The Role Of Autophagy In The Regulation Of Achondroplasia And Chondrogenesis

Endochondral bone formation and intramembranous bone formation are two major forms of bone formation inmammalian. Endochondral ossification is the process that chondrocytes drive from mesenchymal condensations in the growing limb buds, as the elements grow, chondrocytes undergo proliferation, hypertrophy, death, and are replaced by bone, eventually. It is the main forms of bone formation of trunk, limbs, short bones and several irregular bones, which is important for the embryonic development, bone growth and repair of fracture. So far, many signaling pathways including FGF, PTH, BMP and Wnt have been proved to regulate endochondral bone formation.Fibroblast growth factor receptor 3(FGFR3) is a member of the receptor tyrosine kinase(RTK) family and identified to negatively regulate endochondral ossification. Gain-of-function mutations in FGFR3 result in a series of human genetic skeletal disorders, including achondroplasia(ACH), hypochondroplasia(HCH), and thanatophoric dysplasia(TD). Among these skeletal disorders, ACH is the most common type of genetic dwarfism in humans, characterized by short limbs, the large head with frontal bossing and hypoplastic midface. ACH patients showed obvious abnormalities in the growth plate, including disorderly proliferating chondrocyte columns, short proliferating and hypertrophic chondrocyte zone, and premature of growth plates. By using the method of knock in and knock out, Fgfr3 null mice showed the overgrowth of long bones accompanied by the broad proliferative zone and hypertrophic zone, while the gain-of-function Fgfr3 in mice caused dwarf with inhibition of chondrocyte proliferation and hypertrophy. These studies revealed that FGFR3 is a negative regulator of endochondral ossification. Concerning the mechanism of ACH, in addition to ERK/MAPK and STAT, some other signaling pathways, including BMP, IGF, PTHr P and IHH, have been revealed to be involved in the pathogenesis of achondroplasia. However, the detailed mechanism of achondroplasia remains unclearly.Macroautophagy(hereafter referred to as autophagy), an evolutionarily conserved catabolic process in mammalian, is important for the metabolism, development, disease immunity and tumor development. Generally, autophagy maintains the homeostasis of cell under a basal level activity, and protects cell from death induced by a lack of growth factors, oxygen or nutrient, as well as the stress caused by accumulation of damaged organelle and metabolic waste. In growth plate, autophagy protects chondrocytes from apoptosis under stresses such as nutritional deficiencies and hypoxia. Autophagy is indispensable for the maintaining of chondrocyte viability and normal development. Impaired autophagy is involved in the pathogenesis of chondrodysplasia resulted from the loss of Sumf1 or CTGF/CCN2, in which, chondrocyte viability is severely damaged because of the lack of protection from autophagy. All these facts suggest that autophagy is important for the cartilage development, especially the long bone growth.Achondroplasia, the most common type of genetic dwarfism in humans, is characterized by the retarded long bone growth with largely inhibited cell viability of chondrocytes, including suppressive proliferation, slow differentiation and activated apoptosis. The similarity of phenotype between mice with gain-of-function Fgfr3 and loss of CTGF/CCN2 or Sumf1 suggests that inhibited autophagy may participate in the regulation of achondroplasia as well.In our study, we found that activated-FGFR3 decreased autophagic activity of chondrocytes, which was supported by the decreased conversion of endogenous LC3 and LC3 punctation, both in vivo and in vitro. Moreover, we found that autophagy inhibitors(3-MA and chloroquine) significantly inhibited the growth of cultured embryonic cartilage rudiments. The autophagy inhibitor chloroquine decreased the length of proliferative zone and hypertrophy zone in growth plate of cultured embryonic cartilage rudiments, which is similar to the negative effect of FGF/FGFR3 signaling