ERR?-mediated Lipid Catabolic Process Facilitates Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective: This study aims to investigate the effect of estrogen-related receptor ?(ERR?)on regulating lipid catabolic process during osteogenic differentiation of mesenchymal stem cells(MSCs)and to explore the mechanism of ERR? regulates osteogenic differentiation of MSCs.Methods: Firstly,alkaline phosphatase staining and alizarin red S staining were used to determine the differentiation capability of MSCs.The m RNA expression of ERR?,osteogenic markers genes and fatty acid oxidation-related genes were determined by quantitative real-time PCR(q RT-PCR).The protein expression level of Osterix were determined by western blot.Then,MSCs were treated with carnitine palmitoyl transferase 1(CPT1)inhibitor Etomoxir(ETO)to verify the importance of fatty acid oxidation in osteogenic differentiation.Subsequently,lipid droplets(Bodipy,Nile Red staining)and lysosomes(Lysotracker staining)were stained to analyze the changes of lipid droplets during the osteogenic differentiation of MSCs.At the same time,in order to explore the pathway of lipid droplets breakdown,q RT-PCR was used to detect autophagy-related genes and autophagy inhibitors 3-Methyladenine(3-MA)and Chloroquine(CQ),adipose triglyceride lipase inhibitor Atglistatin(ATGLi)were successively treated MSCs during osteogenic differentiation.The senescent MSCs model late passage-MSCs(Lp-MSCs)was further constructed by serial passage in vitro.Then,the osteogenic differentiation ability of Lp-MSCs and the expression of ERR?,fatty acid oxidation-related genes and autophagy-related genes were detected.The differences in lipid droplets utilization were compared with those of normal cells(Early passage-MSCs,Ep-MSCs).Finally,the expression of ERR? in Lp-MSCs was upregulated by ERR?-overexpressing adenovirus,and then the transcriptional expression of fatty acid oxidation-related genes and autophagy-related genes of Lp-MSCs were detected by q RT-PCR.The alizarin red S and alkaline phosphatase staining were used to determine the differentiation capability of Lp-MSCs.Results: We found that ERR? and fatty acid oxidation-related genes were induced upon osteogenic differentiation of MSCs.Furthermore,inhibiting the key rate-limiting enzyme CPT1 of fatty acid oxidation by ETO could inhibit the osteogenic differentiation of MSCs.Lipid droplets were utilized in the process of osteogenic differentiation.By observing the distribution of lipid droplets and lysosomes in cells,it was found that the two co-localized.Using 3-MA and CQ to inhibit autophagy can inhibit the consumption of lipid droplets in MSCs and osteogenesis.However,the lipolysis inhibitor ATGLi had no significant effect on the osteogenic differentiation of MSCs.On the other hand,Compared with Ep-MSCs,Lp-MSCs consumed fewer lipid droplets,ERR?,fatty acid oxidation-related genes,autophagy-related genes,osteogenic marker genes and osteogenic capacity were reduced in Lp-MSCs.Importantly,overexpression of ERR? not only promoted the expression of fatty acid oxidationrelated genes and autophagy-related genes in Lp-MSCs,but also promoted osteogenic differentiation of Lp-MSCs.Conclusions: Our study found that the expression of lipid oxidation-related genes were up-regulated along with the expression of ERR? during the osteogenic differentiation of MSCs,and inhibition of the key rate-limiting enzyme CPT1 of fatty acid oxidation could inhibit the osteogenic differentiation of MSCs.At the same time,the expression of autophagy-related genes were up-regulated during osteogenic differentiation,and lipid droplets were utilized through the lipophagy pathway.What's more,our study demonstrates that decreased osteogenic capacity of MSCs upon aging is associated with down-regulation of ERR? and lipid catabolism.Further mechanistic studies suggest that ERR? restored the osteogenic differentiation ability of Lp-MSCs by promoting the expression of fatty acid oxidation-related genes and autophagy-related genes.Taken together,our mechanistic analysis reveals that ERR? is related to lipid catabolism during the osteogenic differentiation of MSCs,and it can promote osteogenic differentiation and repair the osteogenic differentiation ability of senescent MSCs by regulating lipid catabolism,which may provide a novel research perspective for the prevention and treatment of age-related osteoporosis.