Effects And Mechanisms Of ATRA On Chondrocyte Differentiation In Sika Deer Antlers

Chondrocyte differentiation is essential for endochondral ossification which normally occurs in the morphogenesis of limb buds and growth plates,and in the regeneration and repair of bone injury.Previous studies have evidenced that deer antler is the only mammalian bony organ to be lost and completely regenerated,and its growth also involves endochondral bone formation.Antler regeneration is implicated in several tissues including cartilage where chondrocytes undergo rapidly proliferation without becoming cancerous and differentiation into hypertrophic chondrocytes,and thus provides a valuable model for investigating the mechanisms involved in cartilage development,endochondral ossification and rapid tissue growth.ATRA injection can significantly stimulate the growth of the initial antler,but its regulatory mechanism is unclear.It is reported that ATRA is involved in regulating the proliferation and differentiation of chondrocytes.Therefore,we speculate that ATRA may stimulate the growth of antler by promoting the proliferation and differentiation of antler chondrocytes.In this study,we investigated the effects of ATRA on the proliferation and differentiation of antler chondrocytes and explored its regulatory mechanism by in situ hybridization,flow cytometry,MTS assay,Real-time PCR,gene overexpression and interference,etc.Here we showed that ATRA could promote the proliferation of antler chondrocytes and induce the expression of CCND1,CCND2,CCND3,CCNE1,CDK2,CDK4 and CDK6.The result of Flow cytometry indicated that ATRA treatment could promote the cell transition from G1 phase to S phase in antler chondrocytes.Simultaneously,addition of ATRA induced the expression of COL X and MMP13 which was a well-known differentiation marker for chondrocytes.We found that the RA transporter CRABP2 m RNA signal be located in antler chondrocytes by in situ hybridization.Silencing of CRABP2 could attenuate the induction of ATRA on chondrocyte differentiation,while overexpression of CRABP2 exhibited the opposite effects.ATRA metabolic enzyme CYP26A1 and CYP26B1 were also highly expressed in antler cartilage.Specific si RNA or combined antagonist R115866-mediated inhibition of CYP26A1or/and CYP26B1 enhanced the effect of ATRA on the differentiation of chondrocytes,whereas overexpression of CYP26B1 weakened the susceptibility of antler chondrocytes to ATRA.Further studies have shown that RA receptors RAR?,RAR?,RAR?,RXR? and RXR? were detected in antler cartilage.RAR? agonist Am80 treatment could promote the expression of COL X and MMP13 in antler chondrocytes.In contrast,administration of RAR? antagonist Ro41-5253 or RXR? si RNA repressed the stimulating effect of ATRA on chondrocyte differentiation.Further analysis evidenced that ATRA might induced the differentiation of antler chondrocytes and restrain the expression of BMP2 and WNT4 by activation RAR?/RXR?heterodimers.Knockdown of BMP2 or WNT4 enhanced the induction of ATRA on COL X and MMP13 expression,whereas overexpression of BMP2 or WNT4 abrogated this effectiveness.The transfection of WNT4 si RNA reversed the inhibitory effect of BMP2 overexpression on chondrocyte differentiation.At the same time,WNT4 overexpression blocked the upregulation of COLX and MMP13 expression that was induced by BMP2 si RNA.The expression of WNT4 was detected in antler chondrocytes that were transfected by BMP2 si RNA or overexpression plasmid.The results showed that BMP2 could mediate the regulation of ATRA on WNT4.Administration of ATRA to antler chondrocytes transfected with RUNX1 si RNA failed to induce the differentiation.Conversely,exogenous r RUNX1 strengthened the stimulation of ATRA on COL X and MMP13 expression.Simultaneously,RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte differentiation.In addition,transfection of WNT4 si RNA inhibited the down-regulation of RUNX1 which was caused by overexpression of BMP2 in antler chondrocytes,while WNT4 overexpression could reverse the effect of BMP2 si RNA on RUNX1 expression.Attenuation of BMP2 or WNT4 enhanced the stimulation of ATRA on RUNX1,while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1.Collectively,ATRA might activate RAR?/RXR? and regulate the differentiation of antler chondrocytes through BMP2-WNT4-RUNX1 pathway.CRABP2 enhances the effects of ATRA on the differentiation of antler chondrocytes,while CYP26A1 and CYP26B1 rendered the chondrocytes hyposensitive to ATRA.