Decorin knockdown is beneficial for aged tendons in the presence of biglycan expression

AUTHORS

Zakary M. Beach, Mihir S. Dekhne, Ashley B. Rodriguez, Stephanie N. Weiss, Thomas H. Adams, Sheila M. Adams, Mei Sun, David E. Birk, Louis J. Soslowsky

ABSTRACT

Decorin and biglycan are two major small leucine-rich proteoglycans (SLRPs) present in the tendon extracellular matrix that facilitate collagen fibrillogenesis, tissue turnover, and cell signal transduction. Previously, we demonstrated that knockout of decorin prevented the decline of tendon mechanical properties that are associated with aging. The objective of this study was to determine the effects of decorin and biglycan knockdown on tendon structure and mechanics in aged tendons using tamoxifen-inducible knockdown models. We hypothesized that the knockdown of decorin and compound knockdown of decorin and biglycan would prevent age-related declines in tendon mechanics and structure compared to biglycan knockdown and wild-type controls, and that these changes would be exacerbated as tendon progress towards geriatric ages. To achieve this objective, we created tamoxifen-inducible mouse knockdown models to target decorin and biglycan gene inactivation without the abnormal tendon development associated with traditional knockout models. Knockdown of decorin led to increased midsubstance modulus and decreased stress relaxation in aged tendons. However, these changes were not sustained in the geriatric tendons. Knockdown in biglycan led to no changes in mechanics in the aged or geriatric tendons. Contrary to our hypothesis, the compound decorin/biglycan knockdown tendons did not resemble the decorin knockdown tendons but resulted in increased viscoelastic properties in the aged and geriatric tendons. Structurally, knockdown of SLRPs, except for the 570d I-Dcn-/-/Bgn-/- group, resulted in alterations to the collagen fibril diameter relative to wild-type controls. Overall, this study identified the differential roles of decorin and biglycan throughout tendon aging in the maintenance of tendon structural and mechanical properties and revealed that the compound decorin and biglycan knockdown phenotype did not resemble the single gene decorin or biglycan models and was detrimental to tendon properties throughout aging.

The Emotional Impact of Disrupted Environmental Contexts: Enrichment loss and coping profiles influence stress response recovery in Long-Evans rats

AUTHORS

Molly Kent, Dmitry Kovalev, Ben Hart, Danielle Leserve, Gabriella Handford, Dylan Vavra, Kelly Lambert

ABSTRACT

With increasing rates of anxiety and mood disorders across the world, there is an unprecedented need for preclinical animal models to generate translational results for humans experiencing disruptive emotional symptoms. Considering that life events resulting in a perception of loss are correlated with depressive symptoms, the enrichment-loss rodent model offers promise as a translational model for stress-initiated psychiatric disorders. Additionally, predisposed temperament characteristics such as coping styles have been found to influence an individual's stress response. Accordingly, male rats were profiled as either consistent or flexible copers and assigned to one of three environments: standard laboratory housing; enriched environment; or enriched environment exposure followed by downsizing to standard laboratory cages (i.e., enrichment-loss group). Throughout the study, several behaviors were assessed to determine stress, social, and reward-processing responses. To assess recovery of the stress response, fecal samples were collected following the swim stress in three-hour increments to determine the recovery trajectory of corticosterone (CORT) and dehydroepiandrosterone (DHEA) metabolite levels. Upon death, neural markers of neuroplasticity including doublecortin, glial fibrillary acidic factor, and brain-derived neurotrophic factor were assessed via immunohistochemistry. Results indicated the flexible coping animals in the continuous enriched group had higher DHEA/CORT ratios (consistent with adaptive responses in past research); further, the enrichment-loss animals exhibited a blunted CORT response throughout the assessments and enriched flexible copers had faster CORT recovery rates than consistent copers. Standard housed animals exhibited less exploratory behavior in the open field task and continuous enriched, flexible rats consumed more food rewards than the other groups. No differences in neuroplasticity neural markers were observed. In sum, the current results support past research indicating the disruptive consequences of enrichment-loss, providing evidence that the model represents a valuable approach for the investigation of neurobiological mechanisms contributing to interindividual variability in responses to changing experiential landscapes.

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.