Department of Oral Biology, University at Buffalo, State University of New York, Buffalo, United States.
Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, United States.
Elife. 2022 Feb 9;11:e71403. doi: 10.7554/eLife.71403.
RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice () by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, mice phenocopied mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to mice.
RAGE 是一种可成药的炎症受体，已知其作为寡聚体发挥功能，但确切的寡聚化机制仍知之甚少。我们之前已经表明，硫酸乙酰肝素 (HS) 在 RAGE 寡聚化中发挥积极作用。为了了解 HS 诱导的 RAGE 寡聚化在体内的生理意义，我们通过引入点突变生成了 RAGE 基因敲入小鼠 ()，以特异性破坏 HS-RAGE 相互作用。RAGE 突变体表现出正常的配体结合，但 HS 结合和寡聚化能力受损。值得注意的是， 小鼠在两种不同的病理生理过程中模拟了 小鼠的表型，即骨重塑和中性粒细胞介导的肝损伤，这表明 HS 诱导的 RAGE 寡聚化对于 RAGE 信号传导至关重要。我们的发现表明，通过抑制 HS-RAGE 相互作用阻断 RAGE 信号传导是可能的。为了验证这一点，我们生成了一种针对 RAGE 的 HS 结合位点的单克隆抗体。该抗体在体外和体内阻断了 RAGE 信号传导，重现了 小鼠的表型。通过基因和药理学抑制 HS-RAGE 相互作用，我们的工作验证了拮抗 RAGE 的另一种策略。最后，我们对中性粒细胞和肺进行了 RNA-seq 分析，发现与野生型小鼠相比， 小鼠在这两种组织中的转录组都发生了广泛改变，而 小鼠的转录组变化则更为局限。这一意外发现表明，通过保留 RAGE 蛋白的表达（以显性负形式）， 小鼠可能代表了一种更清洁的遗传模型，用于研究 RAGE 在体内的生理作用，与 小鼠相比。
