Dugan Jae, Griffiths Eric, Snow Paige, Rosenzweig Holly, Lee Ellen, Brown Brieanna, Carr Daniel W, Rose Carlos, Rosenbaum James, Davey Michael P
Portland Veterans Affairs Medical Center, Portland, OR 97239; Department of Medicine, Oregon Health and Sciences University, Portland, OR 97239;
Portland Veterans Affairs Medical Center, Portland, OR 97239;
J Immunol. 2015 Jan 1;194(1):349-57. doi: 10.4049/jimmunol.1402330. Epub 2014 Nov 26.
The biochemical mechanism by which mutations in nucleotide-binding oligomerization domain containing 2 (NOD2) cause Blau syndrome is unknown. Several studies have examined the effect of mutations associated with Blau syndrome in vitro, but none has looked at the implication of the mutations in vivo. To test the hypothesis that mutated NOD2 causes alterations in signaling pathways downstream of NOD2, we created a Nod2 knock-in mouse carrying the most common mutation seen in Blau syndrome, R314Q (corresponding to R334Q in humans). The endogenous regulatory elements of mouse Nod2 were unaltered. R314Q mice showed reduced cytokine production in response to i.p. and intravitreal muramyl dipeptide (MDP). Macrophages from R314Q mice showed reduced NF-κB and IL-6 responses, blunted phosphorylation of MAPKs, and deficient ubiquitination of receptor-interacting protein 2 in response to MDP. R314Q mice expressed a truncated 80-kDa form of NOD2 that was most likely generated by a posttranslational event because there was no evidence for a stop codon or alternative splicing event. Human macrophages from two patients with Blau syndrome also showed a reduction of both cytokine production and phosphorylation of p38 in response to MDP, indicating that both R314Q mice and cells from patients with Blau syndrome show reduced responses to MDP. These data indicate that the R314Q mutation when studied with the Nod2 endogenous regulatory elements left intact is associated with marked structural and biochemical changes that are significantly different from those observed from studies of the mutation using overexpression, transient transfection systems.
核苷酸结合寡聚化结构域包含2（NOD2）中的突变导致布劳综合征的生化机制尚不清楚。几项研究已经在体外检测了与布劳综合征相关的突变的影响，但没有一项研究关注这些突变在体内的意义。为了验证突变的NOD2会导致NOD2下游信号通路改变这一假设，我们创建了一种携带布劳综合征中最常见突变R314Q（相当于人类中的R334Q）的Nod2基因敲入小鼠。小鼠Nod2的内源性调控元件未改变。R314Q小鼠对腹腔注射和玻璃体内注射胞壁酰二肽（MDP）的细胞因子产生反应降低。来自R314Q小鼠的巨噬细胞对MDP的反应显示NF-κB和IL-6反应降低、MAPK磷酸化减弱以及受体相互作用蛋白2的泛素化缺陷。R314Q小鼠表达一种截短的80 kDa形式的NOD2，这很可能是由翻译后事件产生的，因为没有证据表明存在终止密码子或可变剪接事件。两名布劳综合征患者的人类巨噬细胞对MDP的反应也显示细胞因子产生和p38磷酸化均降低，这表明R314Q小鼠和布劳综合征患者的细胞对MDP的反应均降低。这些数据表明，在Nod2内源性调控元件保持完整的情况下研究R314Q突变，其与明显的结构和生化变化相关，这些变化与使用过表达、瞬时转染系统研究该突变所观察到的变化显著不同。
