The Activation Of Vitamin D Receptor In Osteoclast Precursor Affect Mice Osteoclasts Differentiation And Activity

Osteoclast(OC)is a terminal differentiation multinuclear giant cell which was derived from hematopoietic stem cell.Many cytokines and hormones including the macrophage colony stimulating factor(M-CSF),receptor activator for nuclear factor-?B ligand(RANKL)are involved in the differentiation of the osteoclast precursor(OCP),then fuse to generate osteoclast.OC adhered to the surface of the bone matrix and activated to perform the bone resorption function.The abnormal osteoclast number and activity in the body are related to the occurrence of bone metabolism diseases,Such as osteoporosis,bone sclerosis and rheumatoid arthritis.OCP express multiple receptors,including macrophage colony-stimulating factor receptor(c-fms),receptor activator for nuclear factor-?B(RANK)and leucine-rich repeat-containing G-protein coupled receptor 4(LGR4),the interaction of multiple receptors regulates the differentiation of osteoclast precursor into osteoclast.Vitamin D receptor(VDR)is a steroid hormone receptor that is mainly expressed in the nucleus and a little bit of distribution on the cell membrane.Vitamin D receptor was involved in the regulation of multiple systems and cells function,including metabolism,immunity,development.The vitamin D receptor in intestine and kidney can indirectly affect bone metabolism via regulating the reabsorption of calcium and phosphorus.However,the expression of VDR in bone could directly regulate the process of bone reconstruction,osteoclast differentiation and bone absorption.The expression of VDR in osteoblast(OB)and osteocyte impact OC function by promoting the expression of RANKL and inhibiting the expression of osteoprotegerin(OPG),However,the expression of VDR in osteoclast precursor or osteoclast and the effects on osteoclast differentiation and bone absorption are remain largely unknown.The study aims to reveal the effect and mechanism of VDR in OCP in osteoclast differentiation and activation,provide a scientific theory for the role of VDR in the bone metabolism regulation.1.The establishment of osteoclast induce system in vitroThe osteoclast has a short life time,limited number and difficult separate from the bone.In this study,to obtain a large number of osteoclast with bone absorption function,the osteoclast precursor,bone marrow macrophage(BMM)was cultures and induces differentiation into osteoclasts and the co-culture system of osteoclast was established by incubate with osteoblast.In osteoclast direct induction system,BMM are seeded at a density of 1× 104?2×104 and 5×104 cell/well in 96-well plate with 30 ng/ml M-CSF and 50 ng/ml RANKL.The results show that osteoclast was formed after 4-5 days of induction in different cell density.However,the differentiation effciency of osteoclasts decreased with the increase of the bone marrow macrophages number(p<0.01).The largest number of osteoclasts produce in a density of 1 × 104 cell/well.The result indicated that the number of osteoclast precursor is related to the efficiency of osteoclast differentiation.In osteoclast co-culture system,osteoblast were seeded at a density of 1 X 103cell/well in 96-well plate for overnight,then the BMM with cell density as mention above was plant into the 96-well plate with osteoblast in present of prostaglandin E2(PGE2).The results showed that the co-culture of osteoblast and bone marrow macrophages induce osteoclast formation in 6-7 days,and PGE2 could promote the efficiency of osteoclast differentiation.The results showed that the osteoclast co-culture system was established.2.Expression of vitamin D receptor in osteoclast differentiationOsteoclast were directly induced from bone marrow macrophage,the proliferation and activity of cell were detected by RTCA during osteoclast differentiation.Immunofluorescence staining was used to observe the distribution of VDR in osteoclast precursor and osteoclast,RT-PCR was used to detect the expression of relevant marker genes and VDR transcriptional levels during osteoclast differentiation,Western blot was used to detect the expression of key transcription factor NFATc1 and VDR protein during osteoclast differentiation.The results showed that the VDR expressed both in osteoclast precursor and osteoclast,and it was mainly distributed in the nucleus and with a small amount in the cell membrane.The expression of VDR decreased with the increase of the key transcription factor NFATc1 and related marker gene TRAP,CAII and MMP-9 expression in osteoclast differentiation(p<0.01).The dates suggested that VDR in osteoclast precursor may involved in the regulation of osteoclast differentiation and bone absorption function.3.The activation of vitamin D receptor in osteoclast precursor inhibited the differentiation of osteoclastActivated or overexpressed the vitamin D receptor in OCP by using vitamin D receptor activator 1? 