MTORC1 Regulates PTHrP To Coordinate Chondrocyte Growth, Proliferation And Differentiation

IntroductionCartilage is an avascular temporary tissue that only exist in human and other vertebrates. There are three kinds of cartilage that are hyaline cartilage, elastic cartilage and fibrous cartilage. Cartilage is a tissue that consist of chondrocytes, fibers and matrix. In fetus and infant, cartilage tissues are widespread in body, but gradually replaced by bone tissue. The remaining cartilage tissues in adult are in joint, trachea, ear and intervertebral disc. The development of cartilage is a key process in embryo development, cartilage gradually develop into all long bone in body. Thus, the development of cartilage is important to a right shape and length of a bone. And its development is vital to the nerves and muscles which attached to it. Since all the long bone in body are developed from the process of endochondral ossification, a well organized endochondral ossifcation process is essential.The endochondral ossification includes several processes. The first and important process is mesenchyme condensation, and then mesenchyma stem cells differentiate into osteoprogenitor cells, the latter ones differentiate into chondrocytes later. Chondrocytes secrete matrix which is particularly rich in type II collagen, and are embedded in cartilage primordial. The bone first occurs in the middle of the cartilage primordial, which is the bone collar. And then blood vessels invade into the cartilage and bring in osteoclasts and other hematopoietic stem cells. Ossification occurs where the terminal hypertrophic chondrocytes exist and this area is called primary ossification center. Then, the chondrocytes in the end of the cartilage primordial also differentiate into terminal hypertrophic chondrocytes, and terminal hypertrophic chondrocytes attract blood vessels through the production of vascular endothelial growth factor and other factors, and attract osteoclasts to break down cartilage matrix and osteoblasts lay down mineralized matrix. So the secondary ossification center emerged. The remaining cartilage between primary and secondary ossification is growth plate. Chondrocytes are located in growth plate with a well organized pattern. There are several different phases of chondrocytes in growth plate which are resting chondrocytes, proliferative chondrocytes, hypertrophic chondrocytes. The length of body is largely determinate by the organized and coordinately proliferation, hypertrophy, ossification of the growth plate. The molecular mechanism that regulate this process involves several pathways, but the most important one is the IHH-PTHrP axis. IHH is secreted by pre-hypertrophic chondrocytes and stimulate neighboring proliferative chondrocytes to prolierate and stimulate the secretion of PTHrP by resting chondrocytes. The PTHrP, a peptide that close to PTH, is secreted by resting chondrocytes, it binds to its receptor in proliferative chondrocytes and pre-hypertrophic chondrocytes to inhibit its hypertrophic differentiation process.Mechanistic target of rapamycin, mTOR, is a key regulate that integrate the extracellular and intracellular signal to coordinate the growth, proliferation, metabolism and apoptosis of cell. mTOR functions as the catalytic subunit in two functionally distinct signaling complexes:mTORcomplex 1 (mTORC1) and complex 2 (mT0RC2). The complexes are distinct by virtue of specific components, such as Raptor (also known as Rptor) for mTORC1 and Rictor for mTORC2, and by their different downstream effectors. mTORCl is best known for phosphorylating p70 S6 kinase (p70S6K, S6K1; also known as RPS6KB1) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1; also known as EIF4EBP1) to regulate protein synthesis. Global deletion of mTOR or Raptor in the mouse leads to early postimplantation lethality. Subsequent tissue-specific knockout studies have identified crucial roles for mTORC1 in several tissues, but its function in skeletal development has not been examined.Rapamycin has been shown to suppress chondrocyte proliferation and endochondral bone growth in vitro, and in chicken embryo and growing young rats. Recent experiments demonstrated that deletion of either mTOR or Raptor in mouse limb bud mesenchyme using Prxl-Cre leads to diminished embryonic skeletal growth due to reduction