Bayless K J, Meininger G A, Scholtz J M, Davis G E
Microcirculation Research Institute, Texas A & M University Health Science Center, Texas A & M University, College Station, TX 77843-1114, USA.
J Cell Sci. 1998 May;111 ( Pt 9):1165-74. doi: 10.1242/jcs.111.9.1165.
Recent work has shown that osteopontin expression is upregulated at sites of cardiovascular injury. It has been hypothesized that osteopontin provides an adhesive matrix for endothelial and smooth muscle cells during remodeling of the vascular wall following injury. Osteopontin has also been found to be synthesized by monocytes and macrophages within injury sites. Here, we present data showing that osteopontin can promote leukocyte adhesion through the alpha4beta1 integrin. In the presence of physiologic concentrations of Mg2+ and Ca2+, osteopontin purified from bovine milk promoted cell-substrate adhesion of HL-60 and Ramos cells, two model leukocyte cell lines. As with other adhesive ligands, adhesion to osteopontin required leukocyte activation. Under these conditions, no adhesion to control substrates such as bovine serum albumin was observed. Leukocyte adhesion was inhibited by anti-integrin antibodies directed at either the alpha4 or beta1 integrin subunits but not by control antibodies directed to other integrins. Further adhesion experiments revealed that leukocyte binding to osteopontin was completely inhibited by an alpha4beta1-binding peptide containing the leucine-aspartate-valine (LDV) sequence, while a control, non-binding peptide containing leucine-glutamate-valine (LEV) had minimal effects. Affinity chromatography using either surface labeled HL-60 or Ramos cell extracts revealed that the alpha4beta1 integrin specifically bound to osteopontin. Immunoprecipitation of eluted fractions from these columns positively identified the alpha4beta1 integrin. In order to localize potential alpha4beta1-binding sites within osteopontin, the protein was proteolytically cleaved with thrombin. A 30 kDa N-terminal osteopontin fragment purified using fast protein liquid chromatography promoted alpha4beta1 dependent leukocyte adhesion in a manner similar to that of the intact protein. These data collectively demonstrate that the alpha4beta1 integrin is a new adhesion receptor for osteopontin and that an alpha4beta1 binding site exists in the NH2-terminal thrombin fragment of osteopontin.
近期研究表明，骨桥蛋白在心血管损伤部位的表达上调。据推测，在血管壁损伤后的重塑过程中，骨桥蛋白为内皮细胞和平滑肌细胞提供了一个黏附基质。还发现骨桥蛋白由损伤部位的单核细胞和巨噬细胞合成。在此，我们提供的数据表明，骨桥蛋白可通过α4β1整合素促进白细胞黏附。在生理浓度的Mg2+和Ca2+存在下，从牛乳中纯化的骨桥蛋白促进了HL-60和Ramos细胞（两种白细胞模型细胞系）与细胞基质的黏附。与其他黏附配体一样，白细胞与骨桥蛋白的黏附需要白细胞激活。在这些条件下，未观察到细胞与牛血清白蛋白等对照底物的黏附。针对α4或β1整合素亚基的抗整合素抗体可抑制白细胞黏附，但针对其他整合素的对照抗体则无此作用。进一步的黏附实验表明，含有亮氨酸-天冬氨酸-缬氨酸（LDV）序列的α4β1结合肽可完全抑制白细胞与骨桥蛋白的结合，而含有亮氨酸-谷氨酸-缬氨酸（LEV）的对照非结合肽的作用则微乎其微。使用表面标记的HL-60或Ramos细胞提取物进行亲和层析显示，α4β1整合素与骨桥蛋白特异性结合。对这些柱洗脱组分进行免疫沉淀，阳性鉴定出α4β1整合素。为了定位骨桥蛋白内潜在的α4β1结合位点，用凝血酶对该蛋白进行蛋白水解切割。使用快速蛋白质液相色谱法纯化的30 kDa N端骨桥蛋白片段以类似于完整蛋白的方式促进了α4β1依赖性白细胞黏附。这些数据共同表明，α4β1整合素是骨桥蛋白的一种新的黏附受体，且骨桥蛋白的NH2端凝血酶片段中存在α4β1结合位点。
