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

我们之前通过（MRL/lpr x C3H/lpr）F_2小鼠基因组筛选，确定了Opn是MRL/lpr小鼠肾小球肾炎的候选基因，随后进行了MRL和C3H型合成多态Opn肽的功能测定。我们还清楚地表明，MRL-OPN在脾细胞和/或巨噬细胞中诱导更高的免疫球蛋白和细胞因子的表达和产生，包括tnf - α、il - β和ifn - γ。这些发现提示，OPN等位基因多态性导致MRL和C3H株在抗体产生和巨噬细胞活化方面的功能差异，可能参与狼疮性肾炎的发生发展。另一方面，据报道，在体外，自身免疫易感性小鼠的Opn表达水平高于抗病小鼠。为了确定Opn的质量（功能）差异或数量差异是否与自身免疫性疾病的发展有关，我们进行了以下分析：1)通过对两个等位基因间取代氨基酸进行修饰，生成突变型的Opn多肽，并进行细胞结合亲和力的功能分析；2)为了确定Opn在体内表达的等位基因差异，我们在MRL背景下制备了具有C3H等位基因Opn的Opn基因位点的选择性重组基因小鼠；3)然后对重组基因小鼠进行了精确分析。1)通过等位基因多态性的氨基酸替代改变了Opn的细胞结合亲和力。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细胞因子特性的改变有关，包括Th1/Th2平衡。这些结果表明，小鼠Opn等位基因多态性的氨基酸替换以及启动子多态性的氨基酸替换可能在MRL/lpr小鼠肾小球肾炎的发生中起关键作用。

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.