Gao L, Qiu W, Wang Y, Xu W, Xu J, Tong J
Department of Immunology, Nanjing Medical University, 210019 Nanjing, China.
Clin Exp Immunol. 2006 May;144(2):326-34. doi: 10.1111/j.1365-2249.2006.03069.x.
Mesangial cell proliferation is a common cellular response to a variety of different types of glomerular injury. Complement C5b-9 is a prime candidate to mediate mesangial cell proliferation, especially sublytic C5b-9, which can induce the production of multiple inflammatory factors and cytokines. Transforming growth factor (TGF)-beta1 plays a major role in the accumulation of extracellular matrix (ECM), while thrombospondin (TSP)-1 has been identified as an activator of latent TGF-beta1 in an in vitro system. Using rat glomerular mesangial cells (GMCs) as a model system, we assessed the effect of sublytic C5b-9 on the expression of TSP-1 and TGF-beta1 and explored the relevant pathway of signal transduction. First, we ensured the concentrations of anti-Thy1 antibody and complement, which were regarded as a sublytic C5b-9 dose, and examined whether the sublytic C5b-9 induced expression of TSP-1 in rat GMCs which, in turn, activated latent TGF-beta1 by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, we investigated the role of the PI3-k/Akt pathway in sublytic C5b-9-induced TSP-1 production in rat GMCs by Western blot analysis. The addition of sublytic C5b-9 (5% anti-Thy1 antibody and 4% normal serum) to rat GMCs induced activation of latent TGF-beta1 via TSP-1. The addition of sublytic C5b-9 apparently increased the protein of Akt phosphorylation, whereas PI3-k inhibitor LY294002 could clearly reduce the increase of TSP-1 induced by sublytic C5b-9. These results indicate that TSP-1 is an activator of latent TGF-beta1 in sublytic C5b-9-induced rat GMCs; furthermore, the PI3-k/Akt signal transduction pathway may play a key role in sublytic C5b-9-induced TSP-1 production.
系膜细胞增殖是对多种不同类型肾小球损伤的常见细胞反应。补体C5b - 9是介导系膜细胞增殖的主要候选物质，尤其是亚溶解型C5b - 9，它可诱导多种炎症因子和细胞因子的产生。转化生长因子（TGF）-β1在细胞外基质（ECM）积累中起主要作用，而血小板反应蛋白（TSP）-1在体外系统中已被确定为潜伏性TGF -β1的激活剂。我们以大鼠肾小球系膜细胞（GMCs）作为模型系统，评估亚溶解型C5b - 9对TSP -1和TGF -β1表达的影响，并探索相关信号转导途径。首先，我们确定了抗Thy1抗体和补体的浓度，将其视为亚溶解型C5b - 9剂量，并分别通过实时聚合酶链反应（PCR）和酶联免疫吸附测定（ELISA）检测亚溶解型C5b - 9是否诱导大鼠GMCs中TSP -1的表达，进而激活潜伏性TGF -β1。然后，我们通过蛋白质印迹分析研究PI3 - k/Akt途径在亚溶解型C5b - 9诱导大鼠GMCs产生TSP -1中的作用。向大鼠GMCs中添加亚溶解型C5b - 9（5%抗Thy1抗体和4%正常血清）可通过TSP -1激活潜伏性TGF -β1。添加亚溶解型C5b - 9明显增加了Akt磷酸化蛋白，而PI3 - k抑制剂LY294002可明显降低亚溶解型C5b - 9诱导的TSP -1增加。这些结果表明，TSP -1是亚溶解型C5b - 9诱导的大鼠GMCs中潜伏性TGF -β1的激活剂；此外，PI3 - k/Akt信号转导途径可能在亚溶解型C5b - 9诱导的TSP -1产生中起关键作用。
