Effects And Mechanism Of YAP1 On The Differentiation Of ATDC5 Chondroprogenitor Cell Line

The linear growth of most bones and bone repair occur through a process known as endochondral ossification,which is regulated by a variety of endocrine and paracrine hormones?growth factors and transcription factors.YAP1(Yes-associated protein 1)transcriptional coactivator is a downstream gene of the Hippo signaling pathway,which controls cell proliferation and differentiation.YAP1 plays a significant role in the regulation of cartilage and bone development.However,the molecular mechanism by which YAP 1 regulates chondrocyte differentiation remains to be elucidated.Therefore,the present study was carries out as follows.Expression of YAP1 in ATDC5 and the effects and mechanism of YAP1 on proliferation and differentiation of ATDC5 in vitroPart I Endogenous YAP1 expression in ATDC5Experiment I:Endogenous YAP1 expression and subcellular location in ATDC5Objective:To determine the expression and subcellular location of YAP1 in ATDC5.Materials and methods:1.Culture of ATDC5 cells.2.Expression and subcellular location of YAP 1 in ATDC5 by immunofluorescent staining.3.ATDC5 were seeded at two different densities.Expression and subcellular location of YAP1 were detected by immunofluorescent staining.Results:Immunofluorescent staining revealed that YAP1 protein was mainly located in nuclei of ATDC5.The nuclear translocation of YAP1 decreased in ATDC5 cells when cultured at high cell density compared with the low density group.Part II The function of YAP1 on the proliferation,chondrogenic and hypertrophic differentiation of ATDC5Experiment I:Changes of YAP 1 expression in the process of ATDC5 differentiationObjective:To investigate the involvement of YAP1 and TAZ in chondrocyte differentiation.Materials and methods:1.ATDC5 cells were induced to chondrogenic differentiation.2.mRNA changes of YAP 1 and TAZ was detected by qRT-PCR.Results:qRT-PCR showed that YAP1 expression first increased then decreased.YAP1 mRNA levels were significantly upregulated at days 2 and 4.Its paralogue TAZ expression gradually decreased.Experiment ?:YAP1 overexpression and knockdown in ATDC5Objective:To overexpress or knock down YAP1 in ATDC5.Materials and methods:1.pLKO.1-mouse-scrambled,PLX304-human-YAP1-V5,and pLKO.1-mouse-SHYAP1 were each transfected into 293e packaging cells along with pspax2 and pMD2G plasmids.2.Infection efficiency was analyzed by qRT-PCR and Western blot.Results:The YAP1 overexpression and knockdown ATDC5 cells were established.Compared to the control,YAP1 mRNA was overexpressed by over 30-fold and knocked down by over 80%,with concomitant changes in YAP 1 protein levels.Experiment ?:Effect of YAP 1 on cell proliferation,chondrogenic and hypertrophic differentiation of ATDC5Objective:To detect the effect of YAP1 on cell proliferation,chondrogenic and hypertrophic differentiation of ATDC5.Materials and methods:1.The proliferation ability of ATDC5 cells was detected by MTT cell proliferation assay.2.The expression levels of chondrogenic related genes(COL2A1?SOX9)and hypertrophic related genes(COL 10A1?ALP?RUNX2)were measured by qRT-PCR and Western blot.3.Alcian blue staining?ALP staining and ALP activity were conducted.4.qRT-PCR and Alcian blue staining were used to detect the differentiation ability of ATDC5 cells cultured in micromass environment.Results:The MTT assay revealed that YAP1 overexpression promoted cell proliferation,whereas YAP1 knockdown inhibited proliferation.qRT-PCR revealed that YAP1 overexpression significantly decreased the levels of COL2A1 and SOX9 at days 4 and 7.The levels of ALP and RUNX2 also decreased at day 14,Unlike the overexpression group,the YAP 1-knockdown group exhibited an increased expression of COL2A1 at day7 and COL10A1 at days 7 and 14.The important hypertrophic marker ALP was significantly higher in the knockdown group than in the control group at days 7 and 14 of continuous culture.Additionally,the intensities of Alcian blue staining and ALP staining were the highest in the YAP 1-knockdown group,followed by those in the control group.The overexpression group had the lowest intensities.A similar result was obtained from ALP quantification.Further,ATDC5 cells cultured in micromass environment showed corresponding changes.Part ? The mechanism of YAP1 on differentiation of ATDC5Experiment I:The mechanisms by which YAP1 functions in the regulation of ATDC5 cell differentiationObjective:To detect the influence of YAP1 on Wnt/?-catenin signaling pathway.Materials and methods:1.The ?-catenin/TCF/LEF transcriptional activity was detected using a dual-luciferase reporter assay system.2.The expression of the ?-catenin-signaling related proteins(phosphorylated GSK-3??total GSK-3??PDKK1??-catenin)were detected by Western blot in the control and YAP 1-overexpressing groups after stable transfection without induction.Results:YAP1 overexpression increased the luciferase activity of the ?-catenin/TCF/LEF reporter by approximately 1.8-fold.The luciferase activity in the SHYAP1 group was suppressed.Western blot revealed that the Wnt/?-catenin signaling pathway was robustly activated in YAP 1-overexpressing cells.This activation was indicated by a significant increase in phosphorylated GSK-3? and a slight reduction in DKK1.More importantly,the accumulation of ?-catenin was obviously upregulated with respect to that of the control group.Experiment II:The further mechanisms by which YAP1 functions in the regulation of ATDC5 cell differentiationObjective:To further elucidate the functional connection between YAP1 and Wnt/?-catenin signaling in chondrocyte differentiation.Materials and methods:Upon treating YAP1-overexpressing ATDC5 cells with DKK1 for 7 days under induction,or,YAP1-overexpressing ATDC5 were transfected with siRNAs that target ?-catenin,qRT-PCR were used to detect the expression of SOX9?COL2A1?COL10A1.The hypertrophic differentiation ability of three groups were examined by ALP staining.Results:Addition of DKK1 and transfection of siRNAs recaptured the effects of YAP 1 on ATDC5 cell differentiation.Conclusions:Our study demonstrated that YAP1 suppressed ATDC5 cell differentiation,mechanism analysis found YAP1 activated Wnt/?-catenin pathway.The inhibition of Wnt/?-catenin pathway partially reversed the effects of YAP1 on ATDC5 cell differentiation.Moreover,targeted modulation of the YAPI function in chondrocytes may provide a novel strategy for regeneration of bone defects.