25-(OH)2D3 or VDR overexpression virus,then induced with M-CSF and RANKL to generated osteoclast.The effects of vitamin D receptor activator on the proliferation and activity of osteoclast precursor were investigated by RTCA.The formation of osteoclast was observed by TRAP staining.RT-PCR was used to detect the osteoclast marker genes mRNA expression.Western blot was used to detect the expression of marker proteins in osteoclast differentiation.In addition,base on the establishment of osteoclast co-culture model,the effect of VDR expressed in OCP on osteoclast formation was further clarified.The results showed that the activation of VDR had no significant effect on the proliferation and activity of osteoclast precursor(p>0.05).VDR activation or overexpression in OCP inhibited the differentiation of osteoclasts with a concentration-time dependence mamer(p<0.05).VDR activation or overexpression in OCP inhibits the transcription and translation level of osteoclast related gene TRAP,DC-STAMP and Integrin ?3(p<0.01).After the overexpression of VDR in OCP,the number of osteoclast was reduced in the co-culture system(p<0.05).The results demonstrated that the activation or overexpression of VDR in OCP inhibited osteoclast differentiation.4.The molecular mechanism of vitamin D receptor activation in osteoclast precursors inhibits osteoclast differentiationThe vitamin D receptor in OCP was activated by activator during osteoclast induced by M-CSF and RANKL.Western blot was used to detect the effects of VDR activation on early signal MAPKs and Akt signaling pathway and key transcription factor NFATc1 c-fos and VDR expression.The overexpression of c-fos in OCP by using the c-fos overexpression virus,then activated the VDR.The number of osteoclasts were observed by TRAP staining.The expression of the marker gene of osteoclast differentiation was detected by RT-PCR.Add gene transcription and translation inhibitors,then examined the effects of VDR activation on the stability of transcription factor c-fos mRNA and protein by RT-PCR and Western blot.Construct the fusion expression vector of VDR and c-fos with different tag,the interaction between VDR and c-Fos was detected by Western blot and immunofluorescence.The results showed that the activation of VDR in the osteoclast precursors had no significant effect on the early signal of osteoclast differentiation(p>0.05),however,it was inhibited the expression of key transcription factors c-fos and NFATcl(p<0.01).The overexpression of c-fos in osteoclast precursor can eliminate or alleviate the inhibition effect of osteoclast differentiation induced by VDR activation(p<0.01).The activation of VDR in osteoclast precursor attenuate the stability of c-fos and there is interaction between VDR and c-fos(p<0.01).The results revealed that the activation VDR in OCP interact with c-fos,and attenuated the stability of c-fos,then decreased the c-fos and NFATcl expression with the inhibition of osteoclast differentiation.5.The mechanism of vitamin D receptor activation in osteoclast precursors inhibit osteoclast activityThe osteoclasts were cultured on the cortical bone slices induced by M-CSF and RANKL with the activation of VDR in OCP.Osteoclast activity was detected by scanning electron microscopy for bone absorption lacuna.RT-PCR and Western blot were used to detect the expression of osteoclast bone absorption function genes.The osteoclast multi-nucleation and cytoskeleton change were observed by fluorescence microscopy.The expression of key regulation proteins RhoA,Rac1 and Cdc42 in Rho GTPases were detected by Western blot.The results show that the activation of VDR in osteoclast precursor inhibited the osteoclast bone resorption activity and the expression of related functional genes(p<0.01).Meanwhile,the multi-nucleation process of osteoclast and the formation of F-actin ring were inhibited.The key regulation protein Racl and Cdc42 in Rho GTPases were also decreased(p<0.01).The dates illustrated that the Racl and Cdc42 in Rho GTPases participate in the inhibition effect of VDR activation on osteoclast activity in OCP.