The Hippo signalling pathway in bone homeostasis: Under the regulation of mechanics and aging.

The Hippo signalling pathway is a conserved kinase cascade that orchestrates diverse cellular processes, such as proliferation, apoptosis, lineage commitment and stemness. With the onset of society ages, research on skeletal aging-mechanics-bone homeostasis has exploded. In recent years, aging and mechanical force in the skeletal system have gained groundbreaking research progress. Under the regulation of mechanics and aging, the Hippo signalling pathway has a crucial role in the development and homeostasis of bone. We synthesize the current knowledge on the role of the Hippo signalling pathway, particularly its downstream effectors yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), in bone homeostasis. We discuss the regulation of the lineage specification and function of different skeletal cell types by the Hippo signalling pathway. The interactions of the Hippo signalling pathway with other pathways, such as Wnt, transforming growth factor beta and nuclear factor kappa-B, are also mentioned because of their importance for modulating bone homeostasis. Furthermore, YAP/TAZ have been extensively studied as mechanotransducers. Due to space limitations, we focus on reviewing how mechanical forces and aging influence cell fate, communications and homeostasis through a dysregulated Hippo signalling pathway.

Semaphorin3C identified as mediator of neuroinflammation and microglia polarization after spinal cord injury.

Excessive neuroinflammation after spinal cord injury (SCI) is a major hurdle during nerve repair. Although proinflammatory macrophage/microglia-mediated neuroinflammation plays important roles, the underlying mechanism that triggers neuroinflammation and aggravating factors remain unclear. The present study identified a proinflammatory role of semaphorin3C (SEMA3C) in immunoregulation after SCI. SEMA3C expression level peaked 7 days post-injury (dpi) and decreased by 14 dpi. In vivo and in vitro studies revealed that macrophages/microglia expressed SEMA3C in the local microenvironment, which induced neuroinflammation and conversion of proinflammatory macrophage/microglia. Mechanistic experiments revealed that RAGE/NF-κB was downstream target of SEMA3C. Inhibiting SEMA3C-mediated RAGE signaling considerably suppressed proinflammatory cytokine production, reversed polarization of macrophages/microglia shortly after SCI. In addition, inhibition of SEMA3C-mediated RAGE signaling suggested that the SEMA3C/RAGE axis is a feasible target to preserve axons from neuroinflammation. Taken together, our study provides the first experimental evidence of an immunoregulatory role for SEMA3C in SCI via an autocrine mechanism.

Efficacy of EDTA-NS irrigation in eradicating Staphylococcus aureus biofilm-associated infection.

Aims: To investigate the efficacy of ethylenediaminetetraacetic acid-normal saline (EDTA-NS) in dispersing biofilms and reducing bacterial infections. Methods: EDTA-NS solutions were irrigated at different durations (1, 5, 10, and 30 minutes) and concentrations (1, 2, 5, 10, and 50 mM) to disrupt Staphylococcus aureus biofilms on Matrigel-coated glass and two materials widely used in orthopaedic implants (Ti-6Al-4V and highly cross-linked polyethylene (HXLPE)). To assess the efficacy of biofilm dispersion, crystal violet staining biofilm assay and colony counting after sonification and culturing were performed. The results were further confirmed and visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). We then investigated the efficacies of EDTA-NS irrigation in vivo in rat and pig models of biofilm-associated infection. Results: When 10 mM or higher EDTA-NS concentrations were used for ten minutes, over 99% of S. aureus biofilm formed on all three types of materials was eradicated in terms of absorbance measured at 595 nm and colony-forming units (CFUs) after culturing. Consistently, SEM and CSLM scanning demonstrated that less adherence of S. aureus could be observed on all three types of materials after 10 mM EDTA-NS irrigation for ten minutes. In the rat model, compared with NS irrigation combined with rifampin (Ti-6Al-4V wire-implanted rats: 60% bacteria survived; HXLPE particle-implanted rats: 63.3% bacteria survived), EDTA-NS irrigation combined with rifampin produced the highest removal rate (Ti-6Al-4V wire-implanted rats: 3.33% bacteria survived; HXLPE particle-implanted rats: 6.67% bacteria survived). In the pig model, compared with NS irrigation combined with rifampin (Ti-6Al-4V plates: 75% bacteria survived; HXLPE bearings: 87.5% bacteria survived), we observed a similar level of biofilm disruption on Ti-6Al-4V plates (25% bacteria survived) and HXLPE bearings (37.5% bacteria survived) after EDTA-NS irrigation combined with rifampin. The in vivo study revealed that the biomass of S. aureus biofilm was significantly reduced when treated with rifampin following irrigation and debridement, as indicated by both the biofilm bacterial burden and crystal violet staining. EDTA-NS irrigation (10 mM/10 min) combined with rifampin effectively removes S. aureus biofilm-associated infections both in vitro and in vivo. Conclusion: EDTA-NS irrigation with or without antibiotics is effective in eradicating S. aureus biofilm-associated infection both ex and in vivo.

