Transcription Factor NFIB Regulates The Differentiation Of Adipocytes And Osteoblasts Through Wnt/?-catenin Signalling

Objective Bone marrow stromal stem cells(BMSCs)are pluripotent stem cells that have similar differentiation potential to osteoblasts and adipocytes.The imbalance between adipogenic and osteogenic differentiation may lead to various metabolic diseases.It was confirmed that multiple genes and signaling pathways have a regulatory effect on bone metabolism including NFI family members,but whether and how NFIB influences bone metabolism remains unclear.In this study,we explored the function and molecular mechanisms of NFIB in BMSCs differentiation through a series of in vitro and in vivo experiments.Methods 1.The expression of Nfib was quantified in various tissues of mice and in BMSCs and ST2 cells after osteogenic/adipogenic treatment.The expression of Nfib over time during osteogenic and adipogenic differentiation was examined respectively.Nfib was overexpressed or knocked down,then the expression of adipogenesis/osteogenesis/lipid-synthesis related genes was detected by q RT-PCR and Western blotting,and status of cell differentiation were observed by oil red O staining and Alp staining.2.Screen downstream target genes of Nfib by q RT-PCR and WB.Observe the effect of NFIB on nuclear transport by immunofluorescence.Nuclear protein was quantified after overexpression of Nfib.The molecular mechanism by which NFIB regulates target genes was confirmed through dual luciferase reporter gene assay and Ch IP assay.Verify the function of the target gene in the regulation of cell differentiation.Blocking experiments were performed to see if the effect of NFIB in regulating adipogenic/osteogenic differentiation was blocked by target gene knockdown.3.Obesity model induced by high-fat diet was constructed.The body weight was recorded and serum glycolipid metabolism indicators such as total cholesterol,free fatty acid and glucosewa were detected after inject Nfib-sh RNA LV into the tail vein.Qualities and sizes of adipocytes were measured after HE staining.A mouse model of bilateral ovariectomy was constructed,and Nfib si RNA was injected into the medullary cavity for in vivo transfection.One month later,paraffin sections were taken from the tibia,adipocytes were observed after HE staining.Results 1.The expression of Nfib differs in various tissues.In BMSCs and ST2 cells,during adipogenic/osteogenic differentiation,the expression of Nfib changed over time.Overexpress Nfib promoted adipogenesis and lipid synthesis,inhibited osteogenic differentiation.In contrast,Nfib knockdown inhibited adipogenic differentiation and lipid synthesis,and promoted osteogenic differentiation.After infected with Nfibsh RNA LV and inducted,the adipogenesis of BMSCs and ST2 cells was weakened and the osteogenic differentiation was enhanced.2.The expression of Sfrp4 decreased after si RNA was used to reduce Nfib expression.Immunofluorescence showed that the overexpress Nfib prevented the nuclear transport of ?-catenin caused by Wnt3 a.The expression of ?-catenin and TCF7L2 was decreased in nuclear proteins after overexpression of Nfib.The dual luciferase reporter assay and the Ch IP assay demonstrated that Nfib can promote transcription by specifically binding to Sfrp4.Overexpress Sfrp4 promoted adipogenic differentiation and inhibited osteogenic differentiation;Knockdown Sfrp4 inhibited adipogenic differentiation and promoted osteogenic differentiation.Blocking experiments confirmed that reducing Sfrp4 expression attenuated NFIB's ability to regulate the differentiation of BMSCs.3.Intravenous injection of Nfib-sh RNA LV alleviated the weight gain,and inhibited the increase in adipocyte size in HFD-fed mice,reduced the levels of serum free fatty acids,total cholesterol,and glucose.In the mouse model of bilateral ovariectomy,the number of adipocytes in the medullary cavity was significantly reduced after transfection of Nfib si RNA in vivo.Conclusion 1.NFIB can promote the differentiation of BMSCs into adipocytes and inhibit osteogenic differentiation.2.NFIB activates Sfrp4 gene transcription by directly binding to the promoter region of Sfrp4 gene to down-regulate canonical Wnt signaling.3.Nfib-sh RNA LV can alleviate obesity caused by high-fat diet in mice,improve obesity-related glycolipid metabolism disorder.Decreasing Nfib expression can inhibit the accumulation of fat in the bone marrow cavity of ovariectomized mice.