Apert Syndrome Disease Gene FGFR2E731K Mutation On Osteoblast Differentiation And MVs Mineralization

Fibroblast growth factor receptors(FGFRs) family has four tyrosine kinase members (FGFR1-4), they can form dimmers with22kinds of FGFs ligands to activate PLCjy, Ras/Raf/MEK/ERK, RJAK/STAT pathways. They play an important role in bone formation, blood vessel formation, neurogenesis, damage repair and in tumor formation. FGFR2multiple mutations can lead to a rare bone disease-Apert syndrome. Apert syndrome (AS), also known as acroocephalo syyndactyly which is one of the most serious type of Craniosynostosis and is a rare disease, neonatal morbidity between1/160,000and1/65,000. In1906, Apert reported it for the first time, Apert syndrome belongs to the autosomal dominant disease and caused by FGFR2gene mutations, children with this disease is Distributed, caused by new mutations. Recently, Park and others found a new mutation of FGFR2gene in the study of an Apert patients, E731K. In the preliminary study of this new mutation, they found in the absence of FGF ligand binding FGFR2kinase itself can occur phosphorylation to activate ERK-MAP pathway. Apert syndrome is a kind of typical Single-gene-single-point-mutation caused genetic disease. It is a good model for the study of diseases caused by abnormal of bone mineralization. As there is a big difference between in vitro and in vivo environment, the results of many studies in the aspect of bone mineralization in vitro can not be obtained in many cases in vivo validation. In the research on this disease model, We just need to get FGFR2 pathogenic mutations that can make in vitro experimental model occur mineralization anomalies to reduce the impact of environmental differences on the experimental results. To further investigate the causes of Apert syndrome and further elaborated the roles of FGFR2gene in bone formation and elaborate how FGFR2E731K affect matrix vesicles (MVs) mineralization, we designed the following experiment.Objective:This study aims to elaboratehow the causative gene mutation in Apert syndrome, FGFR2E731K mutation, affect osteoblast mineralization in vitro and affect MVs mineralization in able to provide a new idea for further study the function of FGFR2gene and to know the relationship between MVs and Mineralization anomalied Diseases.Methods:In this study, we used human osteosarcoma cell line Saos-2as our cells. FGFR2E731K mtation and FGFR2gene Wild-type are transferred into Saos-2cells by Lentiviral vectors to detect how FGFR2E731K mutations affect osteoblast mineralization and MVs mineralization. First, we used the method of sequencing exon to exclusion the functional mutations of FGFR2gene with Saos-2cells. Then after MT (mutant FGFR2E731K) cells and WT (wild-type FGFR2cells) induced with β-glycerophosphate and ascorbic acid, we examined their alkaline phosphatase (ALP) activity and examined their ability of mineralization by Alizarin red staining. At the same time we examined the mRNA levels of mineralization marker proteins ALP. Runx1、OCN、CollAl by Quantitative PCR in two type of cells. We extracted MVs in two type of cells by ExoQiuck reagents, and we detected their ALP activity and detected their ability of mineralization by Absorbance detection.Results:(1) Sequencing detection proved that FGFR2gene exons of Saos-2cell have no function mutations.(2) We found that in the induction of ascorbic acid and β-glycerol phosphate, the ALP activity and mineralization ability were elevated by the overexpression of FGFR2E731K.(3) ALP、Runx2、OCN、CollA1mRNA levels were elevated by the overexpression by the overexpression of FGFR2E731K.(4) The ALP activity and mineralization ability of MVs were elevated by the overexpression of FGFR2E731K. Conclusion:This study shows that FGFR2E731K mutation can enhance osteoblast mineralization ability and upregulate the expression of ALP, Runx2, OCN, CollAl. In the study of MVs, FGFR2E731K mutation can significantly enhance mineralization ability of MVs which may suggest that FGFR2E731K mutation effect osteoblasts mineralization by MVs, and it also suggest that FGFR2can Regulate the activity of MVs.