Effects And Mechanism Of Peroxisome Prolif-erator Activated Receptorγ On Mineralization In Cementoblasts

Object: PPARγ is a member of nuclear receptor family of transcription factors,which involved in many physiological processes including inflammation regulating and lipid metabolism and having effect on bone rebuilt.Bone shares many similarities with cementum which lines on toot surface,maintains periodontal stability and participates periodontal tissue regeneration.The role of PPARγ in the acquisition and maintenance of bone mass has been scrutinized.However,rare studies explored the effects of PPARγ on cementoblasts differentiation and mineralization.In order to explore the potential approaches in repairing the damaged periodontal tissues especially for cementum,the present study aims to investigate the effects and the regulating mechanism of PPARγ on the mineralization and osteogenic differentiation in cementblasts.Methods: 1.Murine cementoblast cell-lines(OCCM-30)were cultured in basic medium for 48 h to detect the expression of PPARγ after PPARγ agonist and antagonist treatment by qRT-PCR and western blot,and choose the best concentration for further study;2.The cells were cultured in mineralization medium respectively at addition of DMSO,rosiglitazone,GW9662 for 3,7 and 10 days,using qRT-PCR and western blot to evaluate the expression of mineralization genes ALP,OCN and RUNX,detecting ALP activity by ALP staining,and alizarin red staining to observe the formation of mineralization nodules;3.To investigate the expression of β-catenin in total protein and cytoplasm/nucleus protein under treatment rosiglitazone and GW9662 by Western blot.Then cells in basic or mineralization medium were stimulated with Licl,the activator of Wnt/β-catenin signal pathway,detecting the location of β-catenin in cells by immunofluorescence and expression of ALP,OCN and RUNX via qRT-PCR and western blot,and to observing the change of mineralization molecules after treatment with agonist of PPARγ and Licl;4.Finally,to explore the effect of PPARγ on expression of mineralization factors in OCCM-30 cells under inflammatory microenvironment with TNF-α treatment by qRT-PCR.Results: 1.There was PPARγ in cementoblasts,and rosiglitazone improved the expression of PPARγ while antagonist inhibited it;2.Mineralization results showed that the agonist of PPARγ promoted the expression of mineralization genes ALP,RUNX2 and OCN,deepened ALP staining and increased mineralization nodules formation,meanwhile the antagonist presented the converse effect;3.The level of β-catenin decreased after rosiglitazone treatment,and increased at adding inhibitor in total and cytoplasm/nucleus protein.β-catenin accumulated obviously in nucleus and the expression of ALP,RUNX2 and OCN were decreased after adding Licl,an activator of the canonical Wnt pathway.Furthermore,the addition of Licl reversed the up-regulated expression of ALP,RUNX2 and OCN in rosiglitazone treated OCCM-30 cells;4.PPARγ promoted mineralization factors ALP,RUNX2 and OCN expression in inflammatory microenvironment by TNF-α stimuli.Conclusions: Wnt/β-catenin signaling pathway may play a negative feedback role on mineralization and differentiation of cementoblast cell line OCCM-30.2.PPARγ could improve cementoblasts differentiation and mineralization via inhibiting Wnt/β-catenin signaling pathway,which would shed new light on treatment of periodontitis and periodontal tissue regeneration.