Effects Of Caveolin-1/Caveolae On Osteogenic Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells And Its Mechanism

Bone marrow-derived MSCs are multipotent cells,capable of self-renewal and differentiation into at least three lineages(osteogenic,chondrogenic,and adipogenic)when stimulated under appropriate conditions.Due to their differentiation potential and easy acquisition,MSCs are potent in augmenting bone repair and regeneration.To utilize MSCs effectively in regenerative medicine,a clear understanding of how they respond to environmental cues is essential.It is likely that,similar to all other cells,an initial determinant of stem cell responsiveness to external stimuli is the organization of signaling molecules in cell membrane rafts.Membrane rafts are cholesterol and sphingolipid-rich liquid-ordered phases in the cell membrane that allow compartmentalization and clustering of cell surface signaling molecules.Membrane rafts fall into two broad categories,non-caveolar and caveolar,based on the absence or presence,respectively,of caveolin scaffolding proteins.Cav-1 probably dampens pro-osteogenic signaling by sequestering signaling mediators in cell surface caveolae and/or driving their internalization via caveolae endocytosis.In this study we hypothesized that MAPK/ERK1/2 signaling may be pro-osteogenic and post-translationally regulated by Cav-1 and caveolae in mouse MSCs,increased Cav-1 expression during MSCs osteogenesis likely acts as a negative feedback to stabilize the cell phenotype.This study is divided into two stages.The first stage of the study was to investigate the expression of Caveolin-1 in the process of osteogenic differentiation of BMSCsMethod:The rats BMSCs were cultured in vitro and classified into osteoblast-induced groups and non-induced group.After 3、7、10、14days,identification of osteoblasts were identifiedby alizarin red staining and alkaline phosphatase assay,and expression of the Caveolin-1 was detected by real-time reverse transeri ptase-polymerase chain reaction(RT-PCR)and Western Blot.Results:RT-PCR results showed that osteoblast-induced groups had a higher expression level of Caveolin-1 than non-induced group at the same time point,expression of Caveolin-1 was increased in a time-dependent manner in the osteoblast-induced groups.Western Blot test showed that osteoblast-induced groups also had a higher expression level of Caveolin-1 than non-induced group at the same time point.Expression of Caveolin-1 was increased in a time-dependent manner in osteoblast-induced groups.Conclusion:According to the mRNA test and protein test,it is suggested that Caveolin-1 exists in the BMSCs.Caveolin-1 expression increased in the progress of osteogenic differentiation.In the second phase,Caveolin-1 is involved in the regulation of BMSCs osteogenic differentiation.Method:The rats BMSCs were cultured in vitro and were incubated in standard growth medium alone or induced to osteogenically differentiate by the addition ofsupplements(β-glycerophosphate,ascorbic acid,dexamethasone,and 1,25-dihydroxyvitamin D3).The distribution of Caveolin-1 in BMSCs was observed by immunofluorescence.identification of osteoblasts was performed by real-time reverse transeriptase-polymerase chain reaction(RT-PCR)and alizarin red staining.Treat BMSCs with the MARK/ERK1/2 inhibitorPD98059,identification of osteoblastsactivation was performed by alizarin red staining and alkaline phosphatase assay.Treat BMSCs preincubation with methyl-β-cyclodextrin(MβCD),followedby immunoblottingfor total ERK1/2 and phosphorylated ERK1/2.Results:Immunofluorescence results showed that Caveolin-1 was expressed in the cell membrane and cytoplasm of BMSCs;RT–PCR、 alizarin red staining showed that BMSCs differentiation into osteoblastsin vitro.Different doses of PD98059 can inhibit BMSCs differentiating into osteoblasts;Methyl-β-cyclodextrininhibited osteogenesis.Western blotshowed that MβCD suppressed phosphorylation of ERK1/2,but with no change of total ERK1/2 levels.Cholesterol oxidase(CHOD)treatment enhances BMSCsosteogenesis.Western blot showed that phosphorylated ERK1/2 levels increased,while total ERK1/2remained unchanged.Conclusion:MARK/ERK1/2 signaling pathway participates in regulation of BMSCs osteogenic differentiation,Caveolae may be the site of phosphorylation of ERK1/2,Caveolin-1 may inhibit BMSCs differentiating into bone cells by aggregation of ERK1/2 phosphorylated molecules into Caveolae.