on chondrogenesis. Furthermore, chloroquine treatment also decreased viability of RCS cells, inhibited differentiation of ATDC5 cells, and recued the overgrowth of the cultured embryonic cartilage rudiments from Fgfr3 deficient mice. These data demonstrate that FGF/FGFR3 signaling suppresses autophagic activity, which may be subsequently involved in the pathogenesis of achondroplasia.In mammalian, autophagy is a multi-step catabolic process. Several evolutionarily conserved ATG proteins were involved in the formation of autophagosome, among which, ATG12-ATG5 conjugate is the core machinery. The ATG12-ATG5 conjugate binds with ATG16 to form the ubiquitin-like protein system, which is essential for the elongation of phagophore and autophagosome formation. Studies demonstrated that the expression of ATG12-ATG5 conjugate is important for the normal autophagy. Genetic and in vitro studies have found that regulation of the protein level of ATG5(knock out, RNAi or overexpression) effected the activity of autophagy. In our study, we found that FGFR3 signaling pathway negatively regulated the protein level of ATG12-ATG5 conjugate by using primary chondrocytes of ACH, R3 KO and R3CKO: CMV-CreERTmice, meanwhile, overexpression of FGFR3 or treatment of FGF-2 also decreased the protein level of this conjugate. These data suggest that FGFR3 down-regulated the expression of ATG12-ATG5 conjugate, which may be involved in the FGFR3-mediated inhibition of autophagy. What’s more, we found that FGFR3 band ATG12-ATG5 conjugate through its kinase domain and the interaction was strengthened by the treatment of FGF-2. Moreover, the expression patterns of FGFR3, ATG12 and ATG5 in tibia growth plate were overlapped, which further provided the opportunity for their interaction in vivo.Our study showed that FGF/FGFR3 signaling pathway suppressed the activity of autophagy and the protein level of ATG12-ATG5 conjugate, which indicated that ATG12-ATG5-mediated autophagy process may be involved in the pathogenesis of achondroplasia. By employing the lentiviral shRNA against Atg5 to inhibit the activity of ATG12-ATG5-related autophagy, we found that the chondrocyte viability and differentiation were largely inhibited, which resembles the inhibitory function of activated-FGFR3 signaling on chondrogenesis. Furthermore, up-regulation of the expression of ATG12-ATG5 conjugate by increased-expression of ATG5 can partially rescue the inhibition on the viability and differentiation of chondrocytes mediated by activated FGFR3 signaling. These data not only reveal that ATG12-ATG5-mediated autophagy plays animportant role in the chondrogenesis, but also suggest that the suppressed ATG12-ATG5-mediated autophagy may play an important role in the achondroplasia.To further study the role of autophagy in cartilage development, we employed the Atg7 conditional knock-out mice(ATG7flox/flox). Even though ATG7 is different from ATG5, both of them are the core machinery of autophagy, and knock out or knock down one of the two molecules will result in significant block of autophagy process. We got the Atg7 chondrocyte-specific knock-out mice by breeding ATG7flox/flox mice with Col2a-cre transgenetic mice. We found that the RNA and protein level of ATG7 were significantly decreased in the null mice, and the conversion of endogenous LC3 was almost completely blocked with the loss of ATG7. Interestingly, the null mouse showed disproportionate shortened limbs, which is similar to the dwarf phenotype of ACH. Furthermore, we found that the differentiation and proliferation of chondrocytes from null mice were inhibited in growth plate, which may be involved in the pathogenesis of abnormal cartilage develpment caused by impaired autophagy. These results suggest that autophagy plays an important role in the cartilage development in vivo, which further supported our previous founding.In this study, we report that autophagy is essential for the cartilage development, especially the proliferation and differentiation of chondrocytes. Furthermore, suppressed ATG12-ATG5-mediated autophagy may be involved in the pathogenesis of FGFR3-related