Glucocorticoid-induced expansion of classical monocytes contributes to bone loss

AUTHORS

Pei Liu, Youshui Gao, Pengbo Luo, Hongping Yu, Shang Guo, Fuyun Liu, Junjie Gao, Jianzhong Xu, Shengdian Wang & Changqing Zhang

ABSTRACT

Classical monocytes are commonly involved in the innate inflammatory response and are the progenitors of osteoclasts. Excess endogenous glucocorticoids (GCs) can increase the levels of classical monocytes in blood and bone marrow. The role of this cell population in high-dose exogenous GC-induced osteoporosis (GIOP) remains to be elucidated. In this study, GIOP was established in rats and mice by daily methylprednisolone injection, and monocyte subsets were analyzed by flow cytometry. We demonstrated that classical monocytes accumulate in bone marrow during GIOP. Similarly, the monocyte proportion among bone marrow nucleated cells was also increased in patients with steroid treatment history. We sorted classical monocytes and analyzed their transcriptional profile in response to GCs by RNA sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that classical monocytes isolated from GC-treated rats exhibited osteoclast differentiation potential. Deletion of classical monocytes by clodronate liposome treatment prevented GIOP via inhibition of osteoclastogenesis and restoration of CD31HiendomucinHi vessels. Regarding the molecular mechanism, classical monocytes express high levels of glucocorticoid receptors. In vitro treatment with GCs increased both the percentage and absolute number of monocytes and promoted their proliferation. In summary, classical monocytes mediated GC-induced bone loss and are a potential target for therapeutic intervention in GIOP treatment.

Role of chromatin modulator Dpy30 in osteoclast differentiation and function

AUTHORS

Yanfang Zhao, Xiaoxiao Hao, Zhaofei Li, Xu Feng, Jannet Katz, Suzanne M.Michalek, Hao Jiang, Ping Zhang

ABSTRACT

Osteoclasts are the principal bone resorption cells crucial for homeostatic bone remodeling and pathological bone destruction. Increasing data demonstrate a vital role of histone methylation in osteoclastogenesis. As an integral core subunit of H3K4 methyltransferases, Dpy30 is notal as a key chromatin regulator for cell growth and differentiation and stem cell fate determination, particularly in the hematopoietic system. However, its role in osteoclastogenesis is currently unknown. Herein, we generated Dpy30F/F; LysM-Cre+/+ mice, which deletes Dpy30 in myeloid cells, to characterize its involvement in osteoclast differentiation and function. Dpy30F/F; LysM-Cre+/+ mice showed increased bone mass, evident by impaired osteoclastogenesis and defective osteoclast activity, but no alteration of osteoblast numbers and bone formation. Additionally, our ex vivo analysis showed that the loss of Dpy30 significantly impedes osteoclast differentiation and suppresses osteoclast-related gene expression. Moreover, Dpy30 deficiency significantly decreased the enrichment of H3K4me3 on the promoter region of NFATc1. Thus, we revealed a novel role for Dpy30 in osteoclastogenesis through epigenetic mechanisms, and that it could potentially be a therapeutic target for bone destruction diseases.

Toll-like receptor 9 deficiency induces osteoclastic bone loss via gut microbiota-associated systemic chronic inflammation

AUTHORS

Peng Ding, Qiyuan Tan, Zhanying Wei, Qiyu Chen, Chun Wang, Luyue Qi, Li Wen, Changqing Zhang & Chen Yao

ABSTRACT

Toll-like receptors (TLRs) play pivotal roles in inflammation and provide important links between the immune and skeletal systems. Although the activation of TLRs may affect osteoclast differentiation and bone metabolism, whether and how TLRs are required for normal bone remodeling remains to be fully explored. In the current study, we show for the first time that TLR9−/− mice exhibit a low bone mass and low-grade systemic chronic inflammation, which is characterized by the expansion of CD4+ T cells and increased levels of inflammatory cytokines, including TNFα, RANKL, and IL1β. The increased levels of these cytokines significantly promote osteoclastogenesis and induce bone loss. Importantly, TLR9 deletion alters the gut microbiota, and this dysbiosis is the basis of the systemic inflammation and bone loss observed in TLR9−/− mice. Furthermore, through single-cell RNA sequencing, we identified myeloid-biased hematopoiesis in the bone marrow of TLR9−/− mice and determined that the increase in myelopoiesis, likely caused by the adaptation of hematopoietic stem cells to systemic inflammation, also contributes to inflammation-induced osteoclastogenesis and subsequent bone loss in TLR9−/− mice. Thus, our study provides novel evidence that TLR9 signaling connects the gut microbiota, immune system, and bone and is critical in maintaining the homeostasis of inflammation, hematopoiesis, and bone metabolism under normal conditions.

Roxadustat promotes osteoblast differentiation and prevents estrogen deficiency-induced bone loss by stabilizing HIF-1α and activating the Wnt/β-catenin signaling pathway

AUTHORS

Luyao Li, Afang Li, Li Zhu, Liangying Gan & Li Zuo

ABSTRACT

Background

Osteoporosis is a very common skeletal disorder that increases the risk of fractures. However, the treatment of osteoporosis is challenging. Hypoxia-inducible factor-1α (HIF-1α) plays an important role in bone metabolism. Roxadustat is a novel HIF stabilizer, and its effects on bone metabolism remain unknown. This study aimed to investigate the effects of roxadustat on osteoblast differentiation and bone remodeling in an ovariectomized (OVX) rat model.

