Study On The Effect Of ALPL Mutation On ADRB2Gene In The Osteogenesis Of BMMSCs

Hypophosphatasia (HPP) is an inheritable disorder caused by mutations in ALPLgene encoding tissue non-specific alkaline phosphatase (TNAP). The disease ischaracterized by typical insufficiency mineralization of hard tissue such as bone andtooth. The serum alkaline phosphatase (ALP) activity is also low. ALP is recognized as aspecific marker of differentiated osteoblast, and plays a key role in bone development.The molecular mechanisms of HPP were not clear in the prior studies. Therefore, toexplore ALPL gene mutations leading to changes in the cell and molecular mechanisms isuseful to the in-depth understanding of the the HPP pathogenesis and provide newtreatment methods. The classic bone biology theory showed that the bone marrowmesenchymal stem cells (BMMSCs) played a very important role in development ofbone, tissue remodeling and repairation of the bone fracture. Therefore, the BMMSCsfrom HPP patients can be served as the best model of the disease. In this study, byclinical and laboratory examination combined with genetic diagnosis of three childrenwith hypophosphatasia, and the observation of bone development in the Akp2knockout mouse, we plan to confirm that ALPL gene mutations can affect the bone development.BMMSCs from two HPP children and three normal children at the same age wereisolated and cultured. We did some analysis of their biological behaviors to find out theabnormity of the BMMSCs derived from HPP children. For the first time we screenedout the ADRB2gene using microarray analysis, which is closely related to osteogenicdifferentiation of BMMSCs and has different expression levels in normal, and HPPderived BMMSCs. By regulation of ALPL gene in normal or HPP derived BMMSCs, Weaimed to make clear the function of ALPL gene in osteogenesis and ADRB2expression.Finally, we regulated the expression level of ADRB2in normal or HPP derivedBMMSCs and confirmed the role of ADRB2in the osteogenic differentiation ofBMMSCs. The research is divided into the following four parts.Part1. The phenotypes of bone dysplasia result from ALPL gene mutationObjective: To identify the developmental abnormalities of bone tissue with ALPLgene mutation. Methods: By clinical, laboratory tests and genetic analysis, we madeclear the diagnosis and skeleton abnormalities of the3patients. By histologicalobservation of the Akp2+/-mouse bone, we aimed to prove the bone dysplasiaperformance under ALPL gene mutations. Results: All three patients showed lack ofserum ALP activity, decreased long bone density and strength, poor root developmentand premature tooth loss. Genetic analysis found that the three children are all with ALPLgene mutations, therefore they are diagnosed as childhood HPP. Histological observationshowed that Akp2+/-mouse performed skeletal developmental defects Conclusions: Thethree patients are all diagnosed as childhood HPP and exist bone tissue hypoplasia.Akp2+/-mouse also showed bone abnormalities. These results proved that ALPL genemutation can lead to skeletal abnormalities.Part2. Study on the biological characteristics of BMMSCs from HPPpatientsObjective: To explore the abnormal biological behaviors and gene expression ofHPP patients derived BMMSCs and to provide evidence of the pathogenic mechanism of HPP. Methods: After isolation and identification of BMMSCs derived from2HPPpatients and normal children we detected the biological characteristics, including ALPactivity and intracellular distribution, proliferation and differentiation capacity. Withmicroarray detection, we also screened out the different gene expression in osteogenicdifferentiation between normal and HPP patients derived BMMSCs. Results:Mesenchymal-origined stem cells are obtained by in vitro culture of BMMSCs from2HPP patients and3normal children and these stem cells all showed multi-differentiationcapacity. HPP patients derived BMMSCs have the same proliferation ability but reducedosteogenic differentiation capacity than BMMSCs from normal children. By detectionand analysis of gene chip, we found that the expressions of ADRB2gene are significantlydifferent between normal children and HPP patients derived BMMSCs in the process ofosteogenesis. Conclusion: ALPL gene mutation can lead to decreased osteogenicdifferentiation ability of BMMSCs and ADRB2gene expression is changed inosteogenesis in the case of ALPL mutation.Part3. The role ofALPLtoADRB2in the osteogenic differentiation of BMMSCsObjective: To prove the function of ALPL gene in BMMSCs osteogenicdifferentiation capacity and ADRB2gene expression. Methods: After silencing/over-expression of ALPL gene in BMMSCs from normal children/HPP patients, wedetected the differentiation capacity and ADRB2expression level in the regulated stemcells. Results: Osteogenic ability of normal BMMSCs was significantly decreased aftertransfection of siALPL. ADRB2expression was also changed by interfering of ALPL.However, over-expression of ALPL improved the osteogenesis of HPP derived BMMSCsand played a role in the down regulation of ADRB2in the process of osteogenesis.Conclusion: ALPL promotes osteogenic differentiation of BMMSCs but has a negativeregulatory effect on ADRB2expression.Part4. Study on the role of ADRB2in osteogenic differentiation of BMMSCsObjective: To prove the effect of ADRB2on BMMSCs osteogenesis. Methods: The expression of ADRB2gene was silenced/over-expressed in BMMSCs derived fromHPP patient/normal children respectively. After that, we detected the osteogenicdifferentiation capacity of the regulated stem cells. Results: Silencing of ADRB2geneimproved osteogenesis of BMMSCs but had no effect on ALP expression or activity. Onthe contrary, over-expressed ADRB2decreased the osteogenic differentiation, and it hadno effect of ALP either. Conclusion: ADRB2gene suppresses the osteogenicdifferentiation of BMMSCs.