chondrodysplasia. Our study provides novel insights for understanding of the mechanisms of FGFR3-related chondrodysplasia, which may provide a new strategy for the treatment of achondroplasia.METHODS:Part I FGF/FGFR3 signaling suppressed the activity of autophagy1. FGF/FGFR3 signaling suppressed the activity of autophagy in vivo1) The expressions of LC3 in the growth plate of postnatal day 5 ACH mouse and WTlittermate were analyzed by immunohistochemistry.2) The conversions of endogenous LC3 in the primary chondrocytes from ACHmouse and WT littermate were analyzed by Western blotting.3) The conversions of endogenous LC3 in the primary chondrocytes from R3 KOmouse and WT littermate were analyzed by Western blotting.4) The conversion of endogenous LC3 in the primary chondrocytes from R3CKO:CMV-CreERTmouse with the treatment of TM was analyzed by Western blotting, and the TM- untreated group was used as the control.5) The LC3-immunopositive punctate signals of primary chondrocytes from ACHmice and its littermates were analyzed by immunofluorescence. The chondrocytes were cultured with serum-starvation(SS) for 4 h in the presence or absence of E64 d and pepstatin A(PEPS A).6) The LC3-immunopositive punctate signals of primary chondrocytes from R3CKO:CMV-CreERT2 mice were analyzed by immunofluorescence. The chondrocytes were treated with TM for 48 h, and went cultured with serum-starvation for 4 h in the presence or absence of E64 d and pepstatin A.2. FGF/FGFR3 signaling inhibited the autophagic activity in vitro1) The conversions of endogenous LC3 with the overexpression of FGFR3 in RCScells were analyzed by Western blotting, accompanied with the treatment of serum- starvation for 4 h, in the presence or absence of E64 d and pepstatin A.2) ATDC5 cells were serum starvated for 12 h, and followed the treatment of FGF-2for 30 min, in the presence or absence of E64 d and pepstatin A.The conversions of endogenous were analyzed by Western blotting.3) The GFP-LC3 punctate with overexpression FGFR3 in HeLa cells stablyexpressing GFP-LC3 were analyzed by immunofluorescent assay, accompanied with the treatment of serum-starvation for 4 h in the presence or absence of E64 d and pepstatin A.4) The GFP-LC3 punctate with the treatment of FGF-2 under serum-starvation inHeLa cells stably expressing GFP-LC3 were analyzed by immunofluorescent assay.3. Inhibitors of autophagy suppressed the chondrogenesis in vitro1) The effects of autophagic inhibitors in the cartilage development were detected byusing embryonic bone culture system.2) The roles of chloroquine and FGF-2 in the cartilage development were tested byusing embryonic bone culture system. And histological sections of embryonic bone were stained by alcian blue.3) The treatment of chloroquine on the embryonic bone which isolated from R3 KOmice.4) The effects of treatment of chloroquine or FGF-2 on the viability of RCS cells wereanalyzed by Cell counting and MTT assay.5) The effects of treatment of chloroquine or FGF-2 on the differentiation of ATDC5 chondrocytes were analyzed by Alcian blue staining and RT-PCR.Part II FGF/FGFR3 signaling negatively regulated the expression of ATG12-ATG5 conjugate at a posttranscriptional level; FGFR3 interacted with ATG12-ATG5 conjugate by its kinase domain1. FGF/FGFR3 signaling suppressed the expression of ATG12-ATG5 conjugate at aposttranscriptional level1) The RNA levels of Atg5 and Atg12 in chondrocytes from ACH, R3 KO and WTlittermates were tested by RT-PCR.2) The protein levels of ATG12-ATG5 conjugate in the primary chondrocytes fromACH mouse and WT littermates were analyzed by Western blotting.3) The protein levels of ATG12-ATG5 conjugate in the primary chondrocytes fromR3 KO mouse and WT littermates were analyzed by Western blotting.4) The protein level of ATG12-ATG5 conjugate in the primary chondrocytes fromR3CKO: CMV-CreERTmouse with treatment of TM was analyzed by Western blotting, and the TM-untreated group was used as the control.5) The protein levels of ATG12-ATG5 conjugate with the increased amount of FGFR3 in RCS cells were analyzed by Western blotting.6) The protein levels of