Fragile X Messenger Ribonucleoprotein 1(FMR1), a novel inhibitor of osteoblast/osteocyte differentiation, regulates bone formation, mass, and strength in young and aged male and female mice

Fragile X Messenger Ribonucleoprotein 1(FMR1)gene mutations lead to fragile X syndrome,cognitive disorders,and,in some individuals,scoliosis and craniofacial abnormalities.Four-month-old(mo)male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass.However,consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown.We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2-and 9-mo mice.The cancellous bone mass is higher only in females,whereas,cortical bone mass is higher in 2-and 9-mo males,but higher in 2-and lower in 9-mo female FMR1-knockout mice.Furthermore,male bones show higher biomechanical properties at 2mo,and females at both ages.Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro,without affecting osteoclasts in vivo/ex vivo.Thus,FMR1 is a novel osteoblast/osteocyte differentiation inhibitor,and its absence leads to age-,site-and sex-dependent higher bone mass/strength.

Mechanically induced Ca^(2+) oscillations in osteocytes release extracellular vesicles and enhance bone formation

The vast osteocytic network is believed to orchestrate bone metabolic activity in response to mechanical stimuli through production of sclerostin, RANKL, and osteoprotegerin(OPG). However, the mechanisms of osteocyte mechanotransduction remain poorly understood. We've previously shown that osteocyte mechanosensitivity is encoded through unique intracellular calcium (Ca^(2+) ) dynamics. Here, by simultaneously monitoring Ca^(2+) and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca^(2+) transients, which were facilitated by smooth muscle myosin and further confirmed in native osteocytes ex vivo. Actomyosin contractions have been linked to the secretion of extracellular vesicles(EVs), and our studies demonstrate that mechanical stimulation upregulates EV production in osteocytes through immunostaining for the secretory vesicle marker Lysosomal-associated membrane protein 1(LAMP1) and quantifying EV release in conditioned medium, both of which are blunted when Ca^(2+) signaling was inhibited by neomycin. Axial tibia compression was used to induce anabolic bone formation responses in mice, revealing upregulated LAMP1 and expected downregulation of sclerostin in vivo. This load-related increase in LAMP1 expression was inhibited in neomycin-injected mice compared to vehicle.Micro-computed tomography revealed significant load-related increases in both trabecular bone volume fraction and cortical thickness after two weeks of loading, which were blunted by neomycin treatment. In summary, we found mechanical stimulation of osteocytes activates Ca^(2+) -dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca^(2+) signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca^(2+) -mediated signaling in bone adaptation.

Skeletal loading regulates breast cancer-associated osteolysis in a loading intensity-dependent fashion

Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium(CM) and fluid flow-treated conditioned medium(FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition(MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition(EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer(FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumorinduced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFβ levels and the composition of tumor-associated volatile organic compounds in the urine.Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumorosteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.

Overexpression of Lrp5 enhanced the anti-breast cancer effects of osteocytes in bone

Osteocytes are the most abundant cells in bone,which is a frequent site of breast cancer metastasis.Here,we focused on Wnt signaling and evaluated tumor-osteocyte interactions.In animal experiments,mammary tumor cells were inoculated into the mammary fat pad and tibia.The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model.The results revealed that administration of osteocytes or their conditioned medium(CM)inhibited tumor progression and osteolysis.Osteocytes overexpressing Lrp5 or β-catenin displayed strikingly elevated tumor-suppressive activity,accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53.The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound.Notably,silencing Lrp5 in tumors inhibited tumor progression,while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression.Osteocytes exhibited elevated Lrp5 expression in response to tumor cells,implying that osteocytes protect bone through canonical Wnt signaling.Thus,our results suggest that the Lrp5/β-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes.We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.

Suppression of osteosarcoma progression by engineered lymphocyte-derived proteomes

Cancer cells tend to develop resistance to chemotherapy and enhance aggressive-ness.A counterintuitive approach is to tame aggressiveness by an agent that acts opposite to chemotherapeutic agents.Based on this strategy,induced tumor-suppressing cells(iTSCs)have been generated from tumor cells and mesenchymal stem cells.Here,we examined the possi-bility of generating iTSCs from lymphocytes by activating PKA signaling for suppressing the pro-gression of osteosarcoma(OS).While lymphocyte-derived CM did not present anti-tumor capabilities,the activation of PKA converted them into iTSCs.Inhibiting PKA conversely gener-ated tumor-promotive secretomes.In a mouse model,PKA-activated CM suppressed tumorinduced bone destruction.Proteomics analysis revealed that moesin(MSN)and calreticulin(Calr),which are highly expressed intracellular proteins in many cancers,were enriched in PKA-activated CM,and they acted as extracellular tumor suppressors through CD44,CD47,and CD91.The study presented a unique option for cancer treatment by generating iTSCs that secret tumor-suppressive proteins such as MSN and Calr.We envision that identifying these tu-mor suppressors and predicting their binding partners such as CD44,which is an FDA-approved oncogenic target to be inhibited,may contribute to developing targeted protein therapy.