The Roles And Mechanisms Of FGFR2 In Skeletal Development And Bone Formation

Apert syndrome is a severe craniosynostosis caused by gain-of-function mutations in FGFR2.Recently it has been reported that stem cell population decrease can lead to craniosynostosis.We found that Apert mice are characterized with premature aging phenotype with diminished suture stem cells,which indicates that Apert syndrome may result from suture stem cells defects.Autophagy is crucial for maintaining the self-renewal of stem cells,and autophagy dysfunction in stem cells causes senescence.Our studies further showed defect autophagy in Apert mice.These results suggested that activation of FGFR2 may accelerate stem cell senescence by decreasing autophagic activity,which results in craniosynostosis.In this study,we will generate mouse models with Apert mutation alone or Apert p16 deficiency.By analyzing the skull phenotypes of these mice,and using cell culture experiments to study the mechanism of the regulation of FGFR2 on autophagy and stem cell senescence.Finally,we will test autophagy modulation and anti-aging drugs for their effect on the suture stem cells in Apert mice.This study will deepen our understanding of the mechanism of Apert syndrome and skull development in aspect of autophagy and stem cell senescence,and provide experimental basis for development of clinical treatment for Apert.Previous studies have shown that gain-of-function mutations of FGFR2(S252W or P253R)cause skull malformation of human Apert syndrome by affecting both chondrogenesis and osteogenesis,underscoring the key role of FGFR2 in bone development,but the effects of FGFR2 on bone formation in adult have not been fully investigated.A better understanding of the role of FGFR2 in bone formation will help us to develop more effective strategies for managing patients with bone diseases resulting from dysregulated bone homeostasis such as osteoporosis.We generated mice with tamoxifen-inducible expression of the gain-of-function mutant of FGFR2(P253R)at adult stage.The mechanical bone marrow ablation(BMX)was performed in both wild-type(WT)and Fgfr2 mutant(MT)mice.Changes in newly formed trabecular bone were assessed by micro-computed tomography(μCT)and bone histomorphometry.We found that mice with gain-of-function mutation of FGFR2 exhibited increased trabecular bone formation and decreased bone resorption after BMX accompanied with a remarkable increase in bone marrow stromal cell recruitment and proliferation,osteoblast proliferation and differentiation,and enhanced Wnt/β-catenin activity.Furthermore,pharmacologically antagonizing Wnt/β-catenin signaling can partially alleviate the increased trabecular bone formation and decreased bone resorption in Fgfr2 mutant mice after BMX.Our data demonstrated that gain-of-function mutation in FGFR2 exerts a Wnt-dependent anabolic effect on trabecular bone by increasing bone formation and decreasing bone resorption.The attenuated degradation of articular cartilage by cartilage-specific deletion of fibroblast growth factor receptor 1(FGFR1)in adult mice suggests that FGFR1 is a potential target for treating osteoarthritis(OA).The goal of the current study was to investigate the e ect of a novel non-ATP competitive FGFR1 inhibitor,G141,on the catabolic events in human articular chondrocytes and cartilage explants and on the progression of cartilage degradation in a murine model of OA.G141 was screened and identified via cell-free kinase-inhibition assay.In the in vitro study,G141 decreased the mRNA levels of catabolic markers Adamts-5 and Mmp-13,the phosphorylation of ERK1/2,JNK and p38 MAPK,and the protein level of MMP-13 in human articular chondrocytes.In the ex vivo study,proteoglycan loss was markedly reduced in G141 treated human cartilage explants.For the in vivo study,intra-articular injection of G141 attenuated the surgical destabilization of the medial meniscus(DMM)induced cartilage destruction and chondrocyte hypertrophy and apoptosis in mice.Our data suggest that pharmacologically antagonize FGFR1 using G141 protects articular cartilage from osteoarthritic changes,and intra-articular injection of G141 is potentially an effective therapy to alleviate OA progression.Methods:Part I Study on the molecular mechanism of the accelerated suture stem cell senescence resulting from FGFR2 mediated decrease of autophagic activity in Apert syndrome1.CMV-Cre transgenic mice were crossed with Fgfr2+/P253R-neo mice to generate Apert mice,the body length and the body weight were measured