LCN2 Inhibits the BMP9-induced Osteogenic Differentiation through Reducing Wnt/ß-catenin Signaling via Interacting with LRP6 in Mouse Embryonic Fibroblasts.

BACKGROUND: Due to its effective osteogenic ability, BMP9 is a promising candidate for bone regeneration medicine. Whereas, BMP9 can also induce adipogenesis simultaneously. LCN2 is a cytokine associated with osteogenesis and adipogenesis. Reducing the adipogenic potential may be a feasible measure to enhance the osteogenic capability of BMP9. OBJECTIVE: The objective of the study was to explore the role of LCN2 in regulating the BMP9-initialized osteogenic and adipogenic differentiation in mouse embryonic fibroblasts (MEFs), and clarify the possible underlying mechanism. METHODS: Histochemical stain, western blot, real-time PCR, laser confocal, immunoprecipitation, cranial defect repair, and fetal limb culture assays were used to evaluate the effects of LCN2 on BMP9-induced osteogenic and adipogenic differentiation, as well as Wnt/ß-catenin signaling. RESULTS: LCN2 was down-regulated by BMP9. The BMP9-induced osteogenic markers were inhibited by LCN2 overexpression, but the adipogenic markers were increased; LCN2 knockdown exhibited opposite effects. Similar results were found in bone defect repair and fetal limb culture tests. The level of ß-catenin nucleus translocation was found to be reduced by LCN2 overexpression, but increased by LCN2 knockdown. The inhibitory effect of LCN2 overexpression on the osteogenic capability of BMP9 was reversed by ß-catenin overexpression; whereas, the effect of LCN2 knockdown on promoting BMP9 osteogenic potential was almost eliminated by ß-catenin knockdown. LCN2 could bind with LRP6 specifically, and the inhibitory effect of LCN2 on the osteogenic potential of BMP9 could not be enhanced by LRP6 knockdown. CONCLUSION: LCN2 inhibits the BMP9-induced osteogenic differentiation but promotes its adipogenic potential in MEFs, which may be partially mediated by reducing Wnt/ß-catenin signaling via binding with LRP6.

Interleukin 6 promotes BMP9-induced osteoblastic differentiation through Stat3/mTORC1 in mouse embryonic fibroblasts.

Interleukin 6 (IL-6) plays a dual role in regulating bone metabolism, although the concrete mechanism is unclear. Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic inducers, and a promising alternative for bone tissue engineering. The relationship between IL-6 and BMP9 in osteogenic differentiation remains to be elucidated, and the osteoblastic potential of BMP9 needs to be enhanced to overcome certain shortcomings of BMP9. In this study, we used real-time PCR, western blot, immunofluorescent stain, fetal limb culture and cranial defects repair model to explore the IL-6 role in BMP9-induced osteogenic differentiation in mouse embryonic fibroblasts (MEFs). We found that the rat serum level of IL-6 was increased in the dexamethasone-induced osteoporosis model, and IL-6 expression was detectable in several progenitor cells and MEFs. BMP9 upregulated IL-6 in MEFs, and the BMP9-induced osteoblastic markers were elevated by IL-6, but reduced by IL-6 knockdown. BMP9 and/or IL-6 both activated mTOR, and the IL-6 effect on BMP9-induced osteoblastic markers and bone formation were reduced greatly by mTOR inhibition. Raptor was up-regulated by IL-6 and/or BMP9 specifically, and the osteoblastic markers induced by IL-6 and/or BMP9 were reduced by Raptor knockdown. Meanwhile, Stat-3 was activated by IL-6 and/or BMP9, and the increase of Raptor or osteoblastic markers by IL-6 and/or BMP9 were reduced by Stat-3 inhibition. The Raptor promoter activity was regulated by p-Stat-3. Our finding suggested that IL-6 can promote the BMP9 osteoblastic potential, which may be mediated through activating Stat-3/mTORC1 pathway.

Analysis and Validation of Hub Genes in Blood Monocytes of Postmenopausal Osteoporosis Patients.

Osteoporosis is a common systemic bone disease caused by the imbalance between osteogenic activity and osteoclastic activity. Aged women are at higher risk of osteoporosis, partly because of estrogen deficiency. However, the underlying mechanism of how estrogen deficiency affects osteoclast activity has not yet been well elucidated. In this study, GSE2208 and GSE56815 datasets were downloaded from GEO database with 25 PreH BMD women and 25 PostL BMD women in total. The RRA algorithm determined 38 downregulated DEGs and 30 upregulated DEGs. Through GO analysis, we found that downregulated DEGs were mainly enriched in myeloid cell differentiation, cytokine-related functions while upregulated DEGs enriched in immune-related biological processes; pathways like Notch signaling and MAPK activation were found in KEGG/Rectome pathway database; a PPI network which contains 66 nodes and 91 edges was constructed and three Modules were obtained by Mcode; Correlation analysis helped us to find highly correlated genes in each module. Moreover, three hub genes FOS, PTPN6, and CTSD were captured by Cytohubba. Finally, the hub genes were further confirmed in blood monocytes of ovariectomy (OVX) rats by real-time PCR assay. In conclusion, the integrative bioinformatics analysis and real-time PCR analysis were utilized to offer fresh light into the role of monocytes in premenopausal osteoporosis and identified FOS, PTPN6, and CTSD as potential biomarkers for postmenopausal osteoporosis.