H3K36 methyltransferase NSD1 protects against osteoarthritis through regulating chondrocyte differentiation and cartilage homeostasis.

Osteoarthritis (OA) is one of the most common joint diseases, there are no effective disease-modifying drugs, and the pathological mechanisms of OA need further investigation. Here, we show that H3K36 methylations were decreased in senescent chondrocytes and age-related osteoarthritic cartilage. Prrx1-Cre inducible H3.3K36M transgenic mice showed articular cartilage destruction and osteophyte formation. Conditional knockout Nsd1Prrx1-Cre mice, but not Nsd2Prrx1-Cre or Setd2Prrx1-Cre mice, replicated the phenotype of K36M/+; Prrx1-Cre mice. Immunostaining results showed decreased anabolic and increased catabolic activities in Nsd1Prrx1-Cre mice, along with decreased chondrogenic differentiation. Transcriptome and ChIP-seq data revealed that Osr2 was a key factor affected by Nsd1. Intra-articular delivery of Osr2 adenovirus effectively improved the homeostasis of articular cartilage in Nsd1Prrx1-Cre mice. In human osteoarthritic cartilages, both mRNA and protein levels of NSD1 and OSR2 were decreased. Our results indicate that NSD1-induced H3K36 methylations and OSR2 expression play important roles in articular cartilage homeostasis and OA. Targeting H3K36 methylation and OSR2 would be a novel strategy for OA treatment.

MSI2 Modulates Unsaturated Fatty Acid Metabolism by Binding FASN in Bovine Mammary Epithelial Cells.

The regulation of fatty acid metabolism is crucial for milk flavor and quality. Therefore, it is important to explore the genes that play a role in fatty acid metabolism and their mechanisms of action. The RNA-binding protein Musashi2 (MSI2) is involved in the regulation of numerous biological processes and plays a regulatory role in post-transcriptional translation. However, its role in the mammary glands of dairy cows has not been reported. The present study examined MSI2 expression in mammary glands from lactating and dry milk cows. Experimental results in bovine mammary epithelial cells (BMECs) showed that MSI2 was negatively correlated with the ability to synthesize milk fat and that MSI2 decreased the content of unsaturated fatty acids (UFAs) in BMECs. Silencing of Msi2 increased triglyceride accumulation in BMECs and increased the proportion of UFAs. MSI2 affects TAG synthesis and milk fat synthesis by regulating fatty acid synthase (FASN). In addition, RNA immunoprecipitation experiments in BMECs demonstrated for the first time that MSI2 can bind to the 3'-UTR of FASN mRNA to exert a regulatory effect. In conclusion, MSI2 affects milk fat synthesis and fatty acid metabolism by regulating the triglyceride synthesis and UFA content through binding FASN.

Premature aging of skeletal stem/progenitor cells rather than osteoblasts causes bone loss with decreased mechanosensation.

A distinct population of skeletal stem/progenitor cells (SSPCs) has been identified that is indispensable for the maintenance and remodeling of the adult skeleton. However, the cell types that are responsible for age-related bone loss and the characteristic changes in these cells during aging remain to be determined. Here, we established models of premature aging by conditional depletion of Zmpste24 (Z24) in mice and found that Prx1-dependent Z24 deletion, but not Osx-dependent Z24 deletion, caused significant bone loss. However, Acan-associated Z24 depletion caused only trabecular bone loss. Single-cell RNA sequencing (scRNA-seq) revealed that two populations of SSPCs, one that differentiates into trabecular bone cells and another that differentiates into cortical bone cells, were significantly decreased in Prx1-Cre; Z24f/f mice. Both premature SSPC populations exhibited apoptotic signaling pathway activation and decreased mechanosensation. Physical exercise reversed the effects of Z24 depletion on cellular apoptosis, extracellular matrix expression and bone mass. This study identified two populations of SSPCs that are responsible for premature aging-related bone loss. The impairment of mechanosensation in Z24-deficient SSPCs provides new insight into how physical exercise can be used to prevent bone aging.