in chondrocyte cell size and the amount of cartilage matrix, and subsequent severe delay in chondrocyte hypertrophy and bone formationtically. However, how rapamycin treatment affects chondrocytes differentiation is still in debate, and how activation of mTORC1 impacts the series prolifeation, differentiation and ossification still need to be answered.Objects:Our study focus on the following topics:1. The change of mTORC1 activity during the chondrocytes proliferation and differentiation.2. The impacts of rapamycin treatment on the proliferation and differentiation of chondrocytes.3.How does constitutive activation of mTORCl affect the endochondral ossification in mice?4. The underlying mechanism of mTORC1 regulating chondrocytes proliferation and differentiation.Material and methods1.The change of mTORCl activity during the chondrocytes proliferation and differentiation in vivo.The humerus sections of E14.5 mouse and tibia sections of P7, P18 mice were analyzed to study how mTORC1 activity changes during chondrocytes differentiation by visualizing pS6 using immunofluorescence. Western blots of pS6 and differentiation markers were analyzed in the primary cultured chondrocytes. And to answer the question why mTORC1 activity changes during differentiation, the immunofluorescence of TSC1 in section was performed.2. Rapamycin impacts the proliferation and differentiation of chondrocytes.Draw the proliferation curves of rapamycin treated primary cultured chondrocytes. Study the mRNA of several differentiation markers such as Col10al, MMP13, IHH of primary cultured chondrocytes by qPCR. Analyzing the tibia development of PO mice which received 3 days rapamycin treatment with HE staining, immunofluorescence of pS6, in situ hybridization of MMP13.To study how rapamycin affects chondrocytes differentiation, HE staining was quantificated to analyze the length of proliferative zone and hypertrophic zone.3. Analyzing how constitutive activation of mTORC1 in chondrocytes impacts chondrocytes proliferation and hypertrophic differentiation in mice.The chondrocyte specific TSC1 knockout mice (TSC1CKO) was generated by using Cre-Loxp strategy which is crossbreed a Col2al-cre mice line with a TSC1-loxp mice line. The knockout effect was confirmed by western blots of cartilage tissue. The immunofluorescence of pS6 was conducted on the sections of tibias from PO TSC1CKO and TSC1flox/flox mice to evaluate the knockout effect. The lengths of humerus, femur, tibia of 8W TSC1CKO and TSC1flox/flox mice was measured and quantified. Careful analysis of HE staining of P0,2W,4W,8W,12W tibias from TSC1CKO and TSC1flox/flox mice was performed to evaluate the development of TSC1CKO.4. The increased proliferation in chondrocytes of TSC1CKO mice.Analyzing the HE staining of rib cartilage harvested from TSC1CKO and TSC1flox/fiox mice. Brdu immunofluorescence of PO tibias from TSC1CKO and TSClflox/flox mice was performed to study how loss of TSC1 impacts the proliferation of chondrocytes. The expression of Cyclin B1, Cyclin D1 and PCNA in the cartilage tissues of TSC1CKO and TSClflox/flox mice was determined by western blots. The immunohistochemistry of Cyclin B1 in tibias of TSC1CKO and TSC1flox/flox mice was conducted. The proliferation curves of primary cultured chondrocytes harvested from TSC1CKO and TSClflox/flox mice was outlined by using a CCK8 proliferation assay kit.5. The hypertrophic differentiation and the terminal hypertrophic differentiation in TSC1CKO mice are delayed.The in situ hybridization of Col10al in tibias of TSCICKO and TSClflox/flox mice was carefully analyzed. The HE staining of tibias from TSCICKO and TSC1flox/flox mice were studied to determinate the hypertrophic differentiation process. The BrdU chase assay was conducted to visualize the hypertrophic differentiation in vivo in TSC1CKO and TSC1flox/flox mice. The expressions of differentiation markers such as Runx2, Osterix, Col2al and Col10al in tibias from 4W TSCICKO and TSC1flox/flox mice were determined by western blots. The Cyclins and its counterparts such as p21KIP1, p27KIP1 and p57KIP2 in TSCICKO and TSC1flox/flox mice were analyzed by western blots. Also, the terminal differentiation markers like MMP13 and OPN were also studied by western blots. Von Kossa staining was conducted to evaluate the terminal differentiated chondrocytes and