FGFR3-TAK1-p38 MAPK 信号通路在软骨生成中的作用及其机制研究

The gain of function mutation of FGFR3 can result in a series of skeletal dysplasias including achondroplasia, but the precise molecular mechanisms as yet to be elucidated. Accumulated evidences showed that TAK1-MKK3/6-p38 MAPK play a vital role in the regulation of cell proliferation and differention in different cell types, including in the chondrocytes. However, whether FGF/FGFR3 stimulates TAK1 and its downstream pathways and the role and mechanisms of TAK1-MKK3/6-p38 MAPK signaling pathway in the regulation of FGFR3-mediated chondrogenesis are still not well documented. Our preliminary study showed FGF-2 increased the phosphorylation of p38 MAPK in a dose-dependent manner and a TAK1-dependent manner in ATDC5 cells. IP results showed that FGFR3 ditectly binds to TAK1. Furthermore, we found that SB203580, a specific inhibitor for p38 MAPK relieved FGFR3-mediated achondroplasia in a in vitro culture of metatarsal rudiment model. All the data suggested that FGF/FGFR3 can bind to TAK1 and stimulate its downstream pathways. To reveal the mechamisms of FGF/FGFR3-stimulated activation of TAK1 and the role of TAK1-MKK3/6-p38 MAPK pathway in the FGFR3-mediated chondroplasia, the detailed molecular events of FGFR3-TAK1 interaction will be studied by co-immunoprecipitations, GST-pull down and immunofluorescence staining. Mices with FGFR3Flox/flox, TAK1Flox/flox and p38aFlox/flox, FGFR3 knock-in (ACH) and Col2a1-Cre mices will be used to generate Col2a1-Cre;FGFR3Flox/flox, Col2a1-Cre;TAK1Flox/flox, ACH mices with TAK1 gene or p38a deficiency in chondrocytes. By analysing the phenotypes of the mices, intergrating the bone culture, primary chondrocytes culture and inhibitors of TAK1 and p38 MAPK, the role and precised molecular mechanisms of TAK1-MKK3/6-p38 MAPK in the regulation of FGFR3-mediated skeletal dysplasias will be studied in different levels.

FGF/FGFR3与p38 MAPK均在软骨生成中发挥重要作用，但FGF/FGFR3能否激活TAK1-p38 MAPK，并参与FGFR3介导的软骨发育异常尚不清楚。我们发现，FGF-2处理后TAK1磷酸化水平增加，FGFR3与TAK1之间存在相互作用，SB203580可以缓解FGFR3突变所致的骨骼异常。为研究相关机制，本课题拟通过建立软骨特异敲除FGFR3、TAK1和p38α和敲除TAK1或p38α基因的ACH小鼠，通过分析小鼠骨骼发育情况，结合原代胚胎跖骨和软骨细胞分离培养和免疫共沉淀等技术，研究FGF/FGFR3激活TAK1-p38 MAPK信号通路的分子机制，并研究该通路在FGFR3功能增强所致骨骼发育异常中的作用和机制。通过本研究，希望明确FGF/FGFR3激活p38 MAPK通路的分子机制，阐明TAK1-p38 MAPK信号通路在FGFR3功能增强所致的软骨发育不全中的作用。

申报的自然基金“FGFR3-TAK1-p38 MAPK 信号通路在软骨生成中的作用及其机制研究”由一个4年期的面上项目改为1年期的小额资助项目，调整后我们研究工作主要集中在“FGF/FGFR3激活TAK1-MKK3/6-p38 MAPK通路的分子生物学机制”上面。经过一年的工作，我们工作的研究进展主要包括：利用TAK1抑制剂（5Z-7-Oxozeaenol）处理后FGF-2激活的p38 MAPK 、p-MKK3/6、p-TAK1（S412）明显降低，萤火虫荧光素酶报告基因结果也证实，抑制TAK1活性，可以明显抑制RCS细胞中FGF-2激活的pFA-CHOP-Luc活性；FGF-2促进TAK1的磷酸化（p-TAK1-S412增加)；同时我们的研究结果显示，FGF-2促进TAK1的泛素化（K63 的泛素化）；IP的研究结果显示，FGFR3与TAK1之间存在相互作用（不论是转染细胞还是内源细胞均得到相应结果）；Mapping的结果显示，FGFR3通过KD domain与TAK1相互作用；同时在试验中我们发现，FGFR3与TAB1作用，但不与TAB2之间存在相互作用。利用体外跖骨培养系统，我们发现p 38 MAPK信号通路特异阻断剂SB203580可以明显缓解FGFR3突变所致的骨骼发育异常： E18.5的胚胎期TDII小鼠（EIIa-Cre;Fgfr3K644E/+，FGFR3K644E突变持续激活）(以同窝野生型小鼠为对照)双侧下肢中间跖骨，置于48孔板中培养，部分样本加入SB203580处理，培养的0d、7d ，趾骨于IPP7.0软件控制的CCD下测量长度并分析。结果显示：SB203580处理后FGFR3所致的骨骼生长抑制明显缓解，并且这种作用与FGFR3的激活明显相关。另外，我们引进了TAK1flox/flox 小鼠，目前该小鼠正在与FGFR3功能增强型的小鼠ACH（模拟人侏儒）进行交配，我们希望通过获得在软骨细胞中特异敲除TAK1基因的ACH小鼠并分析其表型，明确FGFR3-TAK1-p38 MAPK 信号通路及其在软骨发育分化中的作用和机制。