ATG12-ATG5 conjugate with the treatment of FGF-2 inATDC5 cells were analyzed by Western blotting.7) The protein level of ATG12-ATG5 conjugate with the over-expression of differentforms of FGFR3(WT, Y373 C, K650M) in RCS cells were analyzed by Western blotting.2. FGFR3 interacted with ATG5 by kinase domain in vitro1) Co-immunoprecipitation(Co-IP) was done to analyze the interaction betweenexogenous FGFR3 and ATG5 in 293 T cells(FGFR3 co-immunoprecipitated ATG5 or ATG5 co-immunoprecipitated FGFR3).2) GST pull-down was done to test the interaction between GST-FGFR3-ICD andATG5 in 293 T cells.3) YFP-PCA was done to analyze the interaction between ATG5 and FGFR3 in 293 cells.4) Mapping of the domain of FGFR3 to interact with ATG5.5) Co-immunoprecipitation(Co-IP) was done to analyze the interaction betweenexogenous FGFR3 and ATG5 with the treatment of FGF-2 in 293 T cells.3. FGFR3 interacted with ATG12-ATG5 in vivo1) Co-immunoprecipitation(Co-IP) was performed in HeLa cells with exogenousFGFR3 and endogenous ATG12-ATG5.2) Co-immunoprecipitation(Co-IP) was performed in HeLa cells with endogenousFGFR3 and ATG12-ATG5.3) Co-immunoprecipitation(Co-IP) was performed in primary chondrocytes fromC57Bl/6J mice(FGFR3 co-immunoprecipitated ATG12-ATG5 or ATG5 co-immunoprecipitated FGFR3).4) Immunohistochemistry was performed to analyze the expression of FGFR3 andATG5 in the growth plates of C57Bl/6J mice.5) Immunohistochemistry was performed to analyze the expression of p-ERK andATG5 in the growth plates of postnatal day 5 ACH mouse and WT littermate mouse.6) Immunohistochemistry was performed to analyze the expression of p-ERK andATG5 in the growth plates of postnatal day 5 R3 KO mouse and WT littermate mouse.4. ATG12-ATG5-mediated autophagy maintained the viability and normaldifferentiation of chondrocytes1) The ATDC5 cell line with stably expressed lentiviral shRNA targeting Atg5 wasconstructed.2) The RNA level of Atg5 was tested by RT-PCR.3) The protein levels of ATG5, ATG12-ATG5 conjugate and LC3 were tested byWestern blotting.4) Cell counting and MTT assay were performed to analyze the effect of expression ofRNAi-Atg5 on the viability of ATDC5 cells.5) Alcian blue staining and RT-PCR were performed to analyze the effect ofexpression of RNAi-Atg5 on the differentiation of ATDC5 chondrocytes.6) The RNA levels of Atg5 and Atg12 in the extension of differentiation in ATDC5 cells were tested by RT-PCR.7) Western blotting was done to analyze the protein level of ATG12-ATG5 conjugateand LC3 with overexpression of ATG5, both in RCS cells and ATDC5 cells.8) Cell counting and MTT assay were performed to analyze the effect ofoverexpression of ATG5 on the inhibited chondrocyte proliferation caused by activated-FGFR3 signaling in RCS cells.9) RT-PCR was performed to analyze the effect of overexpression of ATG5 on theinhibited chondrocyte differentiation caused by activated-FGFR3 or FGF-2 treatment in ATDC5 cells.Part III The phenotype of mice lacking Atg7 in chondrocytes was analyzed, as well as the possible mechanism1. ATG7flox/flox: Col2a-cre mice were gained by breeding ATG7 flox/flox mice withCol2a-cre transgenic mice2. The knockout efficiencies of mutant mice were analyzed at RNA and protein level1) RT-PCR was performed to analyze the expression of Atg7 in the primarychondrocytes from mutant mice and its WT littermates.2) Western blotting was performed to analyze the protein level of ATG7 andautophagic activity in the primary chondrocytes from mutant mice and its WT littermates.3. Analyses on the phenotype of mice lacking Atg7 in chondrocytes1) X assay was done to analyze the general phenotype of mice.2) The lengths of tibias, femurs were measured by X assay.3) Histological sections of mice tibia were analyzed by HE-staining.4. Explorations of the mechanism of shortened limbs resulting from lacking Atg7 inchondrocytes1) The effect of ATG7-mediated autophagy on the differentiation of primarychondrocytes was analyzed by Alcian blue staining.2) The expression of PCNA(markers of proliferation) in chondrocytes was analyzedby Immunohistochemistry.3) Co-immunoprecipitation(Co-IP) was performed