Fujita Masaaki, Takada Yoko K, Izumiya Yoshihiro, Takada Yoshikazu
Departments of Dermatology, and Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento, California, United States of America.
PLoS One. 2014 Apr 2;9(4):e93738. doi: 10.1371/journal.pone.0093738. eCollection 2014.
The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvβ3 predicted that mCRP binds to αvβ3 well. We found that mCRP actually bound to integrins αvβ3 and α4β1 well. Antagonists to αvβ3 or α4β1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin β1 mutant (β1-3-1) that has a small fragment from the ligand binding site of β3, we showed that mCRP bound to the classical RGD-binding site in αvβ3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvβ3 and α4β1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvβ3 and α4β1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvβ3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.
典型的急性期反应物C反应蛋白（CRP）不仅是一种标志物，也是炎症性疾病的潜在促成因素。CRP以循环中的天然五聚体CRP（pCRP）和单体异构体（mCRP）的形式存在，mCRP是由pCRP解离过程形成的。mCRP具有高度促炎作用，而pCRP则没有。mCRP促炎作用的机制尚不清楚。我们研究了整合素在mCRP促炎作用中的作用。mCRP与整合素αvβ3相互作用的对接模拟预测mCRP与αvβ3结合良好。我们发现mCRP实际上与整合素αvβ3和α4β1结合良好。αvβ3或α4β1的拮抗剂有效抑制了这种相互作用，表明这种相互作用是特异性的。使用具有来自β3配体结合位点小片段的整合素β1突变体（β1-3-1），我们表明mCRP与αvβ3中的经典RGD结合位点结合。我们研究了整合素在单核细胞U937细胞中CRP信号传导中的作用。整合素αvβ3和α4β1特异性介导mCRP与U937细胞的结合。mCRP在U937细胞中诱导AKT磷酸化，但不诱导ERK1/2磷酸化。值得注意的是，mCRP在U937细胞中诱导强烈的趋化作用，αvβ3和α4β1的拮抗剂以及磷脂酰肌醇3激酶抑制剂，但不是MEK抑制剂，有效抑制了mCRP诱导的U937细胞趋化作用。这些结果表明，整合素和AKT/磷脂酰肌醇3激酶途径在U937细胞中mCRP的促炎作用中发挥作用。相比之下，由于模拟中的空间位阻，预测pCRP与αvβ3的结合有限。与预测一致，pCRP在整合素结合、趋化作用或AKT磷酸化方面的效果要差得多。这些发现表明CRP异构体与整合素结合的能力与其促炎作用有关。
