Roztocil Elisa, Nicholl Suzanne M, Davies Mark G
Vascular Biology and Therapeutics Program, Department of Surgery, University of Rochester, Rochester, New York 14642, USA.
J Surg Res. 2007 Jul;141(1):83-90. doi: 10.1016/j.jss.2007.03.069.
Urokinase plasminogen activator (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to uPA is dependent on both the growth factor binding domain at the aminoterminal end and the kringle (K) domain of the molecule. uPA is readily degraded in vivo into these constitutive domains. The aim of this study was to examine cell signaling during the migration of smooth muscle cell in response to the kringle domain of urokinase.
Murine arterial smooth muscle cells were cultured in vitro. Migration assays were performed in the presence of K with and without the plasmin inhibitors (aprotinin and -aminocaproic acid), the Galphai inhibitor Pertussis toxin, the MMP inhibitor (GM6001), the PI3-K inhibitors, Wortmannin and LY294002, and the MAPK inhibitors PD98089 (MEK1 inhibitor) and SB203580 (p38(MAPK) inhibitor). Western blotting was performed for ERK 1/2 and p38(MAPK) phosphorylation after stimulation with K in the presence and absence of the inhibitors. Statistics were analyzed by one-way ANOVA (n = 6).
The kringle domain (K) induced a plasmin-independent, MMP-dependent increase in cell migration (2-fold, P < 0.05) compared to control. This migratory response to K was Galphai mediated and dependent on both ERK 1/2 and p38(MAPK) activation. K induced time-dependent increases in the phosphorylation of ERK 1/2 (3-fold, P < 0.05) and p38(MAPK) (3-fold, P < 0.05). Activation of p38(MAPK) and ERK 1/2 was completely inhibited by the PI3-K inhibitors. We explored a potential role for the epidermal growth factor receptor (EGFR). K induced EGFR phosphorylation and the presence of AG1478, the EGFR inhibitor, inhibited both cell migration and akt activation in response to K.
Kringle domain of uPA induces smooth muscle cell migration through a G-protein-coupled PI3-K-dependent process involving both ERK 1/2 and p38(MAPK) and is mediated in part through EGFR. Defining the differences in response to key molecular domains of uPA is vital to understand its role in vessel remodeling.
尿激酶型纤溶酶原激活剂（uPA）参与血管重塑并介导平滑肌细胞迁移。对uPA的迁移反应依赖于分子氨基末端的生长因子结合域和kringle（K）域。uPA在体内很容易降解为这些组成域。本研究的目的是研究平滑肌细胞对尿激酶kringle域迁移过程中的细胞信号传导。
体外培养小鼠动脉平滑肌细胞。在有和没有纤溶酶抑制剂（抑肽酶和α-氨基己酸）、Gαi抑制剂百日咳毒素、MMP抑制剂（GM6001）、PI3-K抑制剂渥曼青霉素和LY294002以及MAPK抑制剂PD98089（MEK1抑制剂）和SB203580（p38丝裂原活化蛋白激酶抑制剂）存在的情况下，用K进行迁移试验。在用抑制剂存在和不存在的情况下，用K刺激后，对ERK 1/2和p38丝裂原活化蛋白激酶磷酸化进行蛋白质印迹分析。采用单因素方差分析进行统计学分析（n = 6）。
与对照相比，kringle域（K）诱导了纤溶酶非依赖性、MMP依赖性的细胞迁移增加（2倍，P < 0.05）。这种对K的迁移反应由Gαi介导，并且依赖于ERK 1/2和p38丝裂原活化蛋白激酶的激活。K诱导ERK 1/2（3倍，P < 0.05）和p38丝裂原活化蛋白激酶（3倍，P < 0.05）的磷酸化随时间增加。PI3-K抑制剂完全抑制了p38丝裂原活化蛋白激酶和ERK 1/2的激活。我们探讨了表皮生长因子受体（EGFR）的潜在作用。K诱导EGFR磷酸化，EGFR抑制剂AG1478的存在抑制了对K的细胞迁移和akt激活。
uPA的kringle域通过涉及ERK 1/2和p38丝裂原活化蛋白激酶的G蛋白偶联PI3-K依赖性过程诱导平滑肌细胞迁移，并且部分通过EGFR介导。明确对uPA关键分子域反应的差异对于理解其在血管重塑中的作用至关重要。