how the ossification goes.6. Rapamycin can rescue the phenotypes of TSCICKO mice.Rapamycin treatment started at 3 weeks after born for 4 weeks, the Kaplan-Meier survival curve was drawn to study whether rapamycin can rescue TSCICKO from death. The Alcian blue-Alizarin red staining of skeletons and thorax of TSCICKO and TSC1flox/flox mice was performed to assess the development of cartilage and ossification. HE staining of 4W TSCICKO and TSC1flox/flox mice who received 2 weeks administration of rapamycin was carefully analyzed to study how rapamycin affects the prolifeative zone and hypertrophic zones. The expressions of PCNA, Cyclin D1, and other differentiation markers such as Col2al, CollOal, Runx2, Osterix, MMP13, OPN were studied in the cartilage tissues obtained after rapamycin treatment to study how rapamycin impacts their expressions.7. The underlying mechanism of phenotypes in TSC1CKO mice.The expressions of IHH, PTHrP, Gli1, Patchedl in tibias from TSCICKO and TSC1flox/flox mice were determined by qPCR. Western blots of growth plate tissue from tibias of TSC1CKO and TSC1flox/flox mice were assessed to study how the IHH-PTHrP axis function. The expression of PTHrP in tibias of TSC1CKO and TSC1flox/flox mice was visualized by immunohistochemistry And how rapamycin affects PTHrP expression was evaluated by immunohistochemistry in P14 TSCICKO and TSC1flox/flox mice tibias which received a week rapamycin treatment. The expressions of PTHrP, Gli1 and Patchedl were evaluated by western blots in TSCICKO and TSC1flox/flox mice tibias which received rapamycin treatment. Evaluating the PTHrP expression by transfecting Gli2 plasmid, Gli2 mutants S234A and S234E, and how rapamycin affects the expression of PTHrP under such situation. How rapamcin and IGF-1 stimulation impact the experssions of PTHrP, Gli1 and Patchedl were evaluated by western blots in primary cultured chondrocytes of TSC1CKO and TSC1flox/flox mice. The luciferase assay was conducted by transfecting S6K1 plasmid to see how S6K1 affects the expression of PTHrP and Patchedl in NIH3T3 cell line. The treatments of GDC-0449 and GANT-61 were evaluated to see how these inhibitors impact the proliferation of chondrocytes from TSCICKO and TSC1flox/flox mice expression of PTHrP, Gli1 and Patchedl were evaluated by western blots in primary cultured chondrocytes of TSC1CKO and TSC1flox/flox mice which received GDC-0449 or rapamycin. CHIP assays was conducted to assess the interaction of Gli2 and the promoter of PTHrP in chondrocytes from TSCICKO mice.8. The interaction of Gli2 and mTORCl downstream effector S6K1.The interaction of Gli1, Gli12 and S6K1 in chondrocytes were assessed by immunoprecipitation. The phosphorylation of Gli2 was determined by immunoprecipitation with anti-Gli2 antibody and followed by western blot with anti-phosphorylation-serine antibody. Western blots were performed to evaluate the cyto-nucleus distribution of Gli1 and Gli2 in primary cultured chondrocytes from TSCICKO and TSC1flox/flox mice. And the cyto-nucleus distribution of Gli1 and Gli2 were also determined in ATDC5 transfected with S6K1 plasmid and rapamycin treatment. The similar western blots were conducted in ATDC5 cells treated with IGF-1 and rapamycin. To study the interaction of SuFu and Gli1, Gli2 in TSC1CKO and TSC1flox/flox chondrocytes, immunoprecipitation were conducted. Similar experiment was performed again in ATDC5 cells transfected with Gli2 and its mutants S234A and S234E. The expressions of PTHrP, Gli1, Gli2 in ATDC5 cells transfected with S6K1 were analyzed using western blots. The immunofluorescence of PTHrP and Gli2 in primary cultured chondrocytes from TSCICKO and TSC1flox/flox were performed.Results1. The mTORCl activity was altered during chondrocytes differentiation process.The immunofluorescence of pS6 in E14.5 humerus sections revealed that mTORC1 activity is high in proliferative zone and pre-hypertrophic zone and nearly absent in hypertrophic zone. The same phenomenon was confirmed in the P7 and P18 mice tibias. The ITS inducing medium can induce hypertrophic differentiation of primary cultured chondrocytes and as the differentiation goes on, the TSC1 expression increases whereas the pS6 declines. Both the experiments in vivo and in vitro confirmed that mTORC1 activity alters during differentiation and prolierative/pre-hypertrophic