Study On The Roles Of DEC1 On Bone Growth In Postnatal Mice

Bone remodeling is a highly regulated process mediated by the activities of bone-resorbing osteoclasts and bone-forming osteoblasts.The mature skeleton is remodeled throughout life.Under pathological conditions,this balance is disrupted.High osteoclast activity or low osteoblast activity leads to low bone mass（osteopenia）,while low osteoclast activity or high osteoblast activity leads to high bone mass（osteopetrosis）.Human differentiated embryonic chondrocyte expressed gene 1（DEC1）,a structurally distinct class of basic helix-loop-hellix（bHLH）protein,participates in a large amount of physiological processes including proliferation,differentiation,apoptosis,immune response,chondrogenesis,neurogenesis,lipogenesis and circadian rhythms.Several lines of evidence suggest that DEC1 is essential for bone growth.Moreover,recently reports have showed that DEC1 regulated the Wnt/β-catenin and PI3K/Akt signaling pathways in SaoS2 cells.But how DEC1 deletion affects skeletal bone growth and remolding at different postnatural stages and whether DEC1deficiency affects the expression ofβ-catenin and PI3KCA in mice have not been known.In the present study,we investigated the effects of DEC1 deficiency on long bone growth and remodeling in mice at different stages.Purpose:To investigate the roles of DEC1 deficiency on long bone growth and remodeling in mice at different stages.Methods:1.DEC1 KO mice（RBRC04841）were obtained from RIKEN BioResource Center.The mouse body size,meanwhile the body weight and length of tibia were measured.2.Histological sections of tibia were stained with H&E and Masson to measure the thickness of growth plate.Immuhistochemistry of PCNA was used to analysis the proliferation of chondrocytes in growth plate,Tunel assay was used to test the apoptosis rate of the hypertrophic layer.3.The concentration of calcium and phosphorus in serum was determined by calcium phosphate kit.4.X-Ray and Micro-CT were used to observe the bone structure and analysis bone mass,to make sure the results,Histological sections were stained with HE.HBFP staining was used to observe the total collagen.5.ALP staining and immunohistochemistry of Runx2and osterix was used to observe the osteoblasts activities.6.TRAP staining was used to test the activity of osteoclast,immuhistochemistry of MMP9 and CTSK was used to observe the absorption ability.Western blot was used to make sure the result and test the expression of RANKL,OPG and NFATc1.7.To explore the mechanisms of DEC1’s effect on Bone growth and bone remolding,IHC and WB were used to test the expression ofβ-catenin,DKK1 and SOST.8.WB were used to test the expression of PI3K/Akt/GSK3βsignaling pathway.Result:1.Retarded growth,shorted growth plate and reduced proliferation rate of chonrocytes during endochodral bone formation in 4-week-old DEC1^-/-mice and the retardation was diminished in 24-week-old knockout mice.2.Knockout of DEC1decreased cancellous bone mass.The density of the long boneswas a little decreased in 4 week old DEC1^-/-mice compared with that of control mice.The loss of bone mass in trabecular bone was more obvious in 24 week old DEC1 knockout mice than that in the age matched,as indicated by the decreased BMD,BV/TV,Tb.N,Tb.Th and increased Tb.Sp.3.The expression of ALP,Runx2 and osterix were all reduced in 4-and 24-week old DEC1^-/-mice compared with those in age matched DEC1^+/+mice,suggesting the bone formation decreased in DEC1^-/-mice.4.The number of osteoclasts was a little decreased at 4-week-old DEC1^-/-mice and was significantly decreased at 24-week-old DEC1^-/-compared with that in age matched DEC1^+/+mice,which implied osteoclast differentiation decreased during remolding in DEC1^-/-mice..Immunohistochemistry revealed that the expression of CTSK and MMP9 protein level were significantly reduced in DEC1 KO mice compared with those in WT mice.5.DEC1 up-regulate the Wnt/β-catenin signaling pathway.,IHC analysis displayed significantly lower expression ofβ-catenin protein but higher expression of DKK1 in DEC1^-/-versus DEC1^+/+mice at both early and late stage.The expression of SOST revealed no obvious difference in DEC1^-/-mice relative to wild type.6.Decreased PI3KCA/Akt/GSK3βpathway in DEC1^-/-mice.Western blot showed that the expression of PI3KCA,pGSK-3βand pAkt protein levels were marginally reduced in DEC1^-/-mice compared with those in wildtype littermates.Conclusion:1.DEC1 knockout mice shows retarded growth,shorted growth plate and reduced proliferation rate of chonrocytes during endochodral bone formation.These findings indicate that DEC1 deficiency contributes to the reduced growth plate and the phenotype of impaired linear bone growth in mice.2.The adult DEC1^-/-mice exhibits cancellous lower bone mass,decreased trabecular number than those of age matched DEC1^+/+mice.3.DEC1 deficiency down-regulates the activity of osteoblasts and osteoclasts.4.DEC1 deficiency induces low bone mass in adult mice by down-regulating WNT/β-catenin and PI3KCA/Akt/GSK3βpathway.