Pukac L, Huangpu J, Karnovsky M J
Department of Pathology, Harvard Medical School, Boston, Massachusetts, 02115-5701, USA.
Exp Cell Res. 1998 Aug 1;242(2):548-60. doi: 10.1006/excr.1998.4138.
Vascular smooth muscle cell (VSMC) migration is an important process in the development of vascular occlusive disease. To investigate mitogen regulation of VSMC migration, a cell-layer-scrape assay was used to measure migration 20 h after stimulation of VSMC with platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor I (IGF-I), or phorbol 12-myristate 13-acetate (PMA). The contributions of cell proliferation were eliminated by treatment of VSMC with hydroxyurea, which suppressed DNA synthesis.PDGF-BB stimulated VSMC migration 2.5-fold, while PMA and IGF-I stimulated migration 1.7- and 1.5-fold, respectively. The importance of protein kinase C (PKC), ERK, and phosphoinositide-3' kinase (PI3 kinase) in mitogen-stimulated migration was investigated, using specific inhibitors of these signaling molecules. PDGF-BB-stimulated migration was inhibited by the general PKC inhibitor RO 31-8220 (40%), the MEK inhibitor PD98059 (31%), and the PI3 kinase inhibitor wortmannin (22%) but not by PMA-induced downregulation of conventional and novel PKC isoforms. IGF-I-stimulated migration was inhibited by RO 31-8220 (34%) and wortmannin (37%) but was much less affected by PD98059 (19%) or PKC downregulation (10%). PMA-stimulated migration was inhibited by RO 31-8220 (53%), PD98059 (50%), wortmannin (45%), and PKC downregulation (47%). Western analysis confirmed that ERK was strongly activated by PDGF-BB and PMA but not by IGF-I. To examine potential in vivo negative regulators of VSMC migration, we analyzed the ability of heparin, an analogue of heparan sulfate, and TGFbeta to attenuate mitogen-stimulated migration. Heparin but not TGFbeta inhibited VSMC migration stimulated by all three mitogens. Delayed-addition experiments showed that RO 31-8220 retained substantial inhibitory activity even if added 3 h after PMA or IGF-I stimulation and 5 h after PDGF-BB addition, suggesting that sustained PKC activation is important for migration. The MEK inhibitor retained some effectiveness for 5 h after PDGF-BB stimulation but only 1 h after PMA addition. Western analysis showed ERK activation was transient after PMA treatment but sustained for 6 h after PDGF-BB treatment. Heparin strongly inhibited migration even if added 5-7 h after mitogen stimulation, suggesting that heparin may inhibit both short- and long-term signals necessary for migration. The present studies indicate that PMA and IGF-I activate a limited number of second messengers resulting in moderate stimulation of migration; in contrast PDGF-BB stimulates multiple signaling pathways resulting in strong stimulation of migration and lessened sensitivity to inhibitory signals.
血管平滑肌细胞（VSMC）迁移是血管闭塞性疾病发展过程中的一个重要过程。为了研究有丝分裂原对VSMC迁移的调节作用，采用细胞层刮擦试验来测量在用血小板衍生生长因子-BB（PDGF-BB）、胰岛素样生长因子I（IGF-I）或佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激VSMC 20小时后的迁移情况。通过用羟基脲处理VSMC来消除细胞增殖的影响，羟基脲可抑制DNA合成。PDGF-BB刺激VSMC迁移2.5倍，而PMA和IGF-I分别刺激迁移1.7倍和1.5倍。使用这些信号分子的特异性抑制剂研究了蛋白激酶C（PKC）、细胞外信号调节激酶（ERK）和磷酸肌醇-3'激酶（PI3激酶）在有丝分裂原刺激的迁移中的重要性。PDGF-BB刺激的迁移受到通用PKC抑制剂RO 31-8220（40%）、MEK抑制剂PD98059（31%）和PI3激酶抑制剂渥曼青霉素（22%）的抑制，但不受PMA诱导的传统和新型PKC亚型下调的影响。IGF-I刺激的迁移受到RO 31-8220（34%）和渥曼青霉素（37%）的抑制，但受PD98059（19%）或PKC下调（10%）的影响较小。PMA刺激的迁移受到RO 31-8220（53%）、PD980（50%）、渥曼青霉素（（45%）和PKC下调（47%）的抑制。蛋白质印迹分析证实，ERK被PDGF-BB和PMA强烈激活，但未被IGF-I激活。为了研究VSMC迁移的潜在体内负调节因子，我们分析了硫酸乙酰肝素类似物肝素和转化生长因子β（TGFβ）减弱有丝分裂原刺激的迁移的能力。肝素而非TGFβ抑制了所有三种有丝分裂原刺激的VSMC迁移。延迟添加实验表明，即使在PMA或IGF-I刺激后3小时以及PDGF-BB添加后5小时添加RO 31-8220，其仍保留大量抑制活性，这表明持续的PKC激活对迁移很重要。MEK抑制剂在PDGF-BB刺激后5小时仍保留一定效果，但在PMA添加后仅1小时有效。蛋白质印迹分析表明，PMA处理后ERK激活是短暂的，但PDGF-BB处理后持续6小时。即使在有丝分裂原刺激后5 - 7小时添加肝素，其也能强烈抑制迁移，这表明肝素可能抑制迁移所需的短期和长期信号。目前的研究表明，PMA和IGF-I激活有限数量的第二信使，导致对迁移的适度刺激；相比之下，PDGF-BB刺激多种信号通路，导致对迁移的强烈刺激并降低对抑制信号的敏感性。
