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Osteoclast-specific Plastin 3 knockout in mice fail to develop osteoporosis despite dramatic increased osteoclast resorption activity.

ABSTRACT: PLS3 loss-of-function mutations in humans and mice cause X-linked primary osteoporosis. However, it remains largely unknown how PLS3 mutations cause osteoporosis and which function PLS3 plays in bone homeostasis. A recent study showed that ubiquitous Pls3 KO in mice results in osteoporosis. Mainly osteoclasts were impacted in their function However, it has not been proven if osteoclasts are the major cell type affected and responsible for osteoporosis development in ubiquitous Pls3 KO mice. Here, we generated osteoclast-specific Pls3 KO mice. Additionally, we developed a novel polyclonal PLS3 antibody that showed specific PLS3 loss in immunofluorescence staining of osteoclasts in contrast to previously available antibodies against PLS3, which failed to show PLS3 specificity in mouse cells. Moreover, we demonstrate that osteoclast-specific Pls3 KO causes dramatic increase in resorptive activity of osteoclasts in vitro. Despite these findings, osteoclast-specific Pls3 KO in vivo failed to cause any osteoporotic phenotype in mice as proven by micro-CT and three-point bending test. This demonstrates that the pathomechanism of PLS3-associated osteoporosis is highly complex and cannot be reproduced in a system singularly focused on one cell type. Thus, the loss of PLS3 in alternative bone cell types might contributes to the osteoporosis phenotype in ubiquitous Pls3 KO mice.

SUBMITTER: Maus I

PROVIDER: S-EPMC10971598 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Osteoclast-specific Plastin 3 knockout in mice fail to develop osteoporosis despite dramatic increased osteoclast resorption activity.

Maus Ilka I Dreiner Maren M Zetzsche Sebastian S Metzen Fabian F Ross Bryony C BC Mählich Daniela D Koch Manuel M Niehoff Anja A Wirth Brunhilde B

JBMR plus 20240104 1

PLS3 loss-of-function mutations in humans and mice cause X-linked primary osteoporosis. However, it remains largely unknown how PLS3 mutations cause osteoporosis and which function PLS3 plays in bone homeostasis. A recent study showed that ubiquitous Pls3 KO in mice results in osteoporosis. Mainly osteoclasts were impacted in their function However, it has not been proven if osteoclasts are the major cell type affected and responsible for osteoporosis development in ubiquito ...[more]

PMID: 38549711

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Project description:ObjectiveAutoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption.MethodsAnti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption.ResultsNET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts.ConclusionIgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies.

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

Osteoclast-specific Plastin 3 knockout in mice fail to develop osteoporosis despite dramatic increased osteoclast resorption activity.

Publications

Osteoclast-specific Plastin 3 knockout in mice fail to develop osteoporosis despite dramatic increased osteoclast resorption activity.

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