软骨内PTEN基因特异性敲除对FGFR3功能增强型点突变所致软骨发育不全的影响及机制的初步研究
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摘要
软骨发育不全是一种常染色体遗传病,是人类侏儒最常见的类型,其发生主要是De novo基因突变的结果,目前尚无有效的治疗方法。软骨发育不全主要影响四肢骨、椎骨等长骨(Long Bone)的软骨内成骨(Endochondral Ossification)过程,尤其是软骨生成(Chondrogenesis)过程,包括间充质细胞集聚并分化为软骨细胞,以及随后软骨细胞的增生、肥大和凋亡,但其机制目前并不完全清楚。*
骺生长板软骨细胞增生与分化的调控,对长骨的纵向生长和骨骼的发育至关重要。长骨生长板受多种信号分子的调控,其中成纤维细胞生长因子及其受体(Fibroblast Growth Factors/Fibroblast Growth Factor Receptors,FGFs/FGFRs)在其中起重要作用。目前认为,通过非配体依赖的自主激活,FGFR3的十多种功能增强型(Gain-of-function)点突变可引起多种人类软骨发育障碍性侏儒,包括软骨发育不全(Achondroplasia, ACH)、季肋发育不全(Hypochondroplasia, HCH)、致死性骨发育不全(Thanatophoric Dysplasia, TD)和严重软骨发育不良伴发育迟缓和黑棘皮症(Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans,SADDAN)等,其中95%以上的软骨发育不全患者由FGFR3的功能增强型突变引起。
Deng等发现,FGFR3基因敲除小鼠(FGFR3-/-)有长骨过度生长、生长板增生软骨细胞带和肥大软骨细胞带变宽、软骨细胞增殖活性增加;相反,Chen等人利用基因敲入技术建立了模拟人ACH的FGFR3G369C点突变小鼠模型(相当于人源FGFR3G375C),这种小鼠FGFR3功能增强,个体明显短小,头颅短圆,长骨生长板组织形态结构异常,软骨细胞增生带短稀,增生能力减弱、肥大软骨细胞明显减少伴Collagen X表达减低(即软骨细胞分化能力降低),表明FGFR3是长骨软骨生成的负性调节分子,影响软骨细胞的增殖活性和分化。目前研究FGFR3突变引起的软骨发育异常主要集中于STAT1/p21和ERK-MAPK信号通路上,但抑制此两条信号通路,并不能完全缓解FGFR3突变所致的侏儒表型,提示可能存在其他信号通路参与了对软骨发育异常的调控。
越来越多的研究表明PI3K/AKT信号通路在哺乳动物骨骼发育和细胞增殖分化中起重要作用,成纤维细胞生长因子(FGFs)、胰岛素样生长因子(Insulin-like Growth Factors,IGFs)、胰岛素等对软骨发育和功能重要的生长因子均能激活该通路。PTEN,即第10号染色体同源缺失性磷酸酶-张力蛋白基因(phosphatase and tensin homolog deleted on chromosome ten, PTEN),是PI3K/AKT通路中一个重要的负性调控分子。研究发现在小鼠成-软骨前体细胞中敲除PTEN后影响生长板软骨细胞的增殖与分化。但是,该通路在FGFR3介导的软骨发育不全中的作用目前尚不清楚,需要进一步研究。
鉴于FGFR3在软骨发育不全致病机理中的地位以及PI3K/AKT活性在软骨细胞增生抑制和分化延迟中的重要作用,本研究采用条件性基因敲除技术建立了软骨细胞条件性敲除PTEN基因的FGFR3功能增强型点突变小鼠模型,通过分析相关小鼠的表型,观察在FGFR3功能增强型小鼠软骨细胞特异性敲除PTEN基因后对软骨发育的影响,并初步探讨PTEN基因敲除影响软骨发育的可能的分子机制。
主要实验方法
1.利用基于Cre/LoxP系统的条件性基因敲除技术,建立了软骨细胞特异性敲除PTEN基因的FGFR3功能增强型小鼠模型,PCR鉴定小鼠基因型及免疫荧光鉴定敲除效率;
2.采用X线摄影、全骨架染色和头颅局部摄影,观察小鼠大体形态及颅底软骨联结的变化情况,测定小鼠生长过程中体重、体长、躯干长、尾长和尾椎椎体长度的变化;
3.制备了不同年龄不同基因型小鼠的组织切片,通过HE染色,观察生长板形态和第二骨化中心的发育情况;通过BrdU掺入后免疫组化检测,观察生长板软骨细胞增殖情况;
4.采用定量PCR检测软骨分化相关基因COL2A1、COL10A1和IHH的表达情况;Western Blot检测p-AKT水平,激光共聚焦显微术检测了软骨组织中p-AKT的水平并进行了定位观察;
5.建立胎鼠胫骨及跖骨体外培养模型;采用bpV处理阻断PTEN,观测对跖骨生长率的影响。
主要实验结果
一、软骨细胞特异性敲除PTEN基因的FGFR3功能增强型小鼠的获得
