Lu Min, Munger John S, Steadele Melissa, Busald Christina, Tellier Marinka, Schnapp Lynn M
Pulmonary and Critical Care Medicine, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
J Cell Sci. 2002 Dec 1;115(Pt 23):4641-8. doi: 10.1242/jcs.00145.
The development of fibrosis is a common response to a variety of injuries and results in the net accumulation of matrix proteins and impairment of normal organ function. We previously reported that the integrin alpha8beta1 is expressed by alveolar interstitial cells in normal lung and is upregulated during the development of fibrosis. TGFbeta1 is an important mediator of the inflammatory response in pulmonary fibrosis. TGFbeta1 is secreted as a latent protein that is non-covalently associated with latency-associated peptide (LAP) and requires activation to exert its effects. LAP-TGFbeta1 and LAP-TGFbeta3 contain the tripeptide sequence, arginine-glycine-aspartic acid (RGD), a known integrin recognition motif. The integrin alpha8beta1 binds to several ligands such as fibronectin and vitronectin through the RGD sequence. Recent reports demonstrate that the integrins alphavbeta1, alphavbeta6 and alphavbeta8 adhere to LAP-TGFbeta1 through the RGD site. Therefore, we asked whether LAP-TGFbeta1 might be a ligand for alpha8beta1 and whether this may be important in the development of fibrosis. We found that cell lines transfected with alpha8 subunit were able to spread on and adhere to recombinant LAP-TGFbeta1 significantly better than mock transfected cell lines. alpha8-transfected cells were also able to adhere to LAP-TGFbeta3 significantly better than mock transfected cells. Adhesion to LAP-TGFbeta1 was enhanced by activation of alpha8beta1 by Mn(2+), or 8A2, an integrin beta1 activating antibody. Furthermore, cell adhesion was abolished when we used a recombinant LAP-TGFbeta1 protein in which the RGD site was mutated to RGE. alpha8beta1 binding to LAP-TGFbeta1 increased cell proliferation and phosphorylation of FAK and ERK, but did not activate of TGFbeta1. These data strongly suggest that LAP-TGFbeta1 is a ligand of alpha8beta1 and interaction of alpha8beta1 with LAP-TGFbeta1 may influence cell behavior.
纤维化的发展是对多种损伤的常见反应，会导致基质蛋白的净积累和正常器官功能的受损。我们之前报道过，整合素α8β1在正常肺组织的肺泡间质细胞中表达，并在纤维化发展过程中上调。转化生长因子β1（TGFβ1）是肺纤维化炎症反应的重要介质。TGFβ1以一种潜伏蛋白的形式分泌，该潜伏蛋白与潜伏相关肽（LAP）非共价结合，需要激活才能发挥其作用。LAP - TGFβ1和LAP - TGFβ3含有三肽序列精氨酸 - 甘氨酸 - 天冬氨酸（RGD），这是一个已知的整合素识别基序。整合素α8β1通过RGD序列与几种配体结合，如纤连蛋白和玻连蛋白。最近的报道表明，整合素αvβ1、αvβ6和αvβ8通过RGD位点与LAP - TGFβ1结合。因此，我们探究LAP - TGFβ1是否可能是α8β1的配体，以及这在纤维化发展中是否重要。我们发现，转染了α8亚基的细胞系比 mock 转染的细胞系能更好地在重组LAP - TGFβ1上铺展并黏附。α8转染的细胞也比 mock 转染的细胞能更好地黏附于LAP - TGFβ3。通过用Mn(2+)或整合素β1激活抗体8A2激活α8β1可增强对LAP - TGFβ1的黏附。此外，当我们使用RGD位点突变为RGE的重组LAP - TGFβ1蛋白时，细胞黏附被消除。α8β1与LAP - TGFβ1的结合增加了细胞增殖以及黏着斑激酶（FAK）和细胞外信号调节激酶（ERK）的磷酸化，但未激活TGFβ1。这些数据强烈表明LAP - TGFβ1是α8β1的配体，且α8β1与LAP - TGFβ1的相互作用可能影响细胞行为。
