1.Vitamin D 1α-Hydroxylase Gene Mutations In Patients With Pseudo-Vitamin D-Deficiency Rickets 2.The Effects Of SIP1 On The Osteoblast Specific Transcription Factor Cbfa1 Gene Expression And Its Role In BMP2 Pathway

PartⅠ:Vitamin D 1α-Hydroxylase Gene Mutations in Patients with Pseudo-Vitamin D-Deficiency RicketsObjectivePseudo-Vitamin D-Deficiency Rickets(PDDR) also known as vitamin D-dependent rickets type 1,is a rarely autosomal recessive disorder characterized by the early onset of rickets with severity syndrome of rickets and is caused by mutations of the 25-hydroxyvitamin D 1α-hydroxylase(1α-hydroxylase,CYP27B1) gene.So far, the mutations of CYP27B1 have been found more than thirty,but only two mutations were identified in one Chinese patient.The purpose of the study is to investigate the mutations of CYP27B1 in patients with PDDR.Thereby we can identify the mutation distribution of CYP27B1 gene of PDDR in Chinese Han ethnic group.Methods:1.Clinical data collection:medical history,physical examination,radiological films and laboratory examinations were investigated from 3 probands with PDDR and their family members.2.Genomic DNA extraction:After getting informed consent from all family members,the peripheral venous blood were drown and whole blood genome DNA of three families were extracted by QIAGEN DNA extraction kit.3.PCR amplification:Primers of all 9 exons and exon-intron boundaries of CYP27B1 were designed using with software primer premier 5.The amplification conditions were selected by preliminary experiment.4.Detecting PCR products:PCR products were detected by 2.0%agarose electrophoresis and were sequenced by company.5.PAGE electrophoresis:8%neutral and denatured PAGE electrophoresis were used to confirm deletion and insertion frame shift mutations.6.Clonal sequencing:After purifying of the PCR products with deletion and insertion mutations,then recovered fragments were ligated and transfected into empty E.coli DNA.Screening positive recombinants,preparing plasmids and sequencing,then planked and selected the monoclone.The monoclones were amplified and sent to sequence.Result:1.Clinical data:All three patients had typical manifestations and radiological evidence of rachitic disease,two of three patients had hypocalcemia,higher levels of serum.PTH and ALP.Serum calcemia,PTH and ALP of the other family members were normal.2.Results of sequencing:Four mutations in CYP27B1 were found in these three families,one missense mutation and three deletion or insertion with frame shift. Three mutations are novel.In patient 1 there are a substitution of T for G at p57 in Exon 1(c171G＞V),making Gly change to Val(G57V) and a insertion of 7bp(CCCACCC) at p442 in Exon8(c1325-1332insCCCACCC),causing frame shift and truncated at p466.In patient 2,two mutations are same with those of patient 1.In patient 3 we found two new mutations,one G deletion at p437(437△G) in Exon 8,making frame shift and stoping the translation at 460 that reduced 36 amino acids and one A deletion at p481(481△A) in Exon 9,making frame shift and stoping translation at 539 not at the normal site 509.3.Results of the frame shift mutations:PCR products of Exons which contain frame shift mutations were electrophoresis by PAGE gel.Predicating bands were found in CYP27B1 from 3 probands or their family members.These result supports the findings of mutations in sequence mutation analysis.The results showed the PCR products of all patients and the members of father or mother family had abnormal fragment.The result of PAGE electrophoresis accord with sequencing result.4.Results of clonal sequencing:We found one 7bp insertion in the Exon 8 of patient 1 and 2(c1325-1332insCCCACCC),two deletions in Exon 8 and Exon 9 of respectively patient 3(c1310del and c1442de1A). Conclusion:1.This is the first study in China to investigate the mutations of CYP27B1 in patients of PDDR.Four Mutations in three Chinese Han Families were found.2.Three novel mutations are not reported,one is missense mutation (G57V) in Exon 1,the other two is deletions in Exon 8(437△G)and Exon 9(481△A).3.We confirm again that 7bp insertion in Exon 8 has not obviously ethnic group homology distribution. PartⅡThe effects of SIP1 on the osteoblast specific transcription factor Cbfa1 gene expression and its role in BMP2 pathwayObjectiveOsteoblasts are derived from mesenchymal stem cells and its major effects are to synthesize bone matrix and to regulate bone resorption.They play a specific role in bone formation,modeling and remodeling.Many hormones and cytokines influence the differentiation process from preosteoblasts to osteoblasts,including calcitriol, parathyroid hormone(PTH),calcitionin,gonadal hormone,growth hormone etc. Bone morphogenetic proteins(BMPs) are considered as an most important local regulation factor in the process of osteoblasts differentiation and bone formation. Smad1,Smad5 and Smad8 proteins are phosphorylated by BMPs signaling pathway,after that these phosphorylated proteins enter nuclear to adjust transcription factors including Core binding factorα1(Cbfa1).Cbfa1(Core binding factorα1) is an osteoblast-specific transcription factor essential to develop a mature osteoblast phenotype.It was shown to regulate expression of all major gene expressed by osteoblasts.It is a key factor not only in BMPs signal pathway but also in other osteoblasts regulation