to analyze the interaction betweenexogenous FGFR3 and ATG7 in 293 T cells.4) Co-immunoprecipitation(Co-IP) was performed to analyze the interaction betweenendogenous FGFR3 and ATG7 in RCS cells.5) Co-immunoprecipitation(Co-IP) was performed in primary chondrocytes formC57Bl/6J mice(FGFR3 co-immunoprecipitated ATG7).RESULTS:Part I FGF/FGFR3 signaling negatively regulated the autophagic activity.1. FGF/FGFR3 signaling negatively regulated the autophagic activity of primarychondrocytes in vivo.1) The immunoreactive LC3 particles in the tibia growth plate from ACH mouse weresignificantly reduced comparing with that of WT control.2) The conversion of endogenous LC3 in the primary chondrocytes from ACH micewas decreased compared with that of WT littermate.3) The conversion of endogenous LC3 in the primary chondrocytes from R3 KO micewas increased compared with the WT littermate mouse.4) The conversion of endogenous LC3 in the primary chondrocytes from R3CKO:CMV-CreERTmouse with the treatment of TM was increased compared with that of TM- untreated group.5) The LC3-immunopositive punctate signals of primary chondrocytes from ACHmice were decreased compared with that of the WT littermates.6) The LC3-immunopositive punctate signals of primary chondrocytes from R3CKO:CreERT mouse with the treatment of TM were increased compared with that of TM- untreated group.2. FGF/FGFR3 signaling regulated the autophagic activity of chondrocytes negativelyin vitro1) Overexpression of FGFR3 decreased the conversion of endogenous LC3 in RCScells.2) The treatment of FGF-2 decreased the conversion of endogenous LC3 in ATDC5 cells.3) Overexpression of FGFR3 decreased the GFP-LC3 punctation in HeLa cells stablyexpressing GFP-LC3.4) The treatment of FGF-2 decreased the GFP-LC3 punctation in HeLa cells stablyexpressing GFP-LC3.3. Inhibitors of autophagy suppress chondrogenesis1) The autophagic inhibitors(3MA or chloroquine) inhibited the cartilagedevelopment by using embryonic bone culture system.2) The treatment of chloroquine or FGF-2 suppressed the proliferation anddifferentiation by histological analysis on the embryonic cultured bone.3) The treatment of chloroquine recued the over growth of embryonic bones fromR3 KO mice.4) The treatment of chloroquine inhibited the viability of RCS Cells by counting andMTT assay.5) The treatment of chloroquine inhibited differentiation of ATDC5 chondrocytes byusing alcian blue staining and RT-PCR.Part II FGF/FGFR3 signaling suppressed the expression of ATG12-ATG5 conjugate in vivo and in vitro1) The RNA levels of Atg5 and Atg12 didn’t be changed in chondrocytes from ACHor R3 KO mice, compared with that of WT littermates.2) The protein level of ATG12-ATG5 conjugate was decreased in the primarychondrocytes from ACH compared with that of WT littermate.3) The protein level of ATG12-ATG5 conjugate was increased in the primarychondrocytes from R3 KO compared with that of WT littermate.4) The protein level of ATG12-ATG5 conjugate in the primary chondrocytes fromR3CKO: CMV-CreERT with the treatment of TM was increased compared with that of the TM- untreated group.5) The decreased protein level of ATG12-ATG5 conjugate was enhanced by theincreased amount of FGFR3 in RCS cells.6) The treatment of FGF-2 decreased the protein level of ATG12-ATG5 conjugate inATDC5 cells.7) Gain-of-function forms of FGFR3(Y373C, K650M) promoted theFGFR3-mediated decrease of expression of ATG12-ATG5 conjugate in RCS cells.5. FGFR3 interacted with ATG5 in vitro1) The exogenous FGFR3 interacted with ATG5 in 293 T cells.2) GST-FGFR3-ICD interacted with ATG5 in 293 T cells.3) YFP1-ATG5 interacted with YFP2-FGFR3 in 293 cells.4) FGFR3 interacted with ATG5 by kinase domain.5) The treatment of FGF-2 enhanced the interaction between exogenous FGFR3 andATG5 in 293 T cells.6. FGFR3 interacted with ATG12-ATG5 in vivo1) Exogenous FGFR3 interacted with endogenous ATG12-ATG5 in He La cells.2) Endogenous FGFR3 interacted with endogenous ATG12-ATG5 in He La cells.3) Endogenous FGFR3 interacted with endogenous ATG12-ATG5 in primarychondrocytes from C57Bl/6J mice(FGFR3 co-immunoprecipitated ATG5 or ATG5 