The Effect Of Genetic Variation Of Purinergic P2X7 Receptor On The Susceptibility To Essential Osteoporosis And The Mechanism Involved

Background and Objective:Osteoporosis(OP)is a systemic skeletal disease characterized by low bone mass and structural deterioration of bone tissue,which leads to bone fragility and an increased susceptibility to fractures.P2X7 receptor(P2X7)is ATP-gated cation channel with a wide range of biological effects.It was shown to be expressed in a variety of cells,including osteoblast and osteoclast,and play a significant role in both bone formation and bone loss.Genetic factors are thought to be an important predictor of osteoporosis.Recently,the role of functional single nucleotide polymorphisms(SNPs)in P2X7 gene has attracted great interest concerning on its relation with the risk of osteoporosis.Studies have suggested that P2X7 gene polymorphism were associated with the susceptibility to OP in Caucasian.Considering that the type and frequency of P2X7 genetic polymorphisms may be different in various races,it is necessary to carry out the association analysis of P2X7 genetic polymorphisms with osteoporosis risk in Chinese.Thus,the aim of this study was conducted to search for potential susceptible genes and susceptible SNP loci to OP by our genetic epidemiological investigation in Chinese and identify the association of the P2X7 SNP with OP.Our results will contribute to clarify the genetic mechanisms P2X7 functional SNP underlying OP and provide new drug targets for personalized prevention and treatment of OP.Methods:1、This case-control study was carried out in a population of postmenopausal There were 400 OP patients and 400 controls.Basic material and blood of the sample were collected.P2X7 genetic polymorphisms,including Rs3751143(Glu496Ala),Rs2230911(Thr357Ser),Rs1718119(Ala348Thr),Rs2393799(-762 T<C)and Rs7958311(Arg270His),were detected by polymerase chain reaction restriction-fragment length polymorphism(PCR-RFLP)and allele specific PCR.2、Human P2X7-pEGFP-C1 plasmid was constructed after an association of Rs3751143(A<C)which was previously shown as a SNP leading to loss of P2X7 function with OP risk detected in our case-control study.Bone fragments of OP patients with hip arthroplasty were used for the isolation of primary human osteoblasts according to their genotype of P2X7 Rs3751143(AA and CC).Then the human osteoblasts were divided into AA group,CC group,CC+P2X7 plasmid group and CC+Blank group.1)The mRNA expressions of P2X7,alkaline phosphatase(ALP),osteocalcin(OCN)and collagen I were detected in the osteoblast by using RT-PCR method;2)The protein expression of P2X7 in the osteoblast was assessed via immunocytochemical method;3)ATP-induced Ca2+ signal was also in osteoblast;4)The level of ALP in supernatant of osteoblast was measured.Peripheral blood mononuclear cells were isolated from OP patients according to their genotype of P2X7 Rs3751143(AA and CC),and were induced into osteoclast in vitro by applying osteoclast inducing factors(M-CSF and RANKL).Osteoclasts were divided into AA group,CC group,CC+P2X7 plasmid group and CC+Blank group.1)The mRNA expressions of P2X7 were detected in the osteoclast by using RT-PCR method;2)ATP-induced Ca2+ signal was also in the osteoclast;3)The ability of ATP-induced P2X7 pore forming was detected in osteoclast;4)The levels of cysteine protease 1(caspase 1),lactate dehydrogenase(LDH)and tartrate resistant acid phosphatase(TRAP)in supernatant of osteoclast were measured.The levels of serum calcium,ALP and TRAP according to their P2X7 Rs3751143 genotype(AA,AC and CC)were measured in both OP patients and controls.Results:1.There were significant differences in genotype and allele frequency of P2X7 Rs3751143 polymorphism between OP group and control group(p<0.05).P2X7 Rs3751143-C allele was indicated to be the risk allele for OP;carriers with Rs3751143-C allele were associated with 1.70-fold higher odds of OP compared to carriers with Rs3751143-A allele in additive model(95%CI=1.00-2.87,p=0.040).2.No evidence of significant difference in genotype and allele frequency of P2X7 Rs2230911,Rs1718119,Rs2393799 or Rs7958311 between OP group and control group was found(p>0.05).3.7 common haplotypes(frequency>0.03)constructed by P2X7 Rs3751143-Rs2230911-Rs1718119,i.e.ACA,ACG,AGA,AGG,CCA,CCG and CGG,were found in this study population.Individuals carrying haplotype CGG were associated with increased risk to OP(OR=3.762,95%CI:1.21-11.68,p=0.014).4.Individuals carrying with P2X7 Rs3751143-CC homozygous were found to be associated with decreased osteoblast expressions of P2X7 mRNA(p<0.01),reduced osteoblast ATP-induced[Ca2+]i(p<0.01),decreased osteoblast mRNA expressions of ALP(p<0.05),OCN(p<0.05)and collagen I(p<0.01),and low ALP level in the supernatant of osteoblast(p<0.05).Over-expression of P2X7 plasmid could reverse the aforementioned osteoblast phenotype associated with P2X7 Rs3751143-CC,which alleviated the decreased osteoblast expressions of P2X7 mRNA(p<0.01),enhanced osteoblast ATP-induced[Ca2+]i(p<0.01),ameliorated the decreased osteoblast mRNA expressions of ALP(p<0.05),OCN(p<0.05)and collagen I(p<0.01),and low ALP level in the supernatant of osteoblast(p<0.05).5.Individuals carrying with P2X7 Rs3751143-CC homozygous were found to be associated with decreased osteoclast mRNA expressions of P2X7(p<0.01),reduced osteoclast ATP-induced[Ca2+]i(p<0.01),few osteoclast ATP-induced pore formations(p<0.01),low levels of caspase 1(p<0.05)and LDH(p<0.05)while high level of TRAP in the supernatant of osteoclast(p<0.01).Over-expression of P2X7 plasmid could reverse the aforementioned osteoclast phenotype associated with P2X7 Rs3751143-CC,which alleviated the decreased osteoclast mRNA expressions of P2X7(p<0.01),enhanced osteoclast ATP-induced[Ca2+]i(p<0.01)and ATP-induced pore formation(p<0.01),ameliorated the low levels of caspase 1(p<0.05)and LDH(p<0.05)while decreased the high level of TRAP in the supernatant of osteoclast(p<0.01).6.Individuals carrying with P2X7 Rs3751143-CC homozygous were found to be associated with decreased serum concentrations of Ca2+(p<0.05)and ALP(p<0.05)while increased serum concentration of TRAP(p<0.01).Conclusion:1.This case-control study suggested that P2X7 Rs3751143 A<C variation was associated with increased susceptibility to OP in postmenopausal women,and Rs3751143C-Rs2230911 G-Rs 1718119G P2X7 haplotype was associated with increased susceptibility to OP.2.Our functional experiments in human osteoblasts and osteoclasts showed that P2X7 Rs3751143 A<C reduced P2X7 gene expression in both osteoblasts and osteoclasts,hampered the function of P2X7 channel in the osteoblasts and osteoclasts,suppressed the ability of pore forming in the osteoclasts,inhibited osteoblast expressions of ALP,OCN and collagen I,decreased osteoclast release of caspase 1 and LDH secretion,enhanced the activity of TRAP in the osteoclast.Then decreased serum concentrations of Ca2+ and ALP while increased serum concentration of TRAP were associated with the variation of Rs3751143 A<C.These experiments further confirmed that P2X7 Rs3751143 A<C polymorphism,screened to be associated with OP risk in our case-control study,was functional relevant with an increased susceptibility to OP.