pathway.Cbfa1 may be a molecular switch of many signaling pathway for osteoblast differentiation. Smad-interacting Proteins(SIP1) can bind with smads proteins that many be involved in signal transduction in BMPs pathway.It is thus likely that SIP1 might act as a regulator osteoblasts differentiation.MC3T3-E1 and C2C12 cells are preosteoblasts and premyoblasts,both of them possess a potention to differentiate to mature osteoblast when stimulated by BMP2.In this study we investigated whether SIP1 could be a candidate regulator of Cbfa1 gene expression,and the effects SIP1 on osteoblasts differentiation.The role of SIP1 in BMPs signaling pathway were also studied.Materials and Methods1.The effects of SIP1 on Cbfa1 promoter activity of preosteoblasts and premyoblastsA luciferase reporter plasmid p696-luc was contructed by our laboratory previously, which contaning mouse Cbfa1 promoter(-641bp—+55bp).Dual-Luciferase Reporter Assay System was used to detect the report genes.PcDNA3.0—SIP1 plasmid is a SIP1 expression plasmid that cloned mouse SIP1 cDNA to pcDNA3.0 vector in EcoRI/XbaI site.PGL3-Basic plasmid were used as control and PRL SV40 as endogenuous reference.MC3T3-E1 and C2C12 cells were transiently transfected with P696-Luc.Twenty-four hours after transfection,cells were treated with different concentrations of BMP2,and cultured for an additional six hours.Then the luciferase activations of Cbfa1 promoter expression were detected by luciferase assays.After P696-Luc,pcDNA3.0-SIP1,pGL3-Basic and PRL SV40 were transiently cotransfected into MC3T3-E1 and C2C12 cells for twenty-four hours, cells were treated with BMP2,and cultured for an additional six hours,the luciferase activation,were evaluated.2.The effect of SIP1 on BMP2 signaling pathway of preosteoblasts and premyoblastsMC3T3-E1 and C2C12 cells were transiently transfected with pcDNA3.0-SIP1.24 hours after transfection,cells were treated with BMP2,and cultured for an additional 6 hours.Then total RNA were isolated from MC3T3-E1 and C2C12 cells. Cbfa1,Smad1 and SIP1 gene mRNA level were detected by RT-PCR.Cbfa1 signals were normalized to the GAPDH signal. 3.The effect of SIP1 on ALP expression of preosteoblastsMC3T3-E1 and C2C12 cells were transiently transfected with P696-Luc.Thirty hours after transfection,cells grew almost 80-90%and then were dyed by ALP staining kit.The differences of ALP in cells kytoplasm were observed using inverted microscope.Results1.The effect of SIP1 on Cbfa1 promoter activity of preosteoblasts and premyoblastsC2C12 and MC3T3-E1 cells,After treated with BMP2(300ng/ml) for 6 hour in serum free medium,Cbfa1 promoter activity were increased 1.45(P＜0.01) and 1.45(P＜0.01) times in C2C12 and MC3T3-E1 cells respectively.When transiently transfected with pcDNA3.0-SIP1 in C2C12 cells,Cbfa1 promoter activity were also increased(1.59±0.04,p＜0.01) and it was shown a synergy effects on enhancing Cbfa1 promoter activity between BMP2 and SIP1(3.21±0.22,p＜0.01).Similar results were observed in MC3T3-E1 cells.The Cbfa1 promoter activity improved after transiently transfected with pcDNA3.0-SIP1(1.77±0.13,p＜0.01) and this effect could be augmented by BMP2(2.85±0.49,p＜0.05).So our results showed that both BMP2 and SIP1 could enhance Cbfa1 promoter activity,SIP1 and BMP2 have synergy effects on Cbfa1 promoter activity in C2C12 and MC3T3-E1 cells2.The effect of SIP1 on BMP2 signaling pathway of preosteoblasts and premyoblastsAfter transiently transfected with pcDNA3.0-SIP1 or control plasmids and treated with BMP2,total RNA were isolated from MC3T3-E1 and C2C12 cells and mRNA level were detected by RT-PCR method.In C2C12 cells,Cbfa1,Smadland SIP1 expression were increased after BMP2 treatment(1.40±0.21,p＜0.05;1.49±0.36, p＜0.05 and 2.36±1.10,p＜0.05 respectively) and SIP1 also augment Cbfa1,Smad1and SIP1(1.25±0.12 p＜0.05;1.49±0.61,p=0.10 and 2.56±1.06,p＜0.05 respectively) expression,but there was significance was found in Smad1. MC3T3-E1 cells treated with BMP2,Cbfa1 and Smad1 expression(1.52±0.20,p＜0.05;1.68±0.53,p＜0.05) were sigificantly,but there no markedly changes was found in SIP1 expression(1.12±0.33,p=0.51).The expression of Cbfa1 and SIP1(1.56±0.54,p＜0.05;1.53±0.49,p＜0.05) were promoted after transfected with pcDNA3.0-SIP1 plasmid in MC3T3-E1 cells.Though the Smad1 mRNA expressions (1.92±1.02,P=0.08) were also increased,there was no difference in statistics. These results indicated that BMP2 could enhance the expression of Cbfa1 and Smad1,but its effect on SIP1 was not clear.Overexpression of SIP1 could increase the expression of Cbfa1,but its effect on Smad1 was not determined.3.The effect of SIP1 on ALP expressionALP staining indicated that MC3T3-E1 cells treated with BMP2 showed positive granulas increased significantly.After transiently transfected with pcDNA3.0-SIP1 about thirty hours,ALP positive granulas also increased,it implicated that SIP1 also promote the ALP expression in osteoblasts.Conclusion:1.Cbfa1 promoter activity and Cbfa1 gene expression were up-regulated by SIP1, synergy effects of improving Cbfa1 expression were found between BMP2 and SIP1.2.SIP1 can increase ALP expression in preosteobalsts and bone formation,so it may promote the differentiation of preosteoblasts to osteoblasts.3.BMP2 effects could be augmented by SIP1,but this contribution might be taken place in the downstream of Smad1 pathway signaling.