An integrated analyses on the implication of structural and promoter polymorphism of osteopontin modification

本研究利用（MRL/lpr × C3 H/lpr）F_2小鼠进行了全基因组筛选，并对MRL和C3 H型OPN多肽进行了功能分析，确定了MRL/lpr小鼠中OPN为肾小球肾炎易感基因。我们还清楚地表明，与C3 H等位基因相比，MRL-OPN诱导脾细胞和/或巨噬细胞中免疫球蛋白以及细胞因子（包括TNF-α、IL-1 β和IFN-γ）的更高表达和产生。这些结果表明，OPN的等位基因多态性导致MRL和C3 H株之间抗体产生和巨噬细胞活化的功能差异，可能参与狼疮肾炎的发展。另一方面，据报道，在体外，自身免疫易感小鼠中的Opn表达水平高于疾病抵抗小鼠。以确定是否定性为了确定与自身免疫性疾病的发展有关的Opn的（功能）差异或数量差异，我们进行了如下分析：1）我们产生了突变Opn肽，其在两个等位基因之间的取代氨基酸中被修饰，然后进行细胞结合亲和力的功能分析。2）为了确定体内Opn表达的等位基因差异，我们选择了在MRL背景中具有C3 H等位基因Opn的Opn基因位点的重组同源小鼠，3）然后，进行了重组同类小鼠的精确分析。1）通过等位基因多态性修饰Opn的细胞结合亲和力的氨基酸取代。2）Opn的表达水平受Opn等位基因的控制。3）肾小球肾炎的发病率和严重程度以及个体的存活率可能与Th 1/Th 2细胞因子特性的改变有关。Th 2平衡。这些结果表明，在MRL/lpr小鼠中，小鼠Opn等位基因多态性以及启动子多态性中的氨基酸取代可能在肾小球肾炎的发展中起关键作用。

We previously identified Opn as a candidate gene susceptible to glomerulonephritis in MRL/lpr mice by genome wide screening using (MRL/lpr x C3H/lpr)F_2 mice, followed by a functional assay of synthetic polymorphic OPN peptides of MRL and C3H type. Also we clearly indicated that the MRL-OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF-alpha, IL-lbeta and IFN-gamma in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis. On the other hand, Opn expression level in autoimmune prone mice were reported higher than that in disease resistant mice, in vitro. To determine whether qualitative (functional) difference or quantitative difference of Opn implicated in the development of autoimmune disease, we carried out the analyses as follows: 1) we generated mutant Opn peptides which were modified in substituted amino acid between two alleles followed by functional analyses of cell binding affinity 2) To determine the allelic difference of Opn expression in vivo, we made selective recombinant congenic mice of Opn gene locus which have C3H allele Opn in MRL background, 3) and then, carried out precise analyses of recombinant congenic mice.1) Amino acid substitution by allelic polymorphism modified cell binding affinity of Opn.2) The expression level of Opn was manifested to be controlled by Opn allele.3) Incidence and severity of glomerulonephritis as well as livability of individuals probably via modification of cytokine properties which includes Th1/Th2 balance.These results suggest that an amino acid substitution in allelic polymorphism of murine Opn as well as that in promoter polymorphism may play a critical role for the development of glomerulonephritis in MRL/lpr mice.