Accelerated aging in articular cartilage by ZMPSTE24 deficiency leads to osteoarthritis with impaired metabolic signaling and epigenetic regulation.

Osteoarthritis (OA) is an age-related degenerative disease without disease-modifying therapy. The lack of aging-induced osteoarthritis models makes the discovery of therapeutic drugs more challenging. The deficiency of ZMPSTE24 could induce Hutchinson-Gilford progeria syndrome (HGPS), a genetic disorder of rapid aging. However, the relationship between HGPS and OA remains unclear. Our results found that the expression of Zmpste24 was decreased in the articular cartilage during the aging process. Zmpste24 knockout mice, Prx1-Cre; Zmpste24fl/fl mice and Col2-CreERT2; Zmpste24fl/fl mice displayed OA phenotype. Loss of Zmpste24 in articular cartilage could exacerbate the occurrence and development of osteoarthritis. Transcriptome sequencing revealed that deletion of Zmpste24 or accumulation of progerin affects chondrocyte metabolism, inhibits cell proliferation and promotes cell senescence. Using this animal model, we elucidate the upregulation of H3K27me3 during chondrocyte senescence and discover the molecular mechanism by which lamin A mutant stabilizes EZH2 expression. The construction of aging-induced osteoarthritis models and the elucidation of the signaling pathways and molecular mechanisms of articular chondrocyte senescence would benefit the discovery and development of new drugs for OA.

The RNA-binding protein Musashi2 governs osteoblast-adipocyte lineage commitment by suppressing PPARγ signaling.

Osteoporosis caused by aging is characterized by reduced bone mass and accumulated adipocytes in the bone marrow cavity. How the balance between osteoblastogenesis and adipogenesis from bone marrow mesenchymal stem cells (BMSCs) is lost upon aging is still unclear. Here, we found that the RNA-binding protein Musashi2 (Msi2) regulates BMSC lineage commitment. Msi2 is commonly enriched in stem cells and tumor cells. We found that its expression was downregulated during adipogenic differentiation and upregulated during osteogenic differentiation of BMSCs. Msi2 knockout mice exhibited decreased bone mass with substantial accumulation of marrow adipocytes, similar to aging-induced osteoporosis. Depletion of Msi2 in BMSCs led to increased adipocyte commitment. Transcriptional profiling analysis revealed that Msi2 deficiency led to increased PPARγ signaling. RNA-interacting protein immunoprecipitation assays demonstrated that Msi2 could inhibit the translation of the key adipogenic factor Cebpα, thereby inhibiting PPAR signaling. Furthermore, the expression of Msi2 decreased significantly during the aging process of mice, indicating that decreased Msi2 function during aging contributes to abnormal accumulation of adipocytes in bone marrow and osteoporosis. Thus, our results provide a putative biochemical mechanism for aging-related osteoporosis, suggesting that modulating Msi2 function may benefit the treatment of bone aging.

Msi2-mediated MiR7a-1 processing repression promotes myogenesis.