chondrocytes mTORC1 activity is high while its activity is absent in hypertrophic chondrocytes. Also, the qPCR proved that the terminal hypertrophic differentiation of chondrocytes in ITS inducing medium was much higher than the normal medium since much higher MMP13 expression was detected. In the meanwhile, the immunofluorescence of TSC1 in tibia section revealed that TSC1 expression changes during differentiation.2. Inhibition of mTORC1 by rapamycin promotes terminal hypertrophic differentiation of chondrocytes.Addition of lOnM rapamycin into the medium can inhibit the proliferation of chondrocytes. Details revealed by qPCR showed that rapamycin treatment can accelerate the existing of proliferation of chondrocytes, and promote hypertrophic differentiation and terminal hypertrophic differentiation, as the mRNA of IHH, Col10al, MMP13 which indicates differentiation increased. The HE analysis showed that after rapamycin treatment, the proliferative zone shrink. While the in situ hybridization revealed that MMP13 expressed area expanded in tibia sections from mice received rapamycin treatments. Western blots confirmded that MMP13 expression increased after administration of rapamycin.3. Loss of TSC1 in chondrocytes causes chondrodysplasia in mice.The TSC1CKO mice showed that increased pS6 levels in chondrocytes. The immunofluorescence of pS6 revealed that the normal expression pattern of pS6 in TSCICKO was totally changed, since all the chondrocytes despite its phases were with extremely high level of pS6. The body length and the bone length of TSCICKO mice were shorter than TSC1flox/flox mice. HE analysis revealed that the height of hypertrophic zones of TSCICKO mice gradually increase after born.4. constitutive activation of mTORC1 in chondrocytes leads to increased proliferation.HE staining analysis revealed that more chondrocytes in mitosis were found in the rib cartilage of TSCICKO mice. The BrdU immunofluorescence showed that more BrdU positive chondrocytes in PZ, RZ of tibia section of TSC1CKO mice, even some BrdU positive chondrocytes were discovered in HZ in TSCICKO mice. All the results demonstrated that the proliferation increased after loss of TSC1, and some chondrocytes that should be post-mitosis were still in mitosis. Western blots demonstrated that PCNA, Cyclin D1, Cyclin B1 increased in chondrocytes of TSCICKO mice. The immunohistochemistry revealed that Cyclin B1 increased in chondrocytes of TSCICKO mice. Also, the proliferation curves confirmed that the proliferation of primary cultured chondrocytes increased in TSC1CKO mice.5.The activation of mTORCl inhibits the maturation of chondrocytes.In situ hybridization showed that the zone of Col10al expression in tibia section of 4W TSCICKO mice was much wider than the wild type. Western blots revealed that the differentiation genes such as Runx2, Col10al, Osterix, p21KEPl, p27KIP1 and p57KIP2 were decreased in TSCICKO mice, and so as to the terminal differentiation markers such as OPN and MMP13. The BrdU chase assay showed directly that the hypertrophic differentiation rate was slower in TSCICKO mice than the TSC1flox/flox mice. The immunohistochemistry confirmed that there was a lower expression of p57KIP2 in TSC1CKO mice. The Von kossa staining of TSC1CKO mice showed that there was much less terminal hypertrophic chondrocyte and less ossification than TSC1flox/flox mice.6. Rapamycin rescues the phenotypes of TSCICKO mice.The TSCICKO mice avoided death after 4 weeks administration from 3-week-old. And the Alcian blue-Alizarin red staining of skeletons and thorax of TSC1CKO and TSC1flox/flox mice showed that the TSC1CKO mice were shorter and their rib cartilage failed to ossify. But after rapamycin treatment, the ossification of TSC1CKO mice began. HE analysis showed that the HZ in TSC1CKO mice was higher and rapamycin can reduce the height of HZ. Western blots revealed that the expressions of PCNA and Cyclin D1 increased while the expressions of Col10al, Runx2, MMP13, OPN decreased.7. The mTORCl activity is required by the secretion of PTHrPThe expression of IHH in chondroccytes of TSC1CKO and TSC1flox/flox mice revealed by qPCR showed that loss of TSC1 didn’t change the expression of IHHat all. Whereas, the expression of PTHrP, Gli1, Patchedl increased significantly in chondrocytes of TSC1CKO mice and rapamycin can