Methods

In vitro, primary mouse calvarial osteoblasts were treated with roxadustat. Alkaline phosphatase (ALP) activity and extracellular matrix mineralization were assessed. The mRNA and protein expression levels of osteogenic markers were detected. The effects of roxadustat on the HIF-1α and Wnt/β-catenin pathways were evaluated. Furthermore, osteoblast differentiation was assessed again after HIF-1α expression knockdown or inhibition of the Wnt/β-catenin pathway. In vivo, roxadustat was administered orally to OVX rats for 12 weeks. Then, bone histomorphometric analysis was performed. The protein expression levels of the osteogenic markers HIF-1α and β-catenin in bone tissue were detected.

Results

In vitro, roxadustat significantly increased ALP staining intensity, enhanced matrix mineralization and upregulated the expression of osteogenic markers at the mRNA and protein levels in osteoblasts compared with the control group. Roxadustat activated the HIF-1α and Wnt/β-catenin pathways. HIF-1α knockdown or Wnt/β-catenin pathway inhibition significantly attenuated roxadustat-promoted osteoblast differentiation. In vivo, roxadustat administration improved bone microarchitecture deterioration and alleviated bone loss in OVX rats by promoting bone formation and inhibiting bone resorption. Roxadustat upregulated the protein expression levels of the osteogenic markers, HIF-1α and β-catenin in the bone tissue of OVX rats.

Conclusion

Roxadustat promoted osteoblast differentiation and prevented bone loss in OVX rats. The use of roxadustat may be a new promising strategy to treat osteoporosis.

Motor Impairments and Dopaminergic Defects Caused by Loss of Leucine-Rich Repeat Kinase 2 Function in Mice

AUTHORS

Guodong Huang, Daniel W. Bloodgood, Jongkyun Kang, Anu Shahapal, Phoenix Chen, Konstantin Kaganovsky, Jae-Ick Kim, Jun Ding and Jie Shen

ABSTRACT

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD), but the pathogenic mechanism underlying LRRK2 mutations remains unresolved. In this study, we investigate the consequence of inactivation of LRRK2 and its functional homolog LRRK1 in male and female mice up to 25 months of age using behavioral, neurochemical, neuropathological, and ultrastructural analyses. We report that LRRK1 and LRRK2 double knock-out (LRRK DKO) mice exhibit impaired motor coordination at 12 months of age before the onset of DA neuron loss in the substantia nigra (SNpc). Moreover, LRRK DKO mice develop age-dependent, progressive loss of DA terminals in the striatum. Evoked dopamine release measured by fast-scan cyclic voltammetry in the dorsal striatum is also reduced in the absence of LRRK. Furthermore, LRRK DKO mice at 20–25 months of age show substantial loss of DA neurons in the SNpc. The surviving SNpc neurons in LRRK DKO mice at 25 months of age accumulate large numbers of autophagic and autolysosomal vacuoles and are accompanied with microgliosis. Surprisingly, the cerebral cortex is unaffected, as shown by normal cortical volume and neuron number as well as unchanged number of apoptotic cells and microglia in LRRK DKO mice at 25 months. These findings show that loss of LRRK function causes impairments in motor coordination, degeneration of DA terminals, reduction of evoked DA release, and selective loss of DA neurons in the SNpc, indicating that LRRK DKO mice are unique models for better understanding DA neurodegeneration in PD.

Effects of ferric citrate and intravenous iron sucrose on markers of mineral, bone, and iron homeostasis in a rat model of CKD-MBD

AUTHORS

Annabel Biruete, Corinne E Metzger, Neal X Chen, Elizabeth A Swallow, Curtis Vrabec, Erica L Clinkenbeard, Alexander J Stacy, Shruthi Srinivasan, Kalisha O'Neill, Keith G Avin, Matthew R Allen, Sharon M Moe

ABSTRACT

Background

Anemia and chronic kidney disease-mineral and bone disorder (CKD-MBD) are common and begin early in CKD. Limited studies have concurrently compared the effects of ferric citrate (FC) vs. IV iron on CKD-MBD and iron homeostasis in moderate CKD.

Methods

We tested the effects of 10 weeks of 2% FC vs. IV iron sucrose in rats with moderate CKD (Cy/+ male rat) and untreated normal (NL) littermates. Outcomes included a comprehensive assessment of CKD-MBD, iron homeostasis, and oxidative stress.

Results

CKD rats had azotemia, elevated phosphorus, PTH, and FGF23. Compared to untreated CKD rats, treatment with FC led to lower plasma phosphorus, intact FGF23, and a trend (p = 0.07) towards lower C-terminal FGF23. FC and IV iron equally reduced aorta and heart calcifications to levels similar to NL animals. Compared to NL animals, CKD animals had higher bone turnover, lower trabecular volume, and no difference in mineralization; these were unaffected by either iron treatment. Rats treated with IV iron had cortical and bone mechanical properties similar to NL animals. FC increased transferrin saturation rate compared to untreated CKD and NL rats. Neither iron treatment increased oxidative stress above that of untreated CKD.

Conclusions

Oral FC improved phosphorus homeostasis, some iron-related parameters, and the production and cleavage of FGF23. The intermittent effect of low-dose IV iron sucrose on cardiovascular calcification and bone should be further explored in moderate-to advanced CKD.