Effects And Mechanisms Of Shh Signaling Pathway On Chondrocyte Differentiation In Sika Deer Antlers

It is well known that proliferation and differentiation are two key processes of cartilage ossification,which are essential not only for physiological bone development and growth,but also for the fracture healing and the pathogenesis of osteoarthritis.As a bone organ,sika deer antlers proliferate and differentiate during a series of growth processes to maintain the steady state of antler growth and development without canceration.More importantly,antler chondrocytes provide a good model for studying animal and human bone and joint diseases.Sonic hedgehog(Shh)protein is an important signaling molecule in early stage of embryonic development,Shh protein can regulate the proliferation and differentiation of Bone marrow derived stroma cell(BMSCs)into chondrocytes in tissues and cells,but the mechanism of Shh signaling in antler horn is still unclear.It has been reported that Shh plays an important role in regulating the proliferation and differentiation of chondrocytes.Therefore,we hypothesized that Shh signaling may stimulate the growth of antler by promoting the proliferation and differentiation of antler chondrocytes.In this study,we investigated the effects of Shh signaling pathway on the proliferation and differentiation of antler chondrocytes and explored its regulatory mechanism by in situ hybridization,flow cytometry,Western blot,MTS assay,Real-time PCR and RNA interference,etc.The results showed that Shh mRNA was highly expressed in antler chondrocytes.Shh signaling pathway promoted the proliferation of antler chondrocytes and induced the expression of Cyclin D1(Ccnd1),Ccnd2,Ccnd3,Ccne1,Cdk2,Cdk4 and Cdk6.The result of Flow cytometry indicated that Shh treatment promoted the cell transition from G1 phase to S phase in antler chondrocytes,further revealing the regulation mechanism of Shh promoting the proliferation of antler chondrocytes.Simultaneously,addition of rShh induced the expression of(Collagen X)Col X,(Runt-related transcription factor 2)Runx2 and Alkaline phosphatase(Alpl)which was a well-known differentiation marker for chondrocytes.In situ hybridization results indicated that Shh signaling pathway components Patched1(Ptch1),Smoothened(Smo),Zinc finger transcription factor glioma-associated oncogene homolog 1(Gli1),Gli2 and Gli3 mRNA signal was noted in antler chondrocytes.Ptch1 gene silencing increased the induction of rShh on Smo.rShh increased the expression of Smo,Gli1,Gli2 and Gli3,while specific siRNA attenuated its expression.The Smo inhibitor Cyc attenuated the promoting effect of rShh on chondrocyte proliferation and differentiation.Further studies have found that Gli transcription factor by GANT58 decreased the induction effect of rShh on Col X,Runx2 and Alpl.Consistentwith the above result,silencing of Gli1,Gli2 and Gli3 by the corresponding siRNA resulted in a failure in the induction of rShh on Col X,Runx2 and Alpl.Further studies confirmed that the Shh signaling pathway can affect the proliferation and differentiation of antler chondrocytes through Activin A and(Transforming growth factor beta 1)TGFβ1 signaling pathway.Activin A and TGFβ1 signaling pathway can activate or inhibit the expression of Shh signaling pathway and Foxa family by activating downstream Notch signaling pathway.Activin A,TGFβ1,Notch signaling pathway and Forkhead transcription factor a(Foxa)family mRNA are also expressed in antler chondrocytes.Activin A and TGFβ1 signaling pathway promoted chondrocytes proliferation and differentiation,and accelerated the transition of cell cycle from G1 to S phase,accompanied by activation of Notch and Shh signaling pathways,while Notch signaling pathway inhibitor DAPT and Smo inhibitor Cyc could effectively block induction effect by rActA and rTGFβ1.At the same time,the addition of DAPT resulted in a significant slowdown in cell cycle progression,while the expression of the Shh signaling pathway was decreased.In contrast,the addition of exogenous rShh rescued the delayed action of chondrocyte proliferation and differentiation induced by DAPT,indicating that the Notch signaling pathway was upstream of the Shh signaling pathway.Further analysis demonstrated that DAPT may inhibit the activation of rActA and rTGFβ1 on Shh signaling pathway.At the same time,After transfection with siRNAs targeting Foxa1,Foxa2 or Foxa3 decreased the induction effect of rActA,rTGFβ1 or rShh on Col X,Runx2 and Alpl.In addition,the Shh signaling pathway plays an important role in the crosstalk between Activin A,TGFβ1,Notch signaling pathway and Foxa.In summary,this study was to investigate the role of Activin A,TGFβ1,Notch,Foxa and Shh signaling pathways in the proliferation and differentiation of antler chondrocytes and to elucidate their interactions.Activin A and TGFβ1 might provoke Notch signaling pathway to accelerate the production of Shh.Inactivation of Ptch1 enhanced the induction of Shh on Smo,resulting in the activation of Gli transcription factors to facilitate the expression of downstream target Foxa.