic coupling factor. We have identified Cthrc1, a protein secreted by mature bone-resorbing osteoclasts, that targets stromal cells so as to stimulate osteogenesis. The expression of Cthrc1 is robustly induced when mature osteoclasts are placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increases in a high turnover state, such as that which is induced by RANKL injections in vivo, whereas it decreases with aging or following alendronate treatment, conditions associated with suppressed bone turnover. The targeted deletion of the Cthrc1 gene eliminates Cthrc1 expression in bone, whereas its deficiency in osteoblasts does not exert any significant effect. Osteoclast-specific deletion of the Cthrc1 gene results in osteopenia due to reduced bone formation: it also impairs the coupling process following resorption induced by RANKL injections, with a resultant impairment of bone mass recovery. Thus, Cthrc1 is an osteoclast-secreted “coupling factor” that regulates bone remodeling and hence, skeletal integrity. Total bone marrow cells were prepared from the femurs and tibias of 8-10-week-old C57BL/6 mice and cultured in the presence of M-CSF (100ng/ml) for 3 days as described previously (Takeshita et al., 2000 JBMR 15:1477-1488). Cells were harvested with 0.02% EDTA/PBS and used as bone marrow macrophages (BMMs). These BMMs were cultured in the presence of M-CSF (100 ng/ml) and RANKL (100ng/ml) for 2 days. TRAP positive mononuclear cells were harvested and used as pre-osteoclasts (pOC). These pOC cells were further cultured in the presence of M-CSF and RANKL for 2 days in normal plastic plate or on dentin slices. After 2 days, multinucleated TRAP positive mature osteoclasts were generated as mature osteoclasts on plate (mOCp) and mature resorbing osteoclasts on dentin (mOCd), respectively. RNAs were extracted from four different stages of osteoclast lineage cells; BMMs, pOC, mOCp and mOCd, and used for microarray analysis.

Dataset Information

Microarray studies on genes stimulated by RANKL in bone marrow macrophages in a cyclooxygenase-2-dependent manner

Publications

Serum Amyloid A3 Secreted by Preosteoclasts Inhibits Parathyroid Hormone-stimulated cAMP Signaling in Murine Osteoblasts.

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