co-immunoprecipitated FGFR3).4) The expression patterns of FGFR3 and ATG5 were overlapped in the growth platechondrocyte of C57Bl/6J mice.5) The expression of ATG5 was decreased in the growth plates of postnatal day 5ACH mice compared with that of littermate mice.6) The expression of ATG5 was increased in the growth plates of postnatal day 5R3 KO mice compared with that of littermate mice.7. ATG12-ATG5 conjugate is essential for the chondrogenesis and overexpression ofATG5 partially alleviates FGFR3-mediated suppression of viability and differentiation of chondrocyte.1) The ATDC5 cell line with stably expressed lentiviral shRNA targeting Atg5 wasconstructed.2) The RNA level of Atg5 was significantly decreased in cells expressed withRNAi-Atg5 compared with that of the RNAi-control.3) The protein levels of ATG5, ATG12-ATG5 conjugate and the activity of autophagywere significantly decreased in cells expressed with RNAi-Atg5 compared with that of the RNAi-control.4) The viability of ATDC5 cells expressed with RNAi-Atg5 was significantlydecreased by using cell counting and MTT assay.5) The differentiation of ATDC5 chondrocytes with RNAi-Atg5 was inhibitedcompared with that of the control.6) The RNA levels of Atg5 and Atg12 were increased along with the extendeddifferentiation of ATDC5 cells.7) Overexpression of ATG5 can increase the protein level of ATG12-ATG5 conjugateand autophagic activity, in both RCS cells and ATDC5 cells.8) Overexpression of ATG5 partially recued the inhibited chondrocyte proliferationcaused by activated-FGFR3 in RCS cells.9) Overexpression of ATG5 partially recued the inhibited chondrocyte differentiationcaused by activated-FGFR3 or FGF-2 treatment in ATDC5 cells.Part III Ablation of Atg7 in chondrocytes impaired skeleton growth.1. ATG7flox/flox: Col2a-cre mice were got by breeding ATG7 flox/flox with Col2a-cretransgenic mice.2. ATG7 is significantly decreased in the ATG7flox/flox: Col2a-cre mice.1) The RNA of Atg7 was decreased in the primary chondrocytes from mutant micecompared with that of WT littermates.2) The protein level of ATG7 and autophagic activity were significantly decreased inthe primary chondrocytes from mutant mice compared with that of WT littermates.3. Deletion of Atg7 in chondrocytes impaired skeleton growth.1) The general phenotype of mutant mice was not significant abnormal, comparedwith that of WT littermates.2) The lengths of tibias and femurs of mutant mice were shortened compared withthat of WT littermates.3) Histological sections of tibia from mutant mice revealed the narrowed grow plate,especially the narrowed proliferative zone, compared with WT littermates.4. The proliferation and differentiation were inhibited in the mice lacking Atg7 inchondrocytes.1) Immunohistochemical positive particles of PCNA in growth plate chondrocytesfrom mutant mouse were decreased compared with that of WT littermate.2) Deletion of Atg7 in chondrocytes resulted in the light-colored Alcian blue staining.3) Exogenous FGFR3 interacted with ATG7 in 293 T cells.4) Endogenous FGFR3 interacted with ATG7 in RCS cells.5) Endogenous FGFR3 interacted with ATG7 in primary chondrocytes from C57Bl/6Jmice(FGFR3 co-immunoprecipitated ATG7).CONCLUSIONS:1. FGF/FGFR3 signaling negatively regulates the autophagic activity.2. Inhibitors of autophagy suppress the cartilage development by inhibiting theproliferation and differentiation of chondrocytes.3. FGF/FGFR3 signaling negatively regulates the expression of ATG12-ATG5 conjugate at a posttranscriptional level.4. FGFR3 interacts with ATG12-ATG5 conjugate by binding ATG5 with the kinasedomain.5. The ATG12-ATG5-mediated autophagy is essential for the chondrogenesis,especially for the proliferation and differentiation of chondrocytes.6. Overexpression of ATG5 partially alleviates FGFR3-mediated inhibition ofviability and differentiation of chondrocytes.7. Ablation of Atg7 in chondrocytes results in the significantly decreased autophagicactivity; Length of limbs from mutant mice are shortened compared with that of WT littermates, which resembles the negative regulation of activated-FGFR3 signaling on cartilage development.