reverse these effects. The immunofluorescence of PTHrP in 10 weeks old TSC1CKO and TSC1flox/flox mice confirmed the previously results showed by western blots, rapamycin can also reverse the effects.20nM rapamycin can inhibits the proliferation of chondrocytes from TSC1CKO mice. Overexpression of Gli2 and its mutants S234E can enhance the expression of PTHrP and resist the effects of rapamycin treatment. The stimulation of IGF-1 can also increase the expressions of PTHrP, Gli1 and Patchedl. Luciferase assays conducted showed that the overexpression of S6K1 or S6K1+Gli2 can increase the expression of PTHrP, and co-transfection of Gli2 siRNA can counteract the effects. Addition of GDC-0449 and GANT-61 into the medium can inhibit the proliferation of chondrocyte from TSC1flox/flox mice. While only GANT-61 can inhibit the proliferation of chondroytes from TSC1CKO mice but not GDC-0449. Western blots confirmed that results that GDC-0449 can not inhibit the proliferation of chondrocytes from TSC1CKO mice while rapamycin can inhibit its proliferation. CHIP confirmed that the binding of Gli2 and the promoter of PTHrP was enhanced in TSC1CKO mice and rapamycin inhibited the effect.8. mTORCl/S6K1 increases the nucleus translocation of Gli2 to stimulate the expression of PTHrPThe immunoprecipitaion revealed that S6K1 binds with Glil/2 and these interactions were enhanced in chondrocytes of TSC1CKO mice, and the rapamycin inhibits the binding. Another immunoprecipitation showed that the phosphorylation of Gli2 in chondrocytes of TSC1CKO mice was enhanced, while rapamycin can also inhibits this effect. Western blots of chondrocytes nucleus and cytoplasma extracts showed that more Gli1 and Gli2 were located in nucleus in TSC1CKO mice, and these translocations can also inhibited by rapamycin. The experiments of IGF-1 and overexpression of S6K1 showed similar results. Another immunoprecipitation experiment showed that the binding of SuFu and Gli1/2 was weaken after knockout of TSC1. And the mutants experiments of overexpression of S234A, S234E also showed that the phosphorylation of Gli2 had a impact on the interaction of SuFu and Gli2. And the overexpression of active Gli2 mutant S234E showed that it can increase the expression of PTHrP and Glil, however another mutant S234A showed reverse effects. The immunofluorescence of PTHrP and Gli2 showed that PTHrP is enhanced in primary cultured chondrocytes of TSC1CKO mice and more Gli2 was translocated into the nucleus in TSC1CKO mice.ResultsOur study focus on the regulatory role of mTORC1 in chondrocytes proliferation and differentiation, and the underlying molecular mechanism in PTHrP secretion. Our research reached several conclusions via the experiments of TSC1 knockout mice.The mTORC1 changes during the normal proliferation and hypertrophic differentiation of chondroytes:the resting chondrocytes have a little mTORC1 activity, while extremely high level of mTORC1 activity in proliferative and pre-hypertrophic chondrocytes, and nearly absent of mTORC1 activity in hypertrophic chondrocytes.Rapamycin inhibits the hypertrophic differentiation of chondrocytes especially the terminal hypertrophic differentiation.Loss of TSC1 leads to dwarfism of mice. The main reason of this phenotype was the proliferation of chondrocytes increased, the hypertrophic differentiation and terminal hypertrophic differentiation of chondrocytes were delayed. And rapamycin can reverse the phenomenon.The constitutive activation of mTORCl in chondrocytes regulates PTHrP to impact the normal differentiation of chondrocytes. Activation of mTORCl enhance the expression of PTHrP, which leads to delayed hypertrophic differentiation and ossification.Loss of TSC1 leaded to more active S6K1 binded to Gli2 and promotes its translocation to increase PTHrP. S6K1 phosphorylates Gli2 at Ser234 to inhibit the binding of SuFu with Gli2, promotes the nucleus translocation of Gli2. Thus, making PTHrP expression increased.Our research not only proved that mTORCl activity altered during proliferation and differentiation of chondrocytes. But it proved that a precisely controlled mTORC1 pathway is vital to the normal development of endochondral ossification. And we also elucidated that how mTORCl impacts the secretion of PTHrP, providing a new aspect of understanding the regulation of chondrocytes development.