The Mechanism Of ERK5 Signaling Pathway Regulates Adipogenic Differentiation And Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective: More adipocytes are detected in clinical osteoporotic patients,but the mechanisms underlying it remain largely unknown.Our previous studies have shown that the inactivation of ERK5 signaling pathways resulted in low bone mass,more adipocytes,and less osteoblasts in mice.We postulated that activated ERK5 can maintain the homeostasis of osteogenesis and adipogenesis in bone marrow stromal cells(BMSCs).This study intends to further study the molecular mechanism of ERK5 signaling pathway regulating the adipogenic differentiation and osteogenic differentiation balance of bone marrow mesenchymal stem cells at the cellular level,in order to provide theoretical basis and ideas for clinical treatment of osteoporosis.Methods:(1)BMSCs were infected by lentivirus,inserted into ERK5 interference gene fragments,and then screened by puromycin to obtain mouse BMSCs model with stable and low expression of ERK5;(2)Osteoblast induction was performed on mouse BMSCs after ERK5 gene silencing,alkaline phosphatase(ALP)activity was measured on the 4th,9th,and 14 th days of induction,and the osteocalcin(OCN)content secreted into the culture medium was detected at 0-4d,4-9d and 9-14 d.The mRNA expression levels of ERK5 and osteogenic marker genes which include runt-related transcription factor 2(RUNX-2),ALP and bone morphogenic protein 2(BMP2)were examined by RT-PCR after induction for 10 d.Finally,alizarin red staining was performed after 21 days of induction to observe the formation of calcium nodules.(3)Adipogenic induction of mouse BMSCs after ERK5 gene silencing was performed,the mRNA expression levels of ERK5 and the adipogenic marker genes which include peroxisome proliferator-activated receptor-gamma(PPAR?),CCAAT enhancer binding protein alpha(CEBP?)and fatty acid binding protein 4(ap2)were examined by RT-PCR,and oil red O staining was performed 28 days after adipogenicinduction to observe the formation situation of adipocytes.Results:(1)The infection of Balb/c mouse BMSCs with no-load virus CON077 did not affect the expression of ERK5,while the lentivirus ERK5-RNAi(83235-1)and ERK5-RNAi(83236-1)effectively silenced the ERK5 expression;(2)During osteogenic induction differentiation,ERK5 silencing decreased ALP activity,reduced secreted OCN content,and significantly reduced the expression of osteogenic differentiation marker genes RUNX-2,ALP and BMP2.Intracellular calcium nodules were significantly reduced after alizarin red staining when the 21 days of osteogenic differentiation;(3)In the process of adipogenic induction,ERK5 silencing significantly up-regulated the expression of the adipogenic marker genes PPAR?,C/EBP? and ap2,and became adipogenic.Oil-red O staining was performed on 28 days of adipogenic differentiation,lipid droplets were found to be larger,more and more round.Conclusion:(1)ERK5 signaling pathway promotes the osteogenic differentiation of mouse BMSCs by regulating the osteogenic differentiation marker genes RUNX-2,ALP and BMP2;(2)At the same time,ERK5 signaling pathway inhibit the adipogenic differentiation of mouse BMSCs by regulating the adipogenic differentiation marker genes PPAR?,C/EBP? and ap2.