MKRN1 Promotes Osteogenic Differention Through Ubiquitination And Degradation Of PPARγ In MC3T3-E1 Cells

Objective:Osteoporosis is a systemic bone disease with damaged bone microstructure,compromised bone mineral density and biomechanical properties,which accompanies with pain,bone deformation and bone fracture.With the aging of people,the incidence of osteoporosis and osteoporotic fracture are rising,which has become a serious public health problem.Osteoporosis also has a local impact on the jaw and maxillary bone.Several research indicate there is a relationship between osteoporosis and periodontitis,and osteoporosis may have a negative effect on implant osteointegration.Studies have shown that the main pathophysiological mechanism of osteoporosis is the impaired function of osteoblast proliferation,differentiation and mineralization.Therefore,the study of osteoblast function is important for understanding the pathogenesis of osteoporosis and exploring effective treatments.Studies have proved that peroxisome proliferator-activated receptorγ(PPARγ)is an important gene involved in cell differentiation.It is mainly expressed in adipose tissue and immune system,participates in the regulation of various molecular mechanisms,and is closely related to adipocyte differentiation,body immunity,obesity and insulin resistance.Recently,it has been found that PPARγ also plays a role in the field of bone metabolism.It has been reported that PPARγ can inhibit MSCs differentiation to osteoblasts by inhibiting cbfa1 expression,and can inhibit osteoclast differentiation by upregulating IL-4 activity.PPARγ can interact with the transforming growth factor TGF-β / Smad3 to inhibit the differentiation of osteoblast cells.Animal models of murine maxillary expansion showed that use of the PPARγ agonist pioglitazone could reduced new bone formation with maxillary raphe expansion,whereas PPARγ knockout mice had increased new bone,decreased osteocast numbers and osteoclastogenesis gene expression.In 2014,it was found that PPARγ is regulated by MKRN1.And K184 and K185 are two lysine sites of PPARγ modified.The negatively regulating PPARγ function of MKRN1 protein was further demonstrated in knocked down MKRN1-MEF,3T3-L1 and C3H10T1 / 2 cell lines,suggesting that MKRN1 may be a potential therapeutic target for PPARγ-related diseases.No effects of MKRN1 and PPARγ on osteogenesis have been reported.This study will explore whether MKRN1-mediated PPARγ ubiquitination exists during MC3T3-E1 osteogenic differentiation or not.This study will provide a new protocol and basis for the treatment of osteoporotic diseases.Methods:1.Effects of MKRN1 on osteogenic differentiation of MC3T3-E1 cells1.Effects of MKRN1 on osteogenic differentiation of MC3T3-E1 cellsRT-qPCR and Western Blot were used to detect the correlation between MKRN1 expression and osteogenic differentiation of MC3T3-E1 cells.Adenoviral vectors were constructed to infect MC3T3-E1 cells,and the effect of MKRN1 on osteogenic differentiation of MC3T3-E1 cells was determined by ALP activity assay,RT-qPCR,Western Blot,and alizarin red staining.2.Effects of MKRN1 and PPARγ on osteogenic differentiation of MC3T3-E1 cellsMC3T3-E1 cells were infected with adenovirus vector by interfering the expression of MKRN1 and PPARγ,and osteogenic differentiation was induced.Expression of osteogenic differentiation markers and calcification deposition were determined by RT-qPCR,Western Blot,ALP and alizarin red staining.3.Effects of MKRN1 on PPARγ expression level in MC3T3-E1 cellsWestern Blot was used to detect the influedce of MKRN1 on PPARγ in MC3T3-E1 cells.Co-IP detected the binding of endogenoius MKRN1 and PPARγ in MC3T3-E1 cells and the ubiquitination level of PPARγ protein after overexpression of MKRN1.After addition of MG132,PPARγ expression in MKRN1 overexpression cells was deteced by Western Blot.Results:1.The expression level of MKRN1 mRNA and protein increased with time in MC3T3-E1 cells during osteogenin differentiation(P <0.01).The activity of ALP,expression level of Runx2,COL1A1,OCN and the formation of calcium nodules were all reduced after MKRN1 knockdown in MC3T3-E1 cells.On the other hand,all biomarkers of osteogenic differentistion are increased in MKRN1 overexpression cells.2.Interference of PPARγ expression in MC3T3-E1 cells could reverse the inhibitory effect of MKRN1 knockdown on osteogenic differentiation and mineralization.3.MKRN1 ovrpression alleviated PPARγ protein expression and addition of MG132 reversed the effect.Co-IP confirmed the interaction between endogenous MKRN1 and PPARγ in cells.Overexpression of MKRN1 increased the ubiquitination level of PPARγ.Conclusion:1.The E3 ubiquitin ligase MKRN1 can promote osteogenic differentiation of MC3T3-E1 cells.2.MKRN1 promotes osteogenic differentiation through PPARγ ubiquitination and proteasome-dependant degradation.