respectively,X-ray and Micro-CT were performed to observe morphologic changes of skeleton of Apert mice,western blot was performed to detect the expression of p53、p21、p16 和 γH2AX;2.Senescence-associated β-gal(SA-β-gal)staining was performed to detect senescence cells in suture mesenchyme,EdU long-term labeling was performed to detect label retaining cells,TUNEL staining assay was performed to detect apoptosis cells in suture mesenchyme,suture mesenchymal stem cells(MSCs)were isolated to detect clone formation ability and proliferation ability;3.p16-/-mice were crossed with Fgfr2+/P253R-neo mice and CMV-Cre transgenic mice to generate p16-/-;Apert double mutant mice,X-ray and alcian blue/alizarin red skeletal staining were performed to detect morphologic changes of the skull and the suture;4.GFP-LC3 report mice were crossed with Fgfr2+/P253R-neo mice and CMV-Cre transgenic mice to generate GFP-LC3;Apert double mutant mice,frozen section was performed to detect LC3 puncta in suture mesenchymal stem cells;5.Suture MSCs were treated with rapamycin,SA-β-gal staining was performed to detect senescence cells,western blot was performed to detect p53,p21 and γH2AX protein levels,immunofluorescence was performed to detect nuclear accumulation of 53BP1 and γH2AX;6.LysoTracker,MitoTracker and CellROX staining were performed to detect mitochondria,lysosome and ROS in suture MSCs;7.Suture MSCs were treated with NAC,SA-β-gal staining was performed to detect senescence cells,western blot was performed to detect p53,p21 and γH2AX protein levels,immunofluorescence was performed to detect nuclear accumulation of 53BP1 and γH2AX;Part II Study on the role of FGFR2 in bone formation after BMX during adultstage1.CMV-CreERT2 transgenic mice were crossed with Fgfr2+/P253R-neo mice to generate CMV-CreERT2;Fgfr2+/P253R-neo(hereafter referred to as MT)mice,at 2 months of age,the mice were intraperitoneally injected with tamoxifen(1mg/10 g body weight,daily for 5 days),and then BMX surgery was performed;2.Micro-CT and von Kossa staining were performed to analyze newly formed trabecular bone at 1,2 and 3 weeks after BMX;3.H&E staining and histomorphometric measurement were performed to analyze the number of osteoblasts(N.Ob/B.Pm),quantitative PCR were performed to examine the mRNA levels of osteogenic markers including Runx2,alkaline phosphatase(Alp),collagen type I(Col I),osteopontin(OP)and osteocalcin(OC),in tibia diaphyseal regions at 1,2 and 3 weeks after BMX;4.TRAP staining and histomorphometric measurement were performed to analyze the osteoclast surface(Oc.S/BS),quantitative PCR were performed to examine the mRNA levels of Rankl,Opg and M-Csf,in tibia diaphyseal regions at 1,2 and 3 weeks after BMX;5.EdU incorporation assay was performed to analyze stromal cells and osteoblasts proliferation at 4 and 7 days after BMX,immunohistochemistry staining was performed to detect Runx2 and OC expression levels at 1 week after BMX;6.Immunohistochemistry staining and western blot were performed to detect β-catenin accumulation at 1 week after BMX,quantitative PCR were performed to examine the mRNA levels of a panel of known Wnt/β-catenin target genes including Tcf1,Lef1,CyclinD1 and CD44;7.Newly formed trabecular bone in diaphyseal region of tibia from vehicle-treated and XAV939-treated mice were analyzed at 1 week after BMX by Micro-CT,IHC and histopathological staining.Part III Intra-articular injection of G141 protects against cartilage degradation in a murine model of osteoarthritis1.Kinase inhibition assay was performed to test the specificity of G141 on FGFR1,caliper mobility shift assay to study the competitive relationship between ATP and G141.2.Primary human articular chondrocytes in monolayer were pre-incubated with G141 followed by stimulation with FGF-2,qPCR was performed to examine the mRNA levels of Mmp13,Adamts5,Type II collagen and Aggrecan,western blot was performed to examine the protein levels of ERK1/2,ADAMTS-5 and MMP-13;3.Femoral head cartilage samples were cultured in the absence or presence of FGF-2 and G141 for 14 days,Safranin-O-fast green staining and DMMB assay were performed to examine the proteoglycan depletion;4.DMM surgery in the right knee joints of 10-week-old male C57 mice were performed and then the mice received twice weekly intra-articular injection of 10 μM G141 in PBS or PBS alone for 4 and 8 weeks.Safranin O–fast green staining and OARSI histologic scoring system was applied to detect cartilage degradation.5.Immunohistochemical staining was performed to examine the expressions of