Urokinase-induced smooth muscle cell responses require distinct signaling pathways: A role for the epidermal growth factor receptor
Urokinase-induced smooth muscle cell responses require distinct signaling pathways: A role for the epidermal growth factor receptor
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DOI：
10.1016/j.jvs.2005.01.007
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发表时间：
2005-04-01
影响因子：
4.3
通讯作者：
Davies, MG
中科院分区：
文献类型：
--
作者：
Nichol, SM;Roztocil, E;Davies, MG
目的：尿激酶型纤溶酶原激活物（Urokinase plasminogen activator，uPA）是平滑肌重塑过程中的关键丝氨酸蛋白酶，能诱导平滑肌细胞迁移和增殖。然而，产生这些responses.Methods的信号是知之甚少：早期传代大鼠主动脉平滑肌细胞培养在体外和标准的DNA合成（[H-3]胸苷掺入），细胞增殖（手动细胞计数），迁移（线性创伤试验和Boyden室）的测定被用来研究细胞对uPA的反应。通过蛋白质印迹分析，测定了丝裂原活化蛋白激酶（MAPK）、细胞外信号调节激酶1/2（ERK 1/2）、p38（MAPK）、Akt、MAP激酶/ERK激酶（MEK 1/2）、MAP激酶激酶（MKK）3/6和表皮生长因子受体（EGFR）对uPA的响应，以确定每种激酶的磷酸化形式。在G α i抑制剂百日咳毒素存在下重复这些测定（PTx，100 ng/mL），Ras抑制剂manumycin A（MA，10 μ M），磷脂酰肌醇3 '激酶（PI 3 K）抑制剂渥曼青霉素（WN，11 μ M），EGFR抑制剂AG 1478（AG，10 nM），MEK 1抑制剂PD 98059（PD，10 μ M），p38（MAPK）抑制剂SB 203580（SB，10 μ M），以及纤溶酶抑制剂抑肽酶和ε-氨基己酸。uPA诱导平滑肌细胞迁移增加两倍，并增加平滑肌细胞DNA合成和增殖。ERK 1/2和p38（MAPK）抑制剂PD 98059（PD）和SB 203580（SB）阻断细胞增殖，但仅PD阻断细胞迁移。尽管Gai抑制剂可阻断uPA诱导的ERK 1/2和p38（MAPK）磷酸化，但抑制PI 3 K和Ras可降低uPA诱导的ERK 1/2磷酸化，但对p38（MAPK）无影响。MEK 1/2的激活被Gai和Ras的抑制剂废除，但不被PI 3 K抑制。相反，MKK 3/6的活化被Gai的抑制所消除，但不被Ras或PI 3 K抑制所消除。uPA诱导EGFR的时间依赖性磷酸化，其依赖于纤溶酶活性。EGFR的抑制降低了ERK 1/2和p38（MAPK）的活化。结论：uPA通过ERK 1/2和p38（MAPK）介导的信号通路诱导平滑肌细胞增殖。迁移似乎仅依赖于ERK 1/2活性。似乎需要EGFR的激活。通过uPA对ERK 1/2和p38（MAPK）通路的差异激活允许两种不同的生物反应，这两种生物反应都需要酪氨酸激酶受体反式激活。升高的尿激酶样纤溶酶原激活物（uPA）和降低的纤溶酶原激活物抑制剂-1（派-1）水平是再狭窄的预测因子。基质重塑和平滑肌细胞反应是不可分割的联系。平滑肌细胞迁移和增殖的变化取决于它们所包裹的细胞外基质环境。蛋白酶如uPA可以影响平滑肌细胞并改变基质;它们的活性由一系列抑制剂（如派-1）控制。激活和抑制的平衡形成了血管壁中蛋白水解恒温器的基础。了解蛋白水解恒温器的生物学将允许结构化的治疗干预来控制再狭窄，从而改善患者护理并避免二次干预。我们的研究表明，uPA是能够诱导单独的反应，通过一个以上的信号转导途径，在一定程度上，通过反式激活附近的受体无关的配体表皮生长因子受体（EGFR）。阻断EGFR可以抑制uPA诱导的细胞迁移和增殖。这是第一次描述血管平滑肌细胞中uPA和EGFR之间的相互作用。靶向关键受体（例如EGFR）可以被G蛋白偶联受体和受体酪氨酸激酶反式激活，是控制再狭窄的有吸引力的分子靶点。
Objective: Urokinase plasminogen activator (uPA) a key serine protease during remodeling, is capable of inducing both smooth muscle cell migration and proliferation. However, the signals that produce these responses are poorly understood.Methods: Early passage rat aortic arterial smooth muscle cells were cultured in vitro and standard assays of DNA synthesis ([H-3] thymidine incorporation), cell proliferation (manual cell counting), and migration (linear wound assay and Boyden chamber) were used to study the cells responses to uPA. Activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38(MAPK), Akt, MAP kinase/ERK kinase (MEK1/2), MAP kinase kinase (MKK)3/6, and epidermal growth factor receptor (EGFR) in response to uPA was assayed by Western blot analysis for the phosphorylated form of each kinase. These assays were repeated in the presence of the G alpha i inhibitor pertussis toxin (PTx, 100 ng/mL), the Ras inhibitor manumycin A (MA, 10 mu M), the phosphatidyl-inositol 3 ' kinase (PI3K) inhibitor wortmannin (WN, 1 1 mu M), the EGFR inhibitor AG1478 (AG, 10 nM), the MEK1 inhibitor PD98059 (PD, 10 mu M), the p38(MAPK) inhibitor SB203580 (SB, 10 mu M), and the plasmin inhibitors aprotinin and epsilon-aminocaproic acid.Results: uPA induced a twofold increase in smooth muscle cell migration and increased smooth muscle cell DNA synthesis and proliferation. The ERK1/2 and p38(MAPK) inhibitors PD98059 (PD) and SB203580 (SB) blocked cell proliferation, but only PD blocked cell migration. Although uPA-induced phosphorylation of both ERK1/2 and p38(MAPK) was blocked by Gai inhibition, inhibition of PI3K and Ras decreased the uPA-induced phosphorylation of ERK1/2 but not p38(MAPK). Activation of MEK1/2 was abrogated by inhibitors of Gai and Ras, but not by PI3K inhibition. In contrast, activation of MKK3/6 was abrogated by inhibition of Gai, but not by Ras or PI3K inhibition. uPA induced time-dependent phosphorylation of EGFR, which was dependent on plasmin activity. Inhibition of EGFR reduced both ERK1/2 and p38(MAPK) activation. uPA activation of PI3K and MKK3/6 was EGFR-dependent and that of MEK1 was EGFR-independent.Conclusion: uPA induces smooth muscle cell proliferation through ERK1/2- and p38(MAPK)-mediated pathways. Migration appears to be dependent on ERK1/2 activity alone. Activation of EGFR appears to be required. The differential activation of pathways for ERK1/2 and p38(MAPK) by uPA allows for two distinct biologic responses that both require tyrosine kinase receptor transactivation.Clinical relevance. Elevated urokinase-like plasminogen activator (uPA) and decreased plasminogen activator inhibitor-1 (PAI-1) levels are predictors for restenosis. Matrix remodeling and smooth muscle cell responses are integrally linked. Changes in smooth muscle cell migration and proliferation are dependent on the extracellular matrix environment in which they are encased. Proteases such as uPA can effect smooth muscle cells and alter the matrix; their activity is controlled by a series of inhibitors (eg, PAI-1). The balance of activation and inhibition forms the basis of the proteolytic thermostat in the vessel wall. Understanding the biology of the proteolytic thermostat will allow for structured therapeutic interventions to control restenosis and thus improve patient care and avoid secondary interventions. Our study demonstrates that uPA is capable of inducing separate responses through more than one signaling pathway, in part, by transactivation of a nearby receptor for the unrelated ligand epidermal growth factor receptor (EGFR). Blockade of EGFR can inhibit both cell migration and proliferation induced by uPA. This is the first description of cross talk between uPA and EGFR in vascular smooth muscle cells. Targeting a pivotal receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases, is an attractive molecular target to control restenosis.