Hydroxyurea blunts mitochondrial energy metabolism and osteoblast and osteoclast differentiation exacerbating trabecular bone loss in sickle cell mice

AUTHORS

Ashish Kumar Tripathi, Sadaf Dabeer, Jun Song, Tatyana Vikulina, Susanne Roser-Page, Jessica A. Alvarez, David. R. Archer, M. Neale Weitzmann

ABSTRACT

Sickle cell disease (SCD) is a severe hematological disorder characterized by erythrocyte sickling that causes significant morbidity and mortality. Skeletal complications of SCD include a high incidence of bone loss, especially in vertebrae, leading to fragility fractures that contribute to disease burden. Whether hydroxyurea (HU), a front-line therapy for SCD ameliorates bone disease has not been established. To investigate HU action on SCD-related vertebral defects, we used HU-treated “Townes” mice, an SCD animal model and performed high-resolution micro-computed tomography (µCT) imaging to resolve bone volume and micro-architectural structure of cortical and trabecular bone, the two major compartments contributing to bone mass and strength. Our data revealed that cortical bone was significantly diminished in the vertebrae of skeletally mature (representing adults) and immature (representing children) SCD mice, while only mature mice lost trabecular bone mass. Administration of HU ameliorated cortical bone loss in mature SCD mice, but paradoxically promoted trabecular bone decline in both groups. We further investigated the mechanisms of HU action in wild-type C57BL6/J mice. HU caused dose-dependent trabecular bone loss due to diminished osteoclast and osteoblast function, indicative of a low bone turnover state. Mechanistic investigations in vitro revealed that HU impeded osteoblast-progenitor proliferation and early differentiation, and diminished osteoclastogenic cytokine production, blunting osteoclast formation as well as the activity of mature osteoclasts. HU further, suppressed mitochondrial, but not glycolytic energy metabolism in both differentiating osteoblasts and differentiated osteoclasts. Collectively, these findings reveal that despite ameliorating cortical bone loss, HU inhibits trabecular bone formation and resorption, by suppressing mitochondrial energy metabolism and blunting the differentiation and/or activity of osteoblasts and osteoclasts. Together HU drives a low bone turnover state culminating in trabecular bone loss. Further investigation into HU’s impact on bone in SCD patients is warranted for understanding and managing skeletal complications in this population.

NLRP3 blockade by MCC950 suppressed osteoclastogenesis via NF-κB/c-Fos/NFATc1 signal pathway and alleviated bone loss in diabetes mellitus

AUTHORS

Guoping Cai, Xiaoting Song, Hua Luo, Gaoyuan Dai, Honghao Zhang, Dengteng Jiang, Xinhuan Lei, Haixiao Chen, Liwei Zhang

ABSTRACT

Obesity and type 2 diabetes mellitus (T2DM) are linked to osteoporosis development, with obesity being a significant risk factor for T2DM. T2DM patients with obesity exhibit a higher fracture rate and often have a poor prognosis post-fracture. To address the urgent need for understanding the mechanisms of diabetic osteoporosis (DOP), research is ongoing to explore how obesity and T2DM impact bone metabolism. The NLRP3 inflammasome has been implicated in the pathogenesis of osteoporosis, and MCC950, an NLRP3 inflammasome inhibitor, has shown promise in various diseases but its role in osteoporosis remains unexplored. In this study, BMMs and BMSCs were isolated and cultured to investigate the effects of MCC950 on bone metabolism, and DOP model was used to evaluate the efficacy of MCC950 in vivo. The study demonstrated that MCC950 treatment inhibited osteoclast differentiation, reduced bone resorption capacity in BMMs without suppression for osteoblast differentiation from BMSCs. Additionally, MCC950 suppressed the activation of the NF-κB signaling pathway and downregulated key factors associated with osteoclast differentiation. Additionally, MCC950 alleviated bone loss in DOP mouse. These findings suggest that MCC950, by targeting the NLRP3 inflammasome, may have a protective role in preventing osteoporosis induced by T2DM with obesity. The study highlights the potential therapeutic implications of MCC950 in managing diabetic osteoporosis and calls for further research to explore its clinical application in high-risk patient populations.

TGF-β signaling in the cranial neural crest affects late-stage mandibular bone resorption and length

