The Role Of FGFR3 In Differentiation Of MSCs To Osteoblast

Backgound and ObjectiveThe skeleton is generated by two distinct mechanisms,intramembranous and endochondral ossification;either pathway is modulated by the secreting signaling molecules including FGF,TGF-βand IGF,and cell and extracellular matrix^[1].In endochondral ossification,mesenchymal stem cells differentiate into chondrocytes,and then convert to cartilage,invaded by blood vessels and osteoclasts.Osteoblasts remodel the newly generated cartilage thus forming bone tissue.This mechanism accounts for the formation of the vertebrate and long bones^[2].During intramembranous ossification, mesenchymal stem cells convert directly into bone-forming osteoblasts,which secrete bone matrix proteins.This pathway is responsible for the generation of skull flat bones^[3,4]These two pathways often interact during the bone development.For example,in the process of long bone development,the growth mainly consists of a series of chondrocytes proliferation,differentiation and apoptosis,while the thickness of cortical bone depends on intramembranous ossification.On the contrary,cranial growth mainly depends on intramembranous ossification.Fibroblast growth factor receptor 3(FGFR3) is a member of transmembrane tyrosine kinase receptors family(FGFR1-5),which play an important role in skeletal development. Evidence from human and mouse genetics indicates the role of FGFR3 in bone development.Mutant of the transmembrane domain of FGFR3 results in heritable skeletal dysplasias in human,such as dwarfing chondrodysplasias(hypochondroplasia, achondroplasia,severe achondroplasia with developmental delay and acanthosis nigricans—also known as 'SADDAN'—and thanatophoric dysplasia).Based on mutant models,Several reports confirmed that FGFR3 has negative effects on signal transduction regulating growth,differentiation,migration and apoptosis of chondrocyte.Mouse lacking FGFR3(FGFR3-/-) showed the skeletal overgrowth accompanied by increased chondrocyte proliferation,and hypertrophic chondrocyte zone^[5-7].These results suggest that FGFR3 regulates bone growth by inhibiting the growth of chondrocytes.In 2004,investigations demonstrated FGFR3 gene disruption in adult mice resulted in osteopenic due to the reduction of cortical bone thickness and defective trabecular bone mineralization.FGFR3-/- mice MSCs expressed the markers of differentiated osteoblasts but fewer mineralized nodules developed.Studies on FGFR3 Gly369Cys mutant mouse model revealed that genes relating to osteoblast differentiation, such as osteopontin,osteonectin,and osteocalcin are upregulated,moreover,alizarin Red-S staining exhibited an advanced bone collar in mutant mice.These findings suggest the important role of FGFR3 in the process of osteoblasts differentiation^[8].Other researches from ligand(FGF2,FGF9,and FGF18) knockout mice demonstrated that FGFR3 participates in osteoblast differentiation.Mice lacking FGF9 or FGF18 showed the delayed ossification ^[9-12].FGF2 disruption led to the loss of trabecular bone volume and MSCs defective mineralization.^[13].Inactivating FGFR1 in osteo-chondro-progenitor cells increased the proliferation,and delayed osteoblast differentiation,but accelerated the differentiation of osteoblasts,accompanied with an increased expression of FGFR3^[14].All these findings manifest that FGFR3 signaling impacts on osteoblasts differentiation. However,the function of FGFR3 on osteoblast differentiation remains unclear.Mesenchymal stem cells(MSCs),deriving from mesoderm,have the potential of differentiation into osteoblasts,chondrocytes etc.During bone development and the process of bone repair,MSCs differentiate into precursor,pre-osteoblast and osteoblast by turns.At present,MSCs have been regarded as the main source of ostoblast cells.To explore the role of FGFR3 in differentiation of MSCs to osteoblast will better understand the role of FGFR3 in bone development and remodeling and clarify the function of FGFR3 on osteoblast differentiation.Therefore,adenovirus pAdE-FGFR3 and pAd-DNR3(Dominant-negative FGFR3)were constructed in this study and their expressions were verified using RT-PCR and Western blotting analysis.The transcriptional and translational level of the osteoblast marker genes Cbfaâ… and Collagenâ… were detected after pAdE-FGFR3,pAd-DNR3 and FGFR3RNAi transfected into MSCs.MSCs from gain of function FGFR3 mutant mice were separated and cultured,and the differentiation capability of MSCs to osteoblast was investigated in vitro and in vivo. Methods and ResultsFGFR3 and DNR3 recombinant adenovirus were constructed and then transferred into MSCs.Using the FGFR3 Gly369Cys mutant mouse model,the primary MSCs were cultured and differentiate into osteoblast in vivo and in vitro.The main results are as follows.1.FGFR3 and DNR3 recombinant adenovirus were constructed using the pAdEasy system.FGFR3 and DNR3 were cleaved by restrictive enzyme and inserted into pAdtrack-CMV shuttle vector.Recombinant vector was constructed and transferred into E. coli BJ5183.HEK 293 cell were transfected to pack viral particles.Viral particles infected HT-29/HCT 116 cell lines.The expressions of FGFR3 and DNR3 were analyzed by semi-quantify RT-PCR and western blotting.The titer of virus was detected by TCID50. GFP report gene showed the titer of virus was 3×10⁹ pfu/ml and 3.8×10⁹ pfu/ml, respectively.2.Effect of FGFR3 on the transcriptional and translational level of Cbfaâ… and Collagenâ… in MSCs.FGFR3,DNR3 adenovirus and FGFR3RNAi were transfected into MSCs,the expression of FGFR3 was determined by RT-PCR.The transcriptional and translational level of the osteoblast marker genes,collagenâ… and Cbfaâ… ,were detected using semiquantify RT-PCR,immunofluorescent and western blot.The overexpression of FGFR3 obviously increased the transcriptional and translational level of collagenâ… and Cbfaâ… in MSCs.However,DNR3 adenovirus and FGFR3 RNAi significantly decreased the transcriptional and translational level of collagenâ… and Cbfaâ… in MSCs.These findings indicated that FGFR3 can upregulate the expression of Cbfa 1 and collagenâ… in MSCs.3.The gain of function mutation of FGFR3 accelerated the osteoblast differentiation of MSCs in vitro and in vivo.Mutant mice MSCs were isolated and cultured,the 3th passage(passage 3) cell growth curve was drawed.Compared with the control MSCs,the latency of mutant MSCs was longer,suggesting that mutant cells grew more slowly.Cells with FGFR3 mutation cultured in differentiation medium for 7,14 and 21 days stained more intensely for ALP and contained more alizarin Red-S positive mineralized nodules.Semi-quantitative RT-PCR analysis showed Cbfaâ… expressed at a similar level at day 7(P＞0.05),but at a remarkable higher level at day 14(P＜0.05).Mutant MSCs combined with DBM were implanted into the back subcutaneouly of nude mice.After 8 weeks,X-ray images showed that the formation of bone,histology examination manifested trabecular bone formation.These results indicate that gain of function mutation of FGFR3 accelerated the differentiation of MSCs to osteoblast in vivo.Conclusion1.FGFR3 and DNFGFR3 recombinant adenovirus were prepared and could efficiently infect cells.2.FGFR3 and DNFGFR3 recombinant adenovirus and FGFR3 RNAi were successfully transferred into MSCs3.The overexpression of FGFR3 in MSCs upregulated the transcriptional and translational level of the osteoblast marker genes,collagenâ… and Cbfaâ… .4.The gain of function mutation of FGFR3 accelerated the differentiation of MSCs to osteoblast in vitro and in vivo.This study provides a critical data to clarify the role of FGFR3 signaling in osteoblast differentiation and lay an experimental foundation for gene therapy.