The Effect And Underlying Mechanism Of FGFR3 Inbone Remodeling Regulation

There are four fibroblast growth factor receptors: FGFR1,FGFR2,FGFR3 and FGFR4.The FGFRs signaling cascades are triggered by FGFs binding to the extracellular domain of FGFRs.Then FGFs induce receptors dimerization and phosphorylation intracellularly.Numerous studies indicated that FGFR3 negatively regulates endochondral bone formation.Human gain-function mutation of FGFR3 cause abnormal cartilage formation and skeletal dysplasias characterized by reduced limbs length and craniosynostosis including ACH(Achondroplasia),HCH(Hypochondroplasia)and TD(Thanatophoric dysplasia).Researchers used amount of point gain-of-function mutation of FGFR3 mouse models to investigate underlying mechanisms of FGFR3 in skeletal dysplasia.FGFR3 activations cause blocked endochondral bone formation,deCreased bone volume and reduced chondrocytes differentiation.Interestingly,FGFR3 knock out mice also showed bone mass loss and thinner cortical bone even when the length of growth plates and chondrocytes hypertrophy were both inCreased.In our former work,we found that the paradox of mice with ubiquitous knock out or gain-function of mutations were probably because FGFR3 plays different roles in directly or indirectly affection of osteogenesis.In mammalian skeleton development,the crosstalk between chondrocytes and osteoblasts or osteoclasts is the most important regulator of bone formation.Inhibited chondrocytes hypertrophic differentiation led to abnormal perichondrium bone forming.Our earlier study demonstrated that ablation of FGFR3 in chondrocytes mice exhibited inCreased expression of BMP2,BMP4,BMP7 and deCreased expression of BMP signaling inhibitor Noggin.Meanwhile,IHH expression of conditional knock out mice was higher than wild type.All the results revealed that FGFR3 in chondrocytes regulate osteogenesis,but the mechanisms need to be further discovered.On the other hand,chondrocytes may mediate osteoclasts function by cell-cell reactions.Whether chondrocytic FGFR3 affects osteoclasts in a chondrocyte-osteoclast manner or by chondrocyte-osteoblast-osteoclast interactions is not clear at present.The co-culture system of chondrocytes and osteoblasts could further advance our understanding of FGFR3 in regulating bone remodeling and bone formation.In addition to the role of FGFR3 in chondrogenesis,it was also found that FGFR3 may regulate osteoblastic cell lineage.The osteoblastic cells participating in endochondral bone formation mainly derive from mesenchymal progenitors,sequentially become proosteoblasts,then differentiate into mature osteoblasts,which start producing abundant matrix proteins and depositing osteoid or non-mineralized bone matrix.This osteoid becomes mineralized by accumulation of calcium phosphate in the form of hydroxyapatite.Then mature osteoblasts embedded in mineralized tissue.Gain-of-function mutations of FGFR3(ACH)mice exhibited deCreased osteoblastic proliferation,impaired bone matrix mineralization but enhanced ALP activity.The study further indicated that FGFR3 inhibits BMSC proliferation via P38 pathway,and regulates mineralization via ERK1/2.Col1 promotor induced gain-of-function in FGFR3 at early stage of osteoblast showed loss of bone mass,expression of Col1 was higher than wild type but no significantly change in OP.All the results indicated that FGFR3 affects early osteoblasts.But the mechanisms of FGFR3 regulating mature late stage osteoblasts and further controlling bone remodeling require more evidence.There are three destinies of aging osteoblasts: 1.undergo apoptosis,2.become bone lining cells,3.transform to the osteocyte.Osteocytes comprise over 90% of cells in bone and are ‘‘permanent’’ resident osteoblastic cell population.Recent evidencedemonstrated that osteocyte influence bone remodeling via paracrine way,directly control the differentiation and activity of both osteoclasts and osteoblasts by cell-cell crosstalk.Osteocyte is also defined as mechanosensing cell.Mechanical loading deCreased expression of SOST in osteocytes which inhibited osteoblasts function and differentiation by antagonizing the canonical Wnt pathway.In addition to its role in osteoblast,osteocyte may also be involved in mediating osteoclast.Bone resorption is regulated by RANKL seCreting from osteocyte.FGFR3 was detected in osteocyte derived from mice or MLO-Y4 lineage suggested osteocyte FGFR3 may participates in inducing osteocyte function and controlling bone remodeling.Methods:Part I Explore the role of FGFR3 in osteocyte1.Establish stable osteocyte specific deletion of FGFR3 mouse model: Fgfr3flox/flox and Dmp1-Cre mice were bred together.The generations we used were Dmp1-Cre;Fgfr3flox/floxand Fgfr3flox/flox and sacrificed at the age of 3 and 6 months.Tibiae and femurs were harvested for the study.2.Immunohistochemical analysis was used for OC and FGFR3 expression.Tibiae and femurs were obtained for total bone RNA extraction and further FGFR3 expression.3.The whole bodies images of Dmp1-Cre;Fgfr3flox/floxand Fgfr3flox/flox mice were taken by X-Ray.Femurs were scanned by Micro-CT and the three-dimensional reconstructions and measurement were acquired to evaluate spongiosa and cortical bones.4.We used decalcified five-micrometer-thick sections to evaluate bone mass,osteoblast number and osteoblast surface.Undecalcified five-and ten-micrometer-thick sections were prepared for fluorescence observation and Von kossa staining respectively.5.TRAP staining was performed to identify osteoclasts,assess