Glucose Regulates Tissue-specific Chondro-osteogenic Differentiation Of Human Cartilage Endplate Stem Cells Via O-GlcNAcylation Of Sox9 And Runx2

Background:Degenerative disc disease(DDD)is a n age-related chronic disease that can cause low back pain.The pathogenesis of DDD is still unclear,and the microenvironment of the intervertebral disc may play an important role in the occurrence and development of DDD.The physiological low-glucose microenvironment of the cartilage endplate(CEP)is disrupted in DDD.Glucose influences protein O-Glc NAcylation via the hexosamine biosynthetic pathway(HBP),which is the key to stem cell fate.Based on the first confirmation of the presence of cartilage endplate stem cells(CESCs),we found that increased microenvironmental glucose concentration i n the early stages of cartilage endplate degeneration can induce O-Glc NAcylation modification of Sox9 and Runx2 in CESCs.Sox9 and Runx2 are core factors of stem cell differentiation,and O-Glc NAcylation of Sox9 and Runx2 may be closely related to the differentiation of CESCs.This study will further clarify the function of OGlc NAcylation of Sox9 and Runx2 in CESCs.This study is a new mechanism for the regulation of CESCs differentiation,and provides a new therapeutic target for inhibiting cartilage endp late calcification.Methods:Part Ⅰ: Detection of O-Glc NAcylation and ossification/ chondrification of normal or degenerated CEPWe collected normal and degenerative cartilage endplate samples from patients with spinal fractures and patients with lumbar disc degeneration.The O-Glc NAcylation,ossification marker(COL1)and chondrification marker(COL2)of CEP were detected by RT-PCR,Western Blot,and immunohistochemistry.Part Ⅱ: Investigate the role of glucose on CESCs osteogenic and chondrogenic differentiationCESCs were induced with osteogenic and chondrogenic induction medium under low glucose(1 m M),normal glucose(5 m M)and high glucos e(25 m M).Osteogenic differentiation markers(Runx2,COL1,and ALP)and chondrogenic differentiation markers(Sox9,COL2,and AGN)were detected by RT-PCR and Western Blot.Alizarin red staining and Al cin blue staining were used to detect osteogenic the differentiation marker(calcium nodules)and the chondrogenic differentiation marker(acid mucopolysaccharide).Part Ⅲ: Investigate the role of O-Glc NAcylation on CESCs osteogenic and chondrogenic differentiationCESCs were induced with osteogenic and chondrogenic induction medium under low glucose and high glucose.O-Glc NAcylation inducer(Thiamet-G)and inhibitor(DON)were used to up-or down-regulate the O-Glc NAcylation of protein.Osteogenic differentiation markers(Runx2,COL1,and ALP)and chondrogenic differentiation markers(Sox9,COL2,and AGN)were detected by RT-PCR and Western Blot.Alizarin red staining and Al cin blue staining were used to detect osteogenic the differentiation marker(calcium nodules)and the chondrogenic differentiation ma rker(acid mucopolysaccharide).Part Ⅳ: Investigate the modification and regulation of O-Glc NAcylation on Runx2/Sox9We use immunoprecipitation and immunofluorescence staining to detect whether Runx2 / Sox9 could be modified by O-Glc NAcylation.Thiamet-G and DON were used to up-and down-regulate the O-Glc NAc ylation of Runx2 and Sox9.Then,the expression of Runx2 downstream molecules(ALP,OCN)and Sox9 downstream molecule(COL2)were detected by RT-PCR and Western Blot.Results:Part Ⅰ:Compared with the normal CEP,the protein O-Glc NAcylation and the expression of ossification marker(COL1)of degenerated CEP increased,while the expression of chondrification marker(COL2)decreased.Part Ⅱ:1.Low glucose microenvironment promote d CESCs chondrogenic differentiation while inhibited CESCs osteogenic differentiation.2.Normal and high glucose microenvironment promote d CESCs osteogenic differentiation while inhibited CESCs chondrogenic differentiation.Part Ⅲ:1.In low glucose microenvironment,high protein O-Glc NAcylation(Thiamet-G)inhibited CESCs chondrogenic differentiation while promoted osteogenic differentiation.2.In high glucose microenvironment,low protein O-Glc NAcylation(DON)promoted CESCs osteogenic differentiation while inhibited chondrogenic differentiation.Part Ⅳ:1.Runx2 and Sox9 were modified by O-Glc NAcylation.2.O-Glc NAcylation promoted the expression of Runx2 downstream molecules(COL1 and ALP)and inhibited the expression of Sox9 do wnstream molecule(COL2).Conclusions:1.CEP ossification and protein O-Glc NAcylation increased while chondrification decreased during CEP degeneration.2.During CEP degeneration,the glucose concentration in the microenvironment gradually increased,leading to an increase of intracellular protein O-Glc NAcylation,inhibiting CESCs chondrogenic differentiation while promoting osteogenic differentiation.3.CESCs differentiation core factors Runx2 and Sox9 were modified by O-Glc NAcylation.And the O-Glc NAcylation promoted Runx2 activity while inhibited Sox9 activity.