MECHANISM OF THE ACTIVATION OF LATENT TGF-beta IN THE VASCULAR WALL

TGF-β是影响包括动脉粥样硬化在内的血管疾病进程的最重要的细胞因子之一。由于TGF-β总是以潜伏形式分泌，因此分泌后潜伏TGF-β的活化是表达其活性的关键步骤。本研究者先前已经发现，当内皮细胞（EC）和平滑肌细胞（SMC）接触时，潜伏的TGF-β被激活。这种激活需要在EC上表达的u-PA-纤溶酶活性和与SMC结合的潜在TGF-β。由于平滑肌细胞并不总是与血管壁中的内皮细胞接触，因此需要阐明同型平滑肌细胞中潜伏的TGF-β激活的机制。将外源性u-PA加入SMC培养物中，u-PA与受体结合并在细胞表面表达其活性。在这种条件下，潜伏的TGF-β被有效激活。u-PA对潜伏性TGF-β激活的这种作用被针对潜伏性相关肽（LTP）的单克隆抗体KM-704所消除，KM-704抑制潜伏性TGF-β与SMC的结合。这些结果表明，u-PA和潜伏的TGF-β的同时结合在同型SMC中产生活性TGF-β。进一步表征了外源TGF-β与SMC的结合特性。KM-704的抗原表位定位于大肠杆菌N端，由约80个氨基酸组成。在该区域中没有任何已知的蛋白质-蛋白质相互作用的序列。因此，潜伏的TGF-β通过一个新的结合位点与SMC结合。

TGF-beta is one of the most important cytokines affecting the course of vascular diseases including atherosclerosis. Since TGF-beta is always secreted in a latent from, the activation of latent TGF-beta after secretion is the crucial step expressing its activity. The present investigator has previously found that latent TGF-beta is activated when endothelial cells (ECs) and smooth muscle cells (SMCs) are in contact. This activation requires u-PA-plasmin activities expressed on ECs and latent TGF-beta bound to SMCs. Since SMCs are not always in contact with ECs in the vascular wall, a mechanism for the activation of latent TGF-beta in homotypic SMCs needs to be elucidated.The present study revealed that SMCs expressed urokinase-type PA (u-PA) receptor on the cell surface. When exogenous u-PA was added to SMC cultures, u-PA bound to the receptor and expressed its activity on the cell surface. Under this condition, latent TGF-beta was efficiently activated. This effect of u-PA on the activatipn of latent TGF-beta was abrogated by the monoclonal antibody against latency-assosiated peptide (LAP) ; KM-704, which inhibits the binding of latent TGF-beta to SMCs. These results indicate that the simultaneous bindings of u-PA and latent TGF-beta generates active TGF-beta in homotypic SMCs. The binding property of lanent TGF-beta to SMCs was further characterized. The epitope of KM-704 was found to be located on the N-terminal region of LAP consisted with about 80 amino acids. There is not any known sequences for protein-protein interaction in this region. Thus, latent TGF-beta binds to SMC via a novel binding site to LAP.