Type X collagen,MMP-13 and Cleaved caspase-3 in articular chondrocytes,TUNEL staining assay was performed to detect chondrocyte apoptosis.Results:Part I FGFR2 accelerates suture MSCs senescence by decreasing autophagic activity,which results in craniosynostosis in Apert mice1.Apert mice were characterized with premature aging phenotype;2.FGFR2 P253 R mutation causes early onset of senescence and DNA damage in suture MSCs;3.p16 knockout partially alleviates the premature aging phenotype of Apert mice;4.FGFR2 P253 R mutation negatively regulates autophagic activity;5.Autophagy loss in Apert mice resulted in mitochondrial dysfunction and accumulation of ROS in suture MSCs;6.ROS inhibition prevented senescence in Apert suture MSCs.Part II Inducible activation of FGFR2 in adult mice promotes bone formation after bone marrow ablation1.Micro-CT and von Kossa staining results showed that gain-of-function mutation of FGFR2 resulted in increased new bone formation at 1,2 and 3 weeks after BMX;2.H&E staining and histomorphometric measurement results showed that gain-of-function mutation of FGFR2 resulted in increased osteoblasts number(N.Ob/B.Pm)at 1,2,and 3 weeks after BMX,quantitative PCR results showed that gain-of-function mutation of FGFR2 resulted in increased Runx2,alkaline phosphatase(Alp),collagen type I(Col I),osteopontin(OP)and osteocalcin(OC)mRNA levels at 1,2 and 3 weeks after BMX;3.TRAP staining and histomorphometric measurement results showed that gain-of-function mutation of FGFR2 delayed and decreased osteoclast bone resorption after BMX,quantitative PCR results showed that gain-of-function mutation of FGFR2 resulted in increased Rankl,Opg mRNA levels at 1,2 and 3 weeks after BMX;4.EdU incorporation assay results showed that gain-of-function mutation of FGFR2 resulted in increased stromal cells and osteoblasts proliferation at 4 days and 7 days after BMX,immunohistochemistry staining results showed that gain-of-function mutation of FGFR2 resulted in increased Runx2-positive and OC-positive osteoblasts at 1 week after BMX;5.Immunohistochemistry staining and western blot results showed that gain-of-function mutation of FGFR2 resulted in increased β-catenin protein levels,quantitative PCR results showed that gain-of-function mutation of FGFR2 resulted in increased Tcf1,Lef1,CyclinD1 and CD44 mRNA levels at 1 week after BMX;6.Micro-CT and histopathological staining results showed that Wnt inhibition partially alleviated the increased bone formation and decreased bone resorption in FGFR2 mutant mice after BMX.Part III Intra-articular injection of G141 protects against cartilage degradation in a murine model of osteoarthritis1.Kinase inhibition assay results showed that G141 had high affinity for FGFR1(IC50: 2.7± 0.54 μM)and had a much lower activity against other RTKs,caliper mobility shift assay results showed that G141 suppressed FGFR1 activity in an ATP-independent manner;2.qPCR results showed that G141 treatment resulted in a marked reduction of Mmp13 and Adamts5 mRNA levels and an increasing of Type II collagen and Aggrecan mRNA levels,western blot results showed that G141 treatment decreased the ERK1/2,ADAMTS-5 and MMP-13 protein levels in FGF-2–treated human articular chondrocytes;3.Safranin-O-fast green staining and DMMB assay results showed that G141 treatment markedly decreased the release of GAG into the culture medium from FGF-2–treated human femoral head cartilage samples;4.Safranin O–fast green staining and OARSI score results showed that intra-articular injection of G141 delayed cartilage degradation in mouse knee joint afar DMM surgery;5.Immunohistochemical staining and TUNEL staining assay results showed that intra-articular injection of G141 significantly decreased the number of MMP-13 and type X collagen positive cells and apoptosis cells at 8 weeks after DMM.Conclusion:1.FGFR2 accelerates suture MSCs senescence by decreasing autophagic activity,which results in craniosynostosis in Apert mice;2.Induction of the gain-of-function mutation of FGFR2 in adult mice results in increased bone mass by increasing bone formation and decreasing resorption in BMX model and Wnt/β-catenin pathway is involved in the high bone accrual phenotype in mice with gain-of-function mutation of FGFR2 after BMX.3.Pharmacologically antagonize FGFR1 using G141 protects the knee joint cartilage from degradation in a DMM model of mouse OA,probably by suppressing the production of matrix-degrading enzymes MMP-13 and ADAMTS-5 and preventing articular chondrocytes from hypertrophy and apoptosis.