Secreted-Osteopontin Contributes To Brown-adipogenesis Via A CD44-dependent Pathway

Objective:Osteopontin(OPN),a secreted multifunctional glycoprotein,contributes to many pathophysiological processes including immune response,inflammation,tumor formation and metabolism.OPN exists as two forms,i.e.,secreted-OPN(sOPN)and intracellular-OPN(iOPN).While they might have different biological activities,the effects of sOPN and iOPN on the differentiation of brown adipocytes have not been entirely elucidated.Consequently,the purpose of this study was to investigate the effects of sOPN on white adipocyte browning and the underlying mechanisms,and to explore whether the unmodified recombinant human OPN(rhOPN),as a substitute of iOPN,also has the similar effects and mechanisms as the sOPN.Methods:First,3T3-L1 cells were induced by DMI induction with rhOPN(10,50,100,200?M)for 6 days,respectively.And set the vehicle group treated with phosphate buffered solution(PBS).Then,the cells were divided into three groups this treatment,namely,Ctrl group,Vehicle group and rhOPN group.Meanwhile,another batch of 3T3-L1 cells were infected with Ad-GFP-ap2-OPN and empty adenovirus vector(Vehicle),respectively,and followed by DMI differentiation for 6 days.Besides,three groups were set,namely,Ctrl group,Vehicle group and Ad-GFP-ap2-OPN group.Subsequently,the infected cells were treated with anti-CD44 antibody or immunoglobulin G(Ig G),respectively,and induced by DMI for 6 days as described aboved.Then,the treated cells were grouped into the Ig G group and anti-CD44 antibody group.Finally,the following indicators were subsequently tested:1)the cell viability of 3T3-L1 cells treated with rhOPN was determined by MTS assay;2)the levels of sOPN were detected by ELISA assay;3)oil red O staining was used to observe the lipid accumulation;4)Western Blotting assay was used to detect the brown adipocytes-related protein expression,such as UCP-1,PRDM16,PGC-1?and PPAR?,and protein expression levels of P-PI3K,PI3K,AKT,AKT-pS473 to explore the mechanism.Results:1)The results of oil red O staining showed that the amount of lipid droplets gradually increased and peaked at day 6,which indicated that the adipocytes have basically differentiated to maturation.2)After different titer adenovirus Ad-GFP-ap2-OPN was used to infect 3T3-L1 cells,the cells were observed under fluorescence inverted microscope.It was found that the adipocytes were not damaged in morphology and had the highest green fluorescence when the virus titer was1×10~8PFU for 48 h.Therefore,the optimal titer was 1×10~8PFU and the best infection time was 48 h.3)The result of ELISA assay showed that the level of sOPN obviously increased.4)The result of MTS assay showed that the viability of adipocytes treated with different concentrations rhOPN instead of undermodified iOPN was not significantly affected.5)The results of oil red O staining showed that after upregulation of sOPN by infected with Ad-GFP-ap2-OPN,the cells accumulated quantities of lipid droplets,but there was no obvious accumulation of lipid in the cells treated with rhOPN.6)According to Western Blotting results,the expression levels of UCP-1,PRDM16,PGC-1?and PPAR?in 3T3-L1 cells infected with Ad-GFP-ap2-OPN were significantly increased,while those treated with rhOPN failed to function as above.7)After finding that sOPN could promote browning of white adipocytes,the results of oil red O staining showed that after neutralizing the CD44 receptor with anti-CD44 antibody,the lipid droplets aggregation was notably inhibited.8)The results of Western Blotting assay showed that after neutralizing the cd44 receptor,the expression of browning adipocytes-related proteins were distinctly inhibited,and the phosphorylation of PI3K/AKT was significantly inhibited as well.Conclusion:These data demonstrate that sOPN has the capacity to induce the differentiation of white preadipocytes into brown adipocytes through a CD44-dependent mechanism.Nevertheless,rhOPN fails to have the effects described above.