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Identification of osteoclast coupling factors in humans reveals links between bone remodeling and energy metabolism I

ABSTRACT: Purpose: Identification of osteoclast derived coupling factors using Denosumab as a biological probe Method: harvested RNA from centrifuged bone biopsies (heterogeneous population containing bone lining cells, osteoblasts, osteoclasts and osteocytes, without in vitro culture Results: Identification of genes differentially regulated in denosumab treated participants that correlate with bone remodeling indices Conclusions: Using this approach we identified denosumab regulated genes that correlate with bone remodeling in the untreated participants and may represent novel osteoclast derived coupling factors. Of interest, we identified DPP4 to be suppressed in denosumab treated participants, providing a novel link between bone remodeling and energy metabolism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141595 | GEO | 2019/12/07

REPOSITORIES: GEO

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Project description:The involvement of osteocytes in multiple myeloma (MM)-induced osteoclast formation and the occurrence of bone lesions are still unknown. Osteocytes regulate bone remodeling at least in part through the cell death and apoptosis triggering osteoclast recruitment and formation. In this study, firstly we shown that MM cells increased osteocyte death and affect their transcriptional profile evaluated by microarray analysis up-regulating osteoclastogenic cytokines as interleukin (IL)-11. Consistently we show that the conditioned media of human pre-osteocytes co-cultured with MM cells significantly increased osteoclastogenesis. To translate into a clinical perspective such in vitro evidences, we then performed histological analysis on bone biopsies obtained from MM patients, MGUS and healthy controls. We found a significant reduction in the number of viable osteocytes in MM patients as compared to controls. A significant negative correlation between the number of viable osteocytes and that of osteoclasts was also demonstrated. Moreover, as regards the skeletal involvement, we found that MM patients with bone lesions have a significant lower number of viable osteocyte than those without. Overall, our data suggest a role of osteocytic cell death in MM-induced osteoclast formation in vitro and MM bone disease in vivo in MM patients. This series of microarray experiments contains the gene expression profiles of HOB-01 osteocytes cultured alone and co-cultured with the JJN3 human myeloma cell line. 5 micrograms of total RNA was processed, and in accordance with the manufacturer's protocols, the fragmented biotin-labelled cRNA were hybridized on GeneChip Human Genome U133 Plus 2.0 Arrays (Affymetrix Inc.). The arrays were scanned using the Agilent GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChip Operating System (GCOS) 1.1 software and the probe level data converted to expression values using default GCOS1.1 scaling. Natural-scaled data are provided.

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.

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Identification of osteoclast coupling factors in humans reveals links between bone remodeling and energy metabolism I

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