Preliminary Phenotype Analyses Of The Chondrogenesis Of Mice With Simultaneous Gain-of-function Mutation Of FGFR3 And Chondrocyte-specific Deficiency Of PTEN

Achondroplasia (ACH) is an autosomal inherited abnormality disease, which is the most common type of dwarfism of human being. It's resulted from De novo gene mutation, so far there are no effective therapies. Achondroplasia mainly affects the endochondral ossification, especially the chondrogenesis of long bones in limbs and vertebrae. We still do not fully know the mechanisms underlying the chondrogenesis, which includes mesenchymal cell condensation, differentiation, and consequential chondrocyte proliferation, hypertrophy and apoptosis.The regulation of chondrocyte proliferation and differentiation in epiphyseal growth plate is very important for the normal skeletal development including longitudinal growth of long bones. The growth plate development is regulated by multiple signaling molecules, in which fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) are important. It was reported that more than 10 gain-of-function point mutations of FGFR3,mainly resulting in an ligand-independent autoactivation of FGFR3, result in several types of dwarfism and dyschondroplasia of humans including achondroplasia(ACH), hypochondroplasia (HCH), Thanatophoric dysplasia (TD), and Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN). More than 95% cases of achondroplasia were caused by gain-of-function mutations of FGFR3.Deng et al. reported that FGFR3 knock-out mice (FGFR3-/-) exhibited overgrowth of long bone, wider hypertrophic zone, proliferative zone and enhanced proliferative activity of chondrocytes. On the contrary, FGFR3G369C mutant mice (Fgfr3G369C/+mice,ACH mice)established by Chen et al. have small habius, short and round skull, structural abnormality of growth plate, decreased proliferative activity of chondrocytes, reduced number of hypertrophic chondrocytes accompanied lower Collagen X expression, which demonstrated that FGFR3 negatively regulates chondrogenesis of long bones by affecting the proliferative activity and differentiation of chondrocytes. Recent studies on pathways responsible for the chondrodysplasia resulting from gain-of-function mutation of FGFR3 were mainly focused on the STAT1/p21 and ERK-MAPK signal pathways. But inhibition of these two pathways did not lead to total alleviation of dwarfism phenotype, suggesting some other signaling may participate in the pathogenesis of chondrodysplasia.Emerging evidences have proved that PI3K/AKT pathway plays a crucial role in skeletal development as well as cell proliferation and differentiation in mammalians. Some growth factors including FGFs, Insulin-like growth factors (IGFs), insulin, et al. that have been found to play important roles in the chondrogenesis could activate this pathway. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important negative regulator of PI3K/AKT pathway. When PTEN was conditionally knocked out from osteo-chondroprogenitor, the proliferation and differentiation of chondrocyte growth plate were affected. However, the possible role of PI3K/AKT pathway in FGFR3 mutation induced achondroplasia and its underlying mechanisms are still elusive.Due to the critical roles of FGFR3 in the pathogenesis of achondroplasia and potential inhibition effects of PI3K/AKT activity on chondrocyte proliferation and differentiation, we established mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN using Cre/LoxP strategy. To investigate the effects of PTEN deficiency on the chondrogenesis of mice with gain-of-function of FGFR3, chondrocyte phenotypes of several types of mutants and wild type mice littermates were analyzed. The potential roles of PTEN in the chondrogenesis of ACH mice were preliminarily investigated.METHODS1. Based on Cre/LoxP conditional knockout strategy, mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN were established, genotypes and PTEN deficiency were characterized by PCR and immunofluorescent staining.2. X-ray radiography, whole skeleton staining and skull photography were performed, general shape of mice and cranial synchondrosis were observed, body weight, length of truck, tail, body and caudal vertebrae were measured.3. Long bone epiphyseal sections were prepared, the morphology of growth plate and secondary ossification center were observed under HE slides, chondrocyte proliferation were investigated by BrdU incorporation assay and immunohistochemistry.4. Chondrocyte differentiation associated gene as Collagen II, Collagen X and IHHexpressions were determined by quantitative PCR. p-AKT level in chondrocytes was determined by Western Blot and