AUTHORS

Claire J. Houchen, Saif Ghanem, Vesa Kaartinen, Erin Ealba Bumann

ABSTRACT

Malocclusions are common craniofacial malformations that cause quality of life and health problems if left untreated. Unfortunately, the current treatment for severe skeletal malocclusion is invasive surgery. Developing improved therapeutic options requires a deeper understanding of the cellular mechanisms responsible for determining jaw bone length. We have recently shown that neural crest mesenchyme (NCM) can alter jaw length by controlling the recruitment and function of mesoderm-derived osteoclasts. Transforming growth factor beta (TGF-β) signaling is critical to craniofacial development by directing bone resorption and formation, and heterozygous mutations in the TGF-β type I receptor (TGFBR1) are associated with micrognathia in humans. To identify the role of TGF-β signaling in NCM in controlling osteoclasts during mandibular development, the mandibles of mouse embryos deficient in the gene encoding Tgfbr1, specifically in NCM, were analyzed. Our laboratory and others have demonstrated that Tgfbr1fl/fl;Wnt1-Cre mice display significantly shorter mandibles with no condylar, coronoid, or angular processes. We hypothesize that TGF-β signaling in NCM can also direct late bone remodeling and further regulate late embryonic jaw bone length. Interestingly, analysis of mandibular bone based on micro-computed tomography and Masson’s trichrome revealed no significant difference in bone quality between the Tgfbr1fl/fl;Wnt1-Cre mice and controls, as measured by the bone perimeter/bone area, trabecular rod-like diameter, number and separation, and gene expression of collagen type 1 alpha 1 (Col1α1) and matrix metalloproteinase 13 (Mmp13). Although there was not a difference in localization of bone resorption within the mandible indicated by tartrate-resistant acid phosphatase (TRAP) staining, Tgfbr1fl/fl;Wnt1-Cre mice had approximately three-fold less osteoclast number and perimeter than controls. Gene expression of receptor activator of nuclear factor kappa-β (Rank) and Mmp9, markers of osteoclasts and their activity, also showed a three-fold decrease in Tgfbr1fl/fl;Wnt1-Cre mandibles. Evaluation of osteoblast-to-osteoclast signaling revealed no significant difference between Tgfbr1fl/fl;Wnt1-Cre mandibles and controls, leaving the specific mechanism unresolved. Finally, pharmacological inhibition of Tgfbr1 signaling during the initiation of bone mineralization and resorption significantly shortened jaw length in embryos. We conclude that TGF-β signaling in NCM decreases mesoderm-derived osteoclast number, that TGF-β signaling in NCM impacts jaw length late in development, and that this osteoblast-to-osteoclast communication may be occurring through an undescribed mechanism.

Epimedin A inhibits the PI3K/AKT/NF-κB signalling axis and osteoclast differentiation by negatively regulating TRAF6 expression

AUTHORS

Jun Li, Jia J. Wei, Cen H. Wu, Tao Zou, Hong Zhao, Tian Q. Huo, Cheng J. Wei, Ting Yang

ABSTRACT

Background

Epimedin A (EA) has been shown to suppress extensive osteoclastogenesis and bone resorption, but the effects of EA remain incompletely understood. The aim of our study was to investigate the effects of EA on osteoclastogenesis and bone resorption to explore the corresponding signalling pathways.

Methods

Rats were randomly assigned to the sham operation or ovariectomy group, and alendronate was used for the positive control group. The therapeutic effect of EA on osteoporosis was systematically analysed by measuring bone mineral density and bone biomechanical properties. In vitro, RAW264.7 cells were treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) to induce osteoclast differentiation. Cell viability assays, tartrate-resistant acid phosphatase (TRAP) staining, and immunofluorescence were used to elucidate the effects of EA on osteoclastogenesis. In addition, the expression of bone differentiation-related proteins or genes was evaluated using Western blot analysis or quantitative polymerase chain reaction (PCR), respectively.

Results

After 3 months of oral EA intervention, ovariectomized rats exhibited increased bone density, relative bone volume, trabecular thickness, and trabecular number, as well as reduced trabecular separation. EA dose-dependently normalized bone density and trabecular microarchitecture in the ovariectomized rats. Additionally, EA inhibited the expression of TRAP and NFATc1 in the ovariectomized rats. Moreover, the in vitro results indicated that EA inhibits osteoclast differentiation by suppressing the TRAF6/PI3K/AKT/NF-κB pathway. Further studies revealed that the effect on osteoclast differentiation, which was originally inhibited by EA, was reversed when the TRAF6 gene was overexpressed.

Conclusions

The findings indicated that EA can negatively regulate osteoclastogenesis by inhibiting the TRAF6/PI3K/AKT/NF-κB axis and that ameliorating ovariectomy-induced osteoporosis in rats with EA may be a promising potential therapeutic strategy for the treatment of osteoporosis.

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats

AUTHORS

Lijun Xu, Jie Zhu, Lingjun Rong, Huinan Yang, Bin Wang, Shuai Lu, Lingxiao Zhang, Fuyi Li, Shihua Yang, Zhifang Wang, Chong Li, Xiao Hu, Ruoyun Liu, Lili Zheng, Hongjian Liu, Haohao Zhang, Yanling Liu, Di Zhao, Shuiying Zhao, Lun Zhang, Yingbo Jia, Shiyu Liang, Zhikang Guo, Xixiu Xi, Ruitian Liu, Lixia Zhang

ABSTRACT

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood.

Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3).

Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1β and IL-18 and increasing the levels of OCN and Runx2.

Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects

AUTHORS

Catharina J. Küng, Arezoo Daryadel, Rocio Fuente, Betül Haykir, Martin Hrabĕ de Angelis, Nati Hernando, Isabel Rubio-Aliaga & Carsten A. Wagner

ABSTRACT

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.