Interleukin-1 (IL-1) and tumor necrosis factor (TNF) mediate bone resorption in a variety of diseases affecting bone. Like TNF, IL-1 is secreted by osteoclast precursors (OCPs), but unlike TNF, it does not induce osteoclast formation directly from OCPs in vitro. TNF induces IL-1 expression and activates c-Fos, a transcription factor required in OCPs for osteoclast formation. Here, we examined whether IL-1 can induce osteoclast formation directly from OCPs overexpressing c-Fos and whether interaction with bone matrix affects OCP cytokine expression. We infected OCPs with c-Fos or green fluorescent protein retrovirus, cultured them with macrophage colony-stimulating factor and IL-1 on bone slices or plastic dishes, and assessed osteoclast and resorption pit formation and expression of IL-1 by OCPs. We used a Transwell assay to determine whether OCPs secrete IL-1 when they interact with bone matrix. IL-1 induced osteoclast formation directly from c-Fos-expressing OCPs on plastic. c-Fos-expressing OCPs formed osteoclasts spontaneously on bone slices without addition of cytokines. OCPs on bone secreted IL-1, which induced osteoclast formation from c-Fos-expressing OCPs in the lower Transwell dishes. The bone matrix proteins dentin sialoprotein and osteopontin, but not transforming growth factor-beta, stimulated OCP expression of IL-1 and induced c-Fos-expressing OCP differentiation into osteoclasts. Osteoclasts eroding inflamed joints have higher c-Fos expression compared with osteoclasts inside bone. We conclude that OCPs expressing c-Fos may induce their differentiation directly into osteoclasts by an autocrine mechanism in which they produce IL-1 through interaction with bone matrix. TNF could induce c-Fos expression in OCPs at sites of inflammation in bone to promote this autocrine mechanism and thus amplify bone loss.
"We have also shown that osteoclasts in vivo express inflammasome-related proteins. This is consistent with data in the literature [19-21] and a publically available expression profile database, showing detectable mRNA levels of inflammasome components in osteoclasts differentiated in vitro. However, the function of the inflammasome in osteoclasts is not known yet. "
[Show abstract][Hide abstract] ABSTRACT: Background
This study aims to investigate the inflammasome response in peripheral blood mononuclear cells (PBMCs) and the expression of inflammasome components in bone biopsies from patients with chronic recurrent multifocal osteomyelitis (CRMO).
The expression of inflammasome components mRNAs was evaluated in PBMCs isolated from 15 CRMO patients and 13 healthy controls by quantitative real-time PCR. The Interleukin (IL)-1β released in the medium of PBMC cultures after treatment with lipopolysaccharides (LPS) alone or LPS and ATP was measured by ELISA. Immunohistochemical staining for Apoptosis-associated Speck-like protein (ASC), caspase-1 (CASP-1), Nod-like receptor protein-3 (NLRP3) and IL-1β expression was performed in bone biopsies from CRMO patients.
mRNA levels of ASC, CASP-1 and IL-1β were significantly higher in freshly isolated PBMCs from CRMO patients in active disease than in healthy controls. CASP-1 and IL-1β transcript levels were significantly higher also in PBMCs from CRMO patients in remission compared to healthy controls. PBMCs from CRMO patients in active disease stimulated in vitro with LPS showed a significant increase in IL-1β release compared to healthy control cells. Immunohistochemistry staining of bone tissue revealed the expression of inflammasome components in CRMO osteoclasts.
Our data suggest that an abnormal regulation of IL-1β axis may be involved in CRMO pathogenesis.
"To examine whether MM cells induce OC inhibition via MM cell-monocyte interaction or via MM cell-produced soluble cytokines, we used a transwell coculture system (Figure S1B) that disrupts MM cell-monocyte interaction but does not affect MM cell growth or the ability of MM cells to produce and release cytokines. Monocytes were seeded in culture wells and MM cells were seeded in transwell inserts in the medium with or without RANKL. Transwell cocultures of monocytes with ARP-1 or MM.1S cells (Figure 3A) or other MM cells (data not shown) retained the ability to inhibit OCs. "
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) cells are responsible for aberrant osteoclast (OC) activation. However, when cocultured monocytes, but not OC precursors, with MM cells, we made a novel observation that MM cells inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced increase of OC differentiation, OC gene expression, signaling pathways and bone resorption activity. Our results showed that MM cells produced multiple inhibitory cytokines of osteoclastogenesis, such as IL-10, which activated STAT3 signaling and induce OC inhibition. However, cocultures of bone marrow stromal cells (BMSCs) reversed MM-induced OC inhibition. We found that MM cells increased production of MCP-1 from BMSCs and BMSC-derived MCP-1 enhanced OC formation. Mechanistic studies showed that IL-10 downregulated RANK expression in monocytes and thus, inhibited RANKL-induced OC formation. In contrast, MCP-1 upregulated RANK expression and thus, enhanced OC formation. Overall, our studies for the first time demonstrated that MM cell have inhibitory effects on osteoclastogenesis by producing inhibitory cytokines. Our results further indicate that activation of osteoclastogenesis in bone marrow requests the crosstalk of MM cells, BMSCs and their produced cytokines. Thus, our studies provide evidences that targeting bone marrow microenvironmental cells and/or cytokines may be a new approach to treating MM bone destruction.
PLoS ONE 12/2013; 8(12):e82453. DOI:10.1371/journal.pone.0082453 · 3.23 Impact Factor
"Receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), two factors that regulate osteoclast formation, enable in vitro culturing of osteoclasts . Additionally, RNAKL and M-CSF have been used to induce the differentiation of in vitro cultured spleen, peripheral blood mononuclear, and RAW264.7 cells into large populations of osteoclasts with high purity, thereby establishing a foundation for the study of osteoclast formation and activation mechanisms at the molecular level [35,37]. "
[Show abstract][Hide abstract] ABSTRACT: To investigate 1α,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were combined to induce differentiation of RAW264.7 cells into osteoclasts. Different 1α,25-(OH)2D3 concentrations were included during the culture, and RAW264.7 cell proliferation was determined using the methylthiazol tetrazolium method, while osteoclast formation was confirmed using TRAP staining and bone lacunar resorption. MMP-9 protein expression levels were detected using western blot. We showed that 1α,25-(OH)2D3 inhibited the induction of RAW264.7 cell proliferation by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption activity, and promoted MMP-9 protein expression, all in a concentration-dependent manner. Therefore, 1α,25-(OH)2D3 in the physiological range promoted osteoclast formation, it could regulate osteoclast bone metabolism via increasing MMP-9 protein expression during osteoclast differentiation.
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