osteoclast number and osteoclast surface.Total primary bone RNA was extracted and reverse-transcripted for the quantitative PCR analysis of RankL and Opg.6.Osteocyte using TUNEL labeling were measured to clarify the connection between osteocytic apoptosis and bone resorption.Part II Study of the role of FGFR3 in mature osteoblasts1.Stable osteoblast specific deletion of FGFR3 mouse model were established for the experiments in this study.Body length and weight was measured at 2 month of age.2.We used X-Ray image to evaluate bone structure of OC-Cre;Fgfr3flox/flox mice.Micro-CT analysis was performed to gain an understanding of osteogenesis.3.Undecalcified and ten-micrometer-thick sections were prepared for fluorescence observation and five-micrometer-thick sections were stained for Von kossa,OC immunohistochemical staining was used to evaluate osteoblast function.4.We also used TRAP staining to calculate osteoclast number and osteoclast surface.Part III FGFR3 participates in cross-talk between chondrocyte and other cells during endochondral bone formation1.Co-couture was carried out using Trans-well system.First passage of primary Fgfr3flox/flox(WT)osteoblasts were seeded in a 6-multiwell plate,as primary chondrocytes came from WT and Col2-Cre;Fgfr3flox/flox(MUT)were separately plated in the polycarbonate membrane inserts.2.Alizarin red staining for mineralized osteoblast cultures and ALP staining were performed.We also used ALP activity analysis to evaluate osteoblasts function.3.After 14 days cultured osteoblast RNA was harvested and reverse-transcripted for quantitative PCR analysis of osteoblastic marker expression: Cbfa1,Col1 and OC.4.Total RNA extracted from chondrocytes plated in the polycarbonate membrane inserts together with osteoblast RNA mentioned above was both used to detect expression of RankL and Opg for our understing of deCreased bone resorption in MUT mice.5.Chondrocyt RNA was also reverse-transcripted for the expression of TGF-β1、Wnt2、Wnt3、Wnt4 and Wnt5 b which were chosen according to our former work about ACH chondrocyte gene expression profiling.Results:Part I Explore the role of FGFR3 in osteocyte1.Dmp1 induced Cre recombination efficiently deleted FGFR3 in osteocytes-specific manner.2.Inactivation of FGFR3 in osteocytes led to deCreased bone mass,trabecular thickness and trabecular number,inCreasedtrabecular separation;Thinner cortical bone and deCreased bone volume were also discovered in Dmp1-Cre;Fgfr3flox/floxmice.3.Dmp1-Cre;Fgfr3flox/floxmice exhibited reduced number of osteoblasts attaching to the cancellous bone surface.Interestingly,osteoblasts on intracortical were flatter,revealed that the bone formation was reduced.4.Osteocytes with Fgfr3 deficiency inhibited osteogenesis,distance between two labeling lines was shorter in Dmp1-Cre;Fgfr3flox/floxmice in trabecular bone,deCreased bone formation rate.But no significantly change was detected in cortical bone,bone formation rate was also showed no obviously statistical significance.5.Tartrate-resistant acid phosphatase(TRAP)-positive osteoclasts number and surface in primary trabecular and endo-cortical bone of Dmp1-Cre;Fgfr3flox/flox mice was significantly inCreased compared with those in Fgfr3flox/flox mice.The expression of RankL and Opg were both inCreased as the ratio of RankL/Opg inCreased indicated that bone resorption was enhanced.6.Osteocyte apoptosis was not extremely changed,provide the evidence that enhanced bone resorption was not related to apoptosis.Part II Study of the role of FGFR3 in mature osteoblasts1.Mature osteoblast deletion of FGFR3 led to reduced body length and weight,also bone volume was deCreased using X-Ray image analysis.2.Ablation of FGFR3 in mature osteoblasts resulted in impaired bone formation.Osteoblast number was reduced together with OC expression.Differentiation of osteoblasts in OC-Cre;Fgfr3flox/floxmice was remarkably blocked.3.There was no difference in osteoclast number and osteoclast surface between OC-Cre;Fgfr3flox/floxand WT mice.Part III FGFR3 regulates endochondral bone formation by cell-cell crosstalk1.The results showed that the numbers of crystal violet-staining(ALP positive)cells and ALP activity of osteoblasts co-cultured with MUT chondrocytes were remarkably inCreased after co-culture,and the number of mineralized nodules was also inCreased in osteoblasts co-cultured with MUT chondrocytes.Expressions of osteoblast marker Col1,OC and Cbfa1 were all significantly enhanced in osteoblasts co-cultured with MUT chondrocytes.2.Besides Bmp 2/4/7,Ihh and Noggin expression were detected in our former work,we further found Wnt-4 and Tgf-β1 were both remarkably inCreased.3.The expression levels of both RankL and Opg were significantly up-regulated in osteoblasts co-cultured with MUT chondrocytes.However,there was no remarkable difference between the ratios of RankL / Opg in osteoblasts co-cultured with WT and MUT chondrocytes.In chondrocytes,the mRNA expressions of both Rankl and Opg were also inCreased in MUT chondrocytes,moreover the ratio of Rankl to Opg was significantly deCreased in MUT chondrocytes compared with that of WT chondrocytes.Conclusion:1.Targeted deletion of FGFR3 in osteocytes inhibited bone remolding mainly by promoting bone repsorption induced by osteoclast and patially by osteogenesis indeuced by osteoblast.2.Mature osteoblast with FGFR3 deficiency reduced osteogenesis by inhibiting osteoblast differentiation.3.Cross-talk between FGFR3 deficiency chondrocytes and osteoblast or osteoclast contributed to impaired osteogenesis.