Functional Study Of PhosSNPs Associated With Osteoporosis And Rheumatoid Arthritis

Osteoporosis(OP)is a disease characterized by low bone mass and microstructural degradation of bone tissue.OP is clinically diagnosed by bone mineral density(BMD)measurement.The pathological mechanism of OP remains unclear.Currently,genome-wide association studies(GWAS)have identified a number of single nucleotide polymorphisms(SNPs)associated with BMD/OP.Protein phosphorylation modification is indispensable in protein modification and regulates a wide variety of cellular processes.In view of this,we designed the study to explore the mechanism underlying the association between phosphorylation-related SNPs(phosSNPs)and human BMD.In the first part,based on the existing GWAS results,phosSNPs with significant effects on human BMD were selected,and functions of phosSNPs were predicted by bioinformatics analyses.In the second part,we constructed and cultured stably transfected bone cells(osteoblasts and osteoclasts)that contained target phosSNP.In the third part,we tested effects of target phosSNP on protein phosphorylation and osteoblast functions in vitro.In the fourth part,we tested effects of target phosSNP on protein phosphorylation and osteoclast functions in vitro.ObjectiveSNP rs3755955(major/minor allele:G/A)was reported to be significant for the lumbar spine and femoral neck BMD in humans(P=5.2×10-15,Beta=-0.05,N=77508;P=1.5×10-14,Beta=-0.05,N=83894).This follow-up study was to uncover the underlying association mechanism through molecular and cellular functional assays relevant to bone.MethodsWe searched public resources(dbGap,NHGRI)in Phenotype-Genotype Integrator for phosSNPs significant for OP.The SNP rs3755955 located at gene IDUA,which have been identified as important for the lumbar spine and femoral neck BMD in general human populations,was determined for follow-up molecular and cellular functional analyses.We predicted the the impacts of phosSNP on protein phosphorylation.Using pCDH-CMV-MCS-EF1-copGFP(PCDH)plasmids as vector(control),we constructed the wild-type IDUA(allele G at rs3755955,R105),variant IDUA(allele A at rs3755955,Q105)recombinant plasmids.We transfected human fetal osteoblast cell(hFOB)and mouse mononuclear-macrophage cell(RAW264.7)with the three plasmids,respectively.We tested the effects of phosSNP rs3755955 on protein phosphorylation at total protein level and IDUA protein level in both hFOB and RAW264.7 cells.We tested the effects of phosSNP rs3755955 on osteoblastic maker gene expression,cell proliferation,cell apoptosis and ALP enzyme activity in hFOB cells.We also assayed and tested the effects of phosSNP rs3755955 on trans-endothelial migration,osteoclastic maker gene expression,cell growth,cell apoptosis and TRAP enzyme activity in RAW264.7 cells.ResultsThe stable transfection/over expression of recombinant IDUA gene in osteoblasts(p<0.05)and osteoclasts(p<0.05)were confirmed by fluorescence microscopy and RT-PCR.In both hFOB and RAW264.7 cells,the total protein and IDUA protein phosphorylation levels were significantly reduced by variant IDUA transfection,as compared with that of wild IDUA transfection(p<0.05).In hFOB cells,transfection with variant IDUA-Q105 significantly decreased osteoblastic gene expression(OPN,COL1A1 and RANKL)in hFOB cells(p<0.01),,inhibited cell proliferation(p<0.05),promoted cell apoptosis(p<0.001),and decreased ALP enzyme activity,as compared with that of wild IDUA transfection..In RAW264.7 cells,transfection with variant IDUA significantly promoted cell migration(p<0.0001),increased osteoclastic gene expression(TRAP,RANK,Integrin av and Cathepsine K)(p<0.05),inhibited cell apoptosis(p<0.01),promoted cell growth and increased TRAP enzyme activity(p<0.001),as compared with that of wild IDUA transfection.ConclusionsVariant allele of phosSNP rs3755955 reduces protein phosphorylation,inhibits osteoblast function and promotes osteoclastic activity.The newly identified functions of rs3755955 in bone cells are consistent with and explain the prior findings in general human populations,i.e.,the variant allele is associated with decreased BMD.ObjectivePeripheral blood mononuclear cells(PBMCs)are critical for immunity and participated in multiple human diseases,including rheumatoid arthritis(RA).PhosSNPs are non-synonymous SNPs influencing protein phosphorylation,thus probably modulate cell signaling and gene expression.We aimed to identify phosSNPs-regulated gene network/pathway potentially significant for RA.MethodsWe collected genome-wide phosSNP genotyping data and transcriptome-wide mRNA expression data from PBMCs of a Chinese sample.We discovered and verified with public datasets differentially expressed genes(DEGs)associated with RA,and replicated RA-associated SNPs in our study sample.We performed a targeted expression quantitative trait locus(eQTL)study on significant phosSNPs and DEGs.ResultsWe identified 29 nominally significant eQTL phosSNPs and 85 target genes,and constructed comprehensive regulatory/interaction networks,highlighting the vital effects of 2 eQTL phosSNPs(rs371513 and rs4824675,FDR<0.05)and four critical node genes(HSPA4,NDUFA2,MRPL15,and ATP50).Besides,two node/key genes NDUFA2 and ATP50,regulated by rs371513,were significantly enriched in mitochondrial oxidative phosphorylation pathway.Besides,four pairs of eQTL effects were replicated independently in whole blood and/or transformed fibroblasts.ConclusionsThe findings delineated a potential role of protein phosphorylation and genetic variations in RA and warranted the significant roles of phosSNPs in regulating RA-associated genes expression in PBMCs.The results pointed out the relevance and significance of oxidative phosphorylation pathway to RA.