利用FGFR3功能增强点突变小鼠(Fgfr3G369C/+小鼠,即ACH小鼠),Ptenflox/flox小鼠以及软骨细胞中特异表达Cre重组酶的转基因小鼠(Col2αCre小鼠)设计交配策略,获得了基因型为Col2aCre:Ptenflox/flox:ACH的小鼠。该小鼠为软骨细胞特异性敲除PTEN基因的FGFR3功能增强型小鼠。采用免疫荧光化学对该小鼠软骨细胞中PTEN的表达情况进行了鉴定,证实PTEN在软骨细胞中因为基因敲除而表达显著降低。
二、PTEN基因敲除对ACH小鼠一般生长情况的影响
1.PTEN基因敲除对ACH小鼠体重、体长、尾长、椎体长度的影响:①在观测期3-10w内,CAP小鼠体重增长较AP小鼠明显;②4w的CAP小鼠体长、尾长明显长于AP小鼠,但是两者椎体长度差异不显著。
2.PTEN基因敲除对ACH小鼠头颅及颅底软骨联结的影响:①CAP小鼠头颅圆钝的状况较AP小鼠有所改善;②CAP小鼠的颅底软骨联结闭合时间较AP小鼠延迟。
三、PTEN基因敲除对ACH小鼠软骨内成骨的影响
1.PTEN基因敲除对ACH小鼠软骨细胞增殖的影响:出生5d和14d小鼠BrdU掺入检测后发现,CAP小鼠软骨细胞增殖指数较AP小鼠高,提示:CAP小鼠软骨细胞增殖活性较AP小鼠高;
2.PTEN基因敲除对ACH小鼠软骨细胞分化的影响:观察出生7d、12d小鼠的生长板,发现CAP小鼠的第二骨化中心较AP小鼠的提前出现,16d时CAP小鼠生长板软骨细胞未增殖带明显较AP小鼠窄,提示:CAP小鼠软骨细胞分化较AP小鼠快;
3.荧光定量PCR结果显示:7d时,AP小鼠关节软骨中COL2A1 mRNA比CAP小鼠高,CAP小鼠COL10A1 mRNA表达较AP小鼠高,提示CAP小鼠软骨细胞分化较AP小鼠加快。
4.PTEN基因敲除对ACH小鼠产生影响的可能机制:AP小鼠软骨组织未检测到p-AKT,而CAP小鼠p-AKT水平显著高于AP小鼠,提示:敲除PTEN后对ACH小鼠增殖与分化产生的影响可能是由于AKT磷酸化水平改变引起。
四、胎鼠胫骨体外培养模型的建立及阻断PTEN后对胎鼠跖骨生长的影响
建立了ACH胎鼠(E16.5)胫骨体外培养7d,14d的模型,显示ACH胎鼠胫骨生长率较野生慢。ACH胎鼠(E16.5)跖骨在给予不同浓度bpV后结果显示:处理组跖骨生长率明显较未处理组高,提示:给予bpV处理阻断PTEN后,PI3K/AKT通路活化,促进了软骨的生长。
主要结论
通过上述实验结果分析,我们发现小鼠软骨细胞特异性敲除PTEN基因可部分缓解由FGFR3功能增强型点突变所引起的侏儒表型。主要表现为:
1.软骨细胞特异性敲除PTEN的FGFR3功能增强型小鼠大体表型较FGFR3功能增强型小鼠有所缓解;
2.软骨细胞特异性敲除PTEN的FGFR3功能增强型小鼠生长板软骨细胞增殖活性较FGFR3功能增强型小鼠增高,提示PTEN基因的条件性敲除,可以缓解FGFR3功能增强所致的软骨细胞增殖抑制;
3.软骨细胞特异性敲除PTEN的FGFR3功能增强型小鼠生长板软骨细胞较AP小鼠软骨细胞分化加快,提示PTEN基因敲除可缓解FGFR3功能增强所致的分化抑制;
4.PI3K/AKT信号通路在软骨发育中发挥重要作用,AKT活性降低是FGFR3功能增强所致软骨细胞增殖抑制和分化延迟的重要机制之一。进一步地采用bpV处理ACH胎鼠跖骨也发现阻断PTEN使PI3K/AKT通路活化,促进了软骨的生长。但是,FGFR3功能增强所致软骨细胞中AKT活性降低的机制尚需进一步研究。
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.
METHODS
1. 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.
RESULTS
1. The generation of mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN
Using 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 growth
The effects of PTEN deficiency on body weight, body length, tail length and vertebra length of ACH mice:①the weight gain of CAP mice was more evident than AP mice in observation periods ;②the 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:①it was evident that the round and dull skull shape of AP mice was improved in CAP mice;②the cranial synchondrosis time of CAP mice was delayed compared with AP mice.