its location pattern in grow plate was investigated by Confocal Laser Scanning Microscopy.5. The system of tibial and metatarsal organ culture in vitro was established, metatarsal growth rate were determined and compared with or without PTEN blockade by bpV treatment.RESULTS1. The generation of mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTENUsing FGFR3 gain-of-function mutant (Fgfr3G369C/+ mice, ACH mice), Ptenflox/flox mice and chondrocyte specific Cre recombinase expressing transgenic mice (Col2aCre mice), reproductive copulation strategy was designed and mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN(Col2aCre:Ptenflox/flox:ACH mice) were generated. PTEN expression was evidently reduced after PTEN gene deletion,using PTEN immunofluorescent staining performed on cartilage slides.2. The effects of PTEN deficiency on ACH mice growthThe effects of PTEN deficiency on body weight, body length, tail length and vertebra length of ACH mice:â‘ the weight gain of CAP mice was more evident than AP mice in observation periods ;â‘¡the body length, tail length of CAP mice at 4 week were significantly longer than AP mice, but the length difference of vertebra was not significant. The effects of PTEN knockout on skull and cranial synchondrosis:â‘ it was evident that the round and dull skull shape of AP mice was improved in CAP mice;â‘¡the cranial synchondrosis time of CAP mice was delayed compared with AP mice.3. The effects of PTEN deficiency on endochondral ossification of ACH miceThe effects of PTEN knockout on chondrocyte proliferation: the chondrocyte proliferative index in CAP mice was significantly higher than AP mice when BrdU incorporation assay and IHC were performed on 5d and 14d postnatally. This suggests that the chondrocyte proliferative activity of CAP mice were higher than AP mice.The effects of PTEN knockout on chondrocyte differentiation: the secondary ossification center of CAP mice was anticipated compared to AP mice when growth plate was observed on 7d and 12d postnatally. On postnatal 16d, the state zone of chondrocytes in CAP mice growth plate was evidently narrower than AP mice, indicates that CAP mice have a faster differentiation than AP mice.From the quantitative PCR results, we know that Collagen II mRNA in cartilage was higher in AP mice than CAP mice, but Collagen X mRNA level was reversed, suggesting a faster differentiation of chondrocyte in CAP mice than AP mice.The possible mechanisms underlying in effects of PTEN knockout on ACH mice: there was no p-AKT detected in AP mice cartilage, but p-AKT was detectable in CAP mice, indicates that the influences of PTEN knockout on chondrocyte proliferation and differentiation might due to the alteration of AKT phosphorylation level.4. The establishment of tibia organ culture in vitro and the effects of PTEN blockade on metatarsal growth rate.7 days and 14 days tibia culture system of embryonic ACH mice were established in vitro. Analysis revealed that the tibial growth rate of ACH mice was significantly slowed down than littermate control. The metatarsal growth rate in bpV treated group was significantly higher than littermate control, suggesting that when PTEN was blocked by specific inhibitor bpV, PI3K/AKT pathway was activated and the growth of cultured metatarsal was promoted.CONCLUSIONSGenerally speaking, we found that the dwarfism phenotype resulted from FGFR3 gain-of-function mutation were partially rescued with PTEN specific deficiency in chondrocytes.1. The general phenotype of mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN was improved compared with FGFR3 gain-of-function mutant;2. The proliferative activity of chondrocytes in growth plate of mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN was higher than FGFR3 gain-of-function mutant, indicates that the inhibitory effect on proliferation of chondrocytes caused by FGFR3 gain-of-function mutation could be partially released due to PTEN specific deficiency in chondrocytes;3. The differentiation of mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN was faster than FGFR3 gain-of-function mutant, indicates that the inhibitory effect on differentiation of chondrocyte caused by FGFR3 gain-of-function mutation was partially released;4. PI3K/AKT pathway plays a crucial role in chondrogenesis. One of the important mechanisms underlying in proliferation inhibition and differentiation retard is the reduced AKT activition Using bpV treatment on metatarsal , it was found that PTEN blockade could activate PI3K/AKT and promote cartilage growth. However, the mechanisms of reduced AKT activation caused by FGFR3 gain-of-function mutation still needs further exploration.