BACKGROUND: Most of the microRNAs (MiRs) involved in myogenesis are transcriptional regulated. The role of MiR biogenesis in myogenesis has not been characterized yet. RNA-binding protein Musashi 2 (Msi2) is considered to be one of the major drivers for oncogenesis and stem cell proliferation. The functions of Msi2 in myogenesis have not been explored yet. We sought to investigate Msi2-regulated biogenesis of MiRs in myogenesis and muscle stem cell (MuSC) ageing. METHODS: We detected the expression of Msi2 in MuSCs and differentiated myotubes by quantitative reverse transcription PCR (RT-qPCR) and western blot. Msi2-binding partner human antigen R (HuR) was identified by immunoprecipitation followed by mass spectrometry analysis. The cooperative binding of Msi2 and HuR on MiR7a-1 was analysed by RNA immunoprecipitation and electrophoresis mobility shift assays. The inhibition of the processing of pri-MiR7a-1 mediated by Msi2 and HuR was shown by Msi2 and HuR knockdown. Immunofluorescent staining, RT-qPCR and immunoblotting were used to characterize the function of MiR7a-1 in myogenesis. Msi2 and HuR up-regulate cryptochrome circadian regulator 2 (Cry2) via MiR7a-1 was confirmed by the luciferase assay and western blot. The post-transcriptional regulatory cascade was further confirmed by RNAi and overexpressing of Msi2 and HuR in MuSCs, and the in vivo function was characterized by histopathological and molecular biological methods in Msi2 knockout mice. RESULTS: We identified a post-transcription regulatory cascade governed by a pair of RNA-binding proteins Msi2 and HuR. Msi2 is enriched in differentiated muscle cells and promotes MuSC differentiation despite its pro-proliferation functions in other cell types. Msi2 works synergistically with another RNA-binding protein HuR to repress the biogenesis of MiR7a-1 in an Msi2 dose-dependent manner to regulate the translation of the key component of the circadian core oscillator complex Cry2. Down-regulation of Cry2 (0.6-fold, vs. control, P < 0.05) mediated by MiR7a-1 represses MuSC differentiation. The disruption of this cascade leads to differentiation defects of MuSCs. In aged muscles, Msi2 (0.3-fold, vs. control, P < 0.01) expression declined, and the Cry2 protein level also decreases (0.5-fold, vs. control, P < 0.05), suggesting that the disruption of the Msi2-mediated post-transcriptional regulatory cascade could attribute to the declined ability of muscle regeneration in aged skeletal muscle. CONCLUSIONS: Our findings have identified a new post-transcriptional cascade regulating myogenesis. The cascade is disrupted in skeletal muscle ageing, which leads to declined muscle regeneration ability.

Histone demethylase LSD1 is critical for endochondral ossification during bone fracture healing.

Bone fracture is repaired predominantly through endochondral ossification. However, the regulation of endochondral ossification by key factors during fracture healing remains largely enigmatic. Here, we identify histone modification enzyme LSD1 as a critical factor regulating endochondral ossification during bone regeneration. Loss of LSD1 in Prx1 lineage cells severely impaired bone fracture healing. Mechanistically, LSD1 tightly controls retinoic acid signaling through regulation of Aldh1a2 expression level. The increased retinoic acid signaling in LSD1-deficient mice suppressed SOX9 expression and impeded the cartilaginous callus formation during fracture repair. The discovery that LSD1 can regulate endochondral ossification during fracture healing will benefit the understanding of bone regeneration and have implications for regenerative medicine.

VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription.

VGLL4 has been identified as a YAP inhibitor. However, the exact function of VGLL4 in bone development and bone homeostasis remains unclear. In this study, we demonstrated that VGLL4 breaks TEADs-mediated transcriptional inhibition of RUNX2 to promote osteoblast differentiation and bone development. We found that knockout of VGLL4 in mesenchymal stem cells and preosteoblasts showed osteoporosis and a cleidocranial dysplasia-like phenotype due to osteoblast differentiation disorders. Mechanistically, we showed that the TEAD transcriptional factors severely inhibited osteoblast differentiation in a YAP binding-independent manner. TEADs interacted with RUNX2 to repress RUNX2 transcriptional activity. Furthermore, VGLL4 relieved the transcriptional inhibition of TEADs by directly competing with RUNX2 to bind TEADs through its two TDU domains. Collectively, our studies demonstrate that VGLL4 plays an important role in regulating osteoblast differentiation and bone development, and that TEADs regulate the transcriptional activity of RUNX2, which may shed light on treatment of cleidocranial dysplasia and osteoporosis.

3-(4-Meth-oxy-benzyl-idene)-1,5-dioxa-spiro-[5.5]undecane-2,4-dione.

In the title mol-ecule, C(17)H(18)O(5), which was prepared by the reaction of (R)-1,5-dioxaspiro-[5.5]undecane-2,4-dione and 4-meth-oxy-benzaldehyde with ethanol, the 1,3-dioxane ring is in a distorted envelope conformation with the spiro C atom forming the flap. The crystal structure is stabilized by weak inter-molecular C-H⋯O hydrogen bonds.