3. The effects of PTEN deficiency on endochondral ossification of ACH mice
The 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.
CONCLUSIONS
Generally 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.
骺生长板软骨细胞增生与分化的调控,对长骨的纵向生长和骨骼的发育至关重要。长骨生长板受多种信号分子的调控,其中成纤维细胞生长因子及其受体(Fibroblast Growth Factors/Fibroblast Growth Factor Receptors,FGFs/FGFRs)在其中起重要作用。目前认为,通过非配体依赖的自主激活,FGFR3的十多种功能增强型(Gain-of-function)点突变可引起多种人类软骨发育障碍性侏儒,包括软骨发育不全(Achondroplasia, ACH)、季肋发育不全(Hypochondroplasia, HCH)、致死性骨发育不全(Thanatophoric Dysplasia, TD)和严重软骨发育不良伴发育迟缓和黑棘皮症(Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans,SADDAN)等,其中95%以上的软骨发育不全患者由FGFR3的功能增强型突变引起。
Deng等发现,FGFR3基因敲除小鼠(FGFR3-/-)有长骨过度生长、生长板增生软骨细胞带和肥大软骨细胞带变宽、软骨细胞增殖活性增加;相反,Chen等人利用基因敲入技术建立了模拟人ACH的FGFR3G369C点突变小鼠模型(相当于人源FGFR3G375C),这种小鼠FGFR3功能增强,个体明显短小,头颅短圆,长骨生长板组织形态结构异常,软骨细胞增生带短稀,增生能力减弱、肥大软骨细胞明显减少伴Collagen X表达减低(即软骨细胞分化能力降低),表明FGFR3是长骨软骨生成的负性调节分子,影响软骨细胞的增殖活性和分化。目前研究FGFR3突变引起的软骨发育异常主要集中于STAT1/p21和ERK-MAPK信号通路上,但抑制此两条信号通路,并不能完全缓解FGFR3突变所致的侏儒表型,提示可能存在其他信号通路参与了对软骨发育异常的调控。
越来越多的研究表明PI3K/AKT信号通路在哺乳动物骨骼发育和细胞增殖分化中起重要作用,成纤维细胞生长因子(FGFs)、胰岛素样生长因子(Insulin-like Growth Factors,IGFs)、胰岛素等对软骨发育和功能重要的生长因子均能激活该通路。PTEN,即第10号染色体同源缺失性磷酸酶-张力蛋白基因(phosphatase and tensin homolog deleted on chromosome ten, PTEN),是PI3K/AKT通路中一个重要的负性调控分子。研究发现在小鼠成-软骨前体细胞中敲除PTEN后影响生长板软骨细胞的增殖与分化。但是,该通路在FGFR3介导的软骨发育不全中的作用目前尚不清楚,需要进一步研究。
鉴于FGFR3在软骨发育不全致病机理中的地位以及PI3K/AKT活性在软骨细胞增生抑制和分化延迟中的重要作用,本研究采用条件性基因敲除技术建立了软骨细胞条件性敲除PTEN基因的FGFR3功能增强型点突变小鼠模型,通过分析相关小鼠的表型,观察在FGFR3功能增强型小鼠软骨细胞特异性敲除PTEN基因后对软骨发育的影响,并初步探讨PTEN基因敲除影响软骨发育的可能的分子机制。
主要实验方法
1.利用基于Cre/LoxP系统的条件性基因敲除技术,建立了软骨细胞特异性敲除PTEN基因的FGFR3功能增强型小鼠模型,PCR鉴定小鼠基因型及免疫荧光鉴定敲除效率;
2.采用X线摄影、全骨架染色和头颅局部摄影,观察小鼠大体形态及颅底软骨联结的变化情况,测定小鼠生长过程中体重、体长、躯干长、尾长和尾椎椎体长度的变化;
3.制备了不同年龄不同基因型小鼠的组织切片,通过HE染色,观察生长板形态和第二骨化中心的发育情况;通过BrdU掺入后免疫组化检测,观察生长板软骨细胞增殖情况;
4.采用定量PCR检测软骨分化相关基因COL2A1、COL10A1和IHH的表达情况;Western Blot检测p-AKT水平,激光共聚焦显微术检测了软骨组织中p-AKT的水平并进行了定位观察;
5.建立胎鼠胫骨及跖骨体外培养模型;采用bpV处理阻断PTEN,观测对跖骨生长率的影响。
主要实验结果
一、软骨细胞特异性敲除PTEN基因的FGFR3功能增强型小鼠的获得
利用FGFR3功能增强点突变小鼠(Fgfr3G369C/+小鼠,即ACH小鼠),Ptenflox/flox小鼠以及软骨细胞中特异表达Cre重组酶的转基因小鼠(Col2αCre小鼠)设计交配策略,获得了基因型为Col2aCre:Ptenflox/flox:ACH的小鼠。该小鼠为软骨细胞特异性敲除PTEN基因的FGFR3功能增强型小鼠。采用免疫荧光化学对该小鼠软骨细胞中PTEN的表达情况进行了鉴定,证实PTEN在软骨细胞中因为基因敲除而表达显著降低。
二、PTEN基因敲除对ACH小鼠一般生长情况的影响
