The Function And Mechanisms Of OSMR In Osteoblast Differtation And Bone Homestasis

Objective: Oncostatin M(OSM)is a member of the cytokine Interleukin(IL-6)family.Previous Studies found that OSM function as a regulator that has a close relationship with cell survival,proliferation and differentiation.A lot of research has confirmed that OSM can promote bone formation.Oncostatin M receptor(OSMR)is one of the receptors of OSM,and it is widely distributed in different cells.Recent findings domenstrate that the OSM-OSMR signaling pathway mainly regulates bone metabolism by mediating the formation of osteoclasts and bone resorption.The change of bone mass are closely related to the activity of osteoclasts and osteoblasts.At present,there are few studies on the regulatory effect of OSMR on the mesenchymal stem cells,and its regulation mechanism is not completely clear.This study mainly explores the effects of OSMR on osteoblast differentiation and bone homeostasis regulation,and preliminarily explores the mechanism about osteogenic differentiation.Methods: 1.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the changes of Osmr expression during the induction of adipogenic /osteogenic differentiation.2.Osmr plasmid/si RNA were transfeced into bone marrow stromal cell line ST2 to increase or inhibite the expression of Osmr,the effects of Osmr on adipogenesis / osteogenic differentiation of ST2 cells were examined using q RT-PCR,western blotting,and oil red O staining / ALP staining.2.OSMR knock-down lentiviral plasmid was constructed and infected mouse BMSCs,then induced into adipogenesis / osteogenesis,The technique of q RT-PCR,western blotting and oil red O staining / ALP staining were used to detect the effecttions of OSMR on adipogenic / osteogenic differentiation of BMSCs.3.Using q RT-PCR,Western Blotting technology and blocking experiments to detect the expression changes of autophagy signaling pathway-related factors and MAPK signaling pathway after increasing or decreasing Osmr expression,and explore the mechanism of how OSMR plays a regulatory role.4.A 8-week-old C57 BL / 6J female mouse was used to construct ovariectomy(OVX)mouse models,and the Sham model was used as a control,q RT-PCR was used to detect changes in Osmr expression.5.BMSCsallogeneic transplantation was performed on OVX model mice,which were divided into 3 groups: Sham+Ctrl LV?OVX+Ctrl LV?OVX+Osmr-KD LV.Three months after modeling,the changes of bone mass in the control and experimental groups were detected by X-ray,micro-CT,and tissue section tachylimin-eosin(HE)staining.Results: 1.During the adipogenic differentiation of bone marrow mesenchymal stem cells,the expression of Osmr increased firstly and then decreased,and the expression was highest on the second day of adipogenic induction;during the osteogenic differentiation,the expression of Osmr generally increased and then decreased,the decline is more obvious on days 5-9 of osteogenic induction.2.After increasing the expression of Osmr in ST2 cells,the cell adipogenic differentiation was promoted and the osteogenic differentiation was supressed.The expression of adipogenesis-related factor genes such as PPAR?,C/EBP?,a P2 and adipsin were significantly increased.The expression of osteogenesis-related factor genes such as Runx2,OSX,ALP and OPN were obviously decreased.In contrast,decreasing the expression of Osmr in ST2 cells and BMSCs can inhibit adipogenesis and promote osteogenesis.3.After overexpression the Osmr,the autophagy-related genes like LC3 and P62,the LC3?/LC3 ? ratio was decreased,and P62 protein level was increased,and m RNA and protein expressions of ULK-1,Beclin-1,Atg5,and Atg7 were all obviously decreased.In contrast,after decreasing Osmr expression,the LC3 ? / LC3 ? ratio was increased,and P62 was decreased,at the same time the m RNA and protein expressions of ULK-1,Beclin-1,Atg5,and Atg7 were significantly increased.Further more we detected MAPK signaling pathway by Western Blotting,revealed that its key factor extracellularly regulated protein kinases(ERK)phosphorylation level was significantly enhanced.Then ST2 cells were treated with the ERK inhibitor U0126,the results showed that Osmr si RNA promotes the phosphorylation of ERK,thereby increasing the conversion of LC3 ? to LC3 ?,and ultimately affecting on the osteogenic differentiation.4.After modeling 3 months,the Osmr m RNA expression in bone tissue was significantly increased.5.micro-CT and HE showed that the tibia bone mass of OVX+Ctrl LV group was significantly reduced.The tibia bone mass of OVX+ Osmr-KD LV group was reduced compared with the Sham+Ctrl LV group,but the bone loss was relieved compared with the OVX+Ctrl LV group.Conclusion: 1.Osmr expression was significantly increased in the bone tissue of OVX model mice,suggesting a correlation between OSMR and postmenopausal osteoporosis.2.OSMR can promote the differentiation of bone marrow stromal cell line ST2 and BMSCs into adipocytes and inhibit their differentiation into osteoblasts.3.Osmr si RNA positively regulates autophagy by accelerating the phosphorylation of ERK,then enchancing the osteoblast differentiation.4.BMSCs were infected by Osmr-KD LV,then allogeneic transplantated into postmenopausal osteoporosis mice model can slow down the bone loss,and promote osteogenic differentiation of BMSCs.