1.PTEN基因敲除对ACH小鼠体重、体长、尾长、椎体长度的影响:①在观测期3-10w内,CAP小鼠体重增长较AP小鼠明显;②4w的CAP小鼠体长、尾长明显长于AP小鼠,但是两者椎体长度差异不显著。
2.PTEN基因敲除对ACH小鼠头颅及颅底软骨联结的影响:①CAP小鼠头颅圆钝的状况较AP小鼠有所改善;②CAP小鼠的颅底软骨联结闭合时间较AP小鼠延迟。
三、PTEN基因敲除对ACH小鼠软骨内成骨的影响
1.PTEN基因敲除对ACH小鼠软骨细胞增殖的影响:出生5d和14d小鼠BrdU掺入检测后发现,CAP小鼠软骨细胞增殖指数较AP小鼠高,提示:CAP小鼠软骨细胞增殖活性较AP小鼠高;
2.PTEN基因敲除对ACH小鼠软骨细胞分化的影响:观察出生7d、12d小鼠的生长板,发现CAP小鼠的第二骨化中心较AP小鼠的提前出现,16d时CAP小鼠生长板软骨细胞未增殖带明显较AP小鼠窄,提示:CAP小鼠软骨细胞分化较AP小鼠快;
3.荧光定量PCR结果显示:7d时,AP小鼠关节软骨中COL2A1 mRNA比CAP小鼠高,CAP小鼠COL10A1 mRNA表达较AP小鼠高,提示CAP小鼠软骨细胞分化较AP小鼠加快。
4.PTEN基因敲除对ACH小鼠产生影响的可能机制:AP小鼠软骨组织未检测到p-AKT,而CAP小鼠p-AKT水平显著高于AP小鼠,提示:敲除PTEN后对ACH小鼠增殖与分化产生的影响可能是由于AKT磷酸化水平改变引起。
四、胎鼠胫骨体外培养模型的建立及阻断PTEN后对胎鼠跖骨生长的影响
建立了ACH胎鼠(E16.5)胫骨体外培养7d,14d的模型,显示ACH胎鼠胫骨生长率较野生慢。ACH胎鼠(E16.5)跖骨在给予不同浓度bpV后结果显示:处理组跖骨生长率明显较未处理组高,提示:给予bpV处理阻断PTEN后,PI3K/AKT通路活化,促进了软骨的生长。
主要结论
通过上述实验结果分析,我们发现小鼠软骨细胞特异性敲除PTEN基因可部分缓解由FGFR3功能增强型点突变所引起的侏儒表型。主要表现为:
1.软骨细胞特异性敲除PTEN的FGFR3功能增强型小鼠大体表型较FGFR3功能增强型小鼠有所缓解;
2.软骨细胞特异性敲除PTEN的FGFR3功能增强型小鼠生长板软骨细胞增殖活性较FGFR3功能增强型小鼠增高,提示PTEN基因的条件性敲除,可以缓解FGFR3功能增强所致的软骨细胞增殖抑制;
3.软骨细胞特异性敲除PTEN的FGFR3功能增强型小鼠生长板软骨细胞较AP小鼠软骨细胞分化加快,提示PTEN基因敲除可缓解FGFR3功能增强所致的分化抑制;
4.PI3K/AKT信号通路在软骨发育中发挥重要作用,AKT活性降低是FGFR3功能增强所致软骨细胞增殖抑制和分化延迟的重要机制之一。进一步地采用bpV处理ACH胎鼠跖骨也发现阻断PTEN使PI3K/AKT通路活化,促进了软骨的生长。但是,FGFR3功能增强所致软骨细胞中AKT活性降低的机制尚需进一步研究。
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.
METHODS
1. 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.
RESULTS
1. The generation of mice with simultaneous gain-of-function mutation of FGFR3 and chondrocyte-specific deficiency of PTEN
Using 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 growth
The effects of PTEN deficiency on body weight, body length, tail length and vertebra length of ACH mice:①the weight gain of CAP mice was more evident than AP mice in observation periods ;②the 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:①it was evident that the round and dull skull shape of AP mice was improved in CAP mice;②the cranial synchondrosis time of CAP mice was delayed compared with AP mice.
3. The effects of PTEN deficiency on endochondral ossification of ACH mice
The 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.
CONCLUSIONS
Generally 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.
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