Ali S, Davis M G, Becker M W, Dorn G W
Department of Medicine/Cardiology, University of Cincinnati College of Medicine, Ohio 45267.
J Biol Chem. 1993 Aug 15;268(23):17397-403.
We have shown previously that thromboxane A2 stimulates hypertrophy of cultured rat aortic smooth muscle cells defined as protooncogene expression and protein synthesis without DNA synthesis or cellular proliferation (Dorn, G.W., II, Becker, M.W., Davis, M.G. (1992) J. Biol. Chem. 267, 24897-24905). Since endogenous growth modulators could possibly regulate vascular smooth muscle growth to this vasoconstrictor, we tested the hypothesis that thromboxane-stimulated vascular smooth muscle hypertrophy was due to increased expression of endogenously produced basic fibroblast growth factor (bFGF). The thromboxane mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM) increased cultured rat aorta derived smooth muscle cell immunoreactive bFGF content by 331 +/- 40% over untreated controls after 24 h. Co-incubation of vascular smooth muscle cells with a specific antisense oligodeoxynucleotide (AS) against codon 60 of bFGF coding sequence reduced thromboxane-stimulated bFGF expression by 72 +/- 5% and prevented thromboxane-stimulated hypertrophy (nonsense oligonucleotide had no effects). Addition of exogenous bFGF (20 ng/ml) restored growth to AS-treated/thromboxane-stimulated vascular smooth muscle cells. Furthermore, addition to the culture medium of neutralizing antibody against bFGF inhibited U46619-stimulated vascular smooth muscle hypertrophy by 69 +/- 17%, whereas nonimmune IgG had no effect. Since protein tyrosine phosphorylation is a cell signal associated with growth, thromboxane-stimulated tyrosine phosphorylation was also examined. Exposure to 1 microM U46619 for 10 min increased vascular smooth muscle immunoreactive phosphotyrosine content of 130-144-, 86-, 80-, 75-, and 58-kDa proteins. The tyrosine kinase inhibitor herbimycin A (5 microM) prevented thromboxane-stimulated tyrosine phosphorylation, but not thromboxane-stimulated hypertrophy, suggesting that tyrosine phosphorylation was not required for thromboxane-stimulated vascular smooth muscle growth. These results indicate that increased expression and release of endogenous bFGF, but not direct tyrosine phosphorylation, mediates the hypertrophic vascular smooth muscle response to thromboxane.
我们先前已经表明，血栓素A2可刺激培养的大鼠主动脉平滑肌细胞肥大，这种肥大表现为原癌基因表达和蛋白质合成增加，而无DNA合成或细胞增殖（多恩，G.W.，二世，贝克尔，M.W.，戴维斯，M.G.（1992年）《生物化学杂志》267卷，24897 - 24905页）。由于内源性生长调节剂可能会调节血管平滑肌对这种血管收缩剂的生长反应，我们检验了以下假设：血栓素刺激的血管平滑肌肥大是由于内源性产生的碱性成纤维细胞生长因子（bFGF）表达增加所致。血栓素类似物（15S） - 羟基 - 11α，9α - （环氧亚甲基）前列腺 - 5Z，13E - 二烯酸（U46619）（1微摩尔）作用24小时后，培养的大鼠主动脉来源的平滑肌细胞免疫反应性bFGF含量比未处理的对照增加了331±40%。将血管平滑肌细胞与针对bFGF编码序列第60密码子的特异性反义寡脱氧核苷酸（AS）共同孵育，可使血栓素刺激的bFGF表达降低72±5%，并阻止血栓素刺激的肥大（无义寡核苷酸无此作用）。添加外源性bFGF（20纳克/毫升）可使经AS处理/血栓素刺激的血管平滑肌细胞恢复生长。此外，向培养基中添加抗bFGF中和抗体可使U46619刺激的血管平滑肌肥大受到69±17%的抑制，而非免疫IgG则无此作用。由于蛋白质酪氨酸磷酸化是一种与生长相关的细胞信号，因此我们也研究了血栓素刺激的酪氨酸磷酸化情况。暴露于1微摩尔U46619 10分钟可使血管平滑肌中130 - 144 kDa、86 kDa、80 kDa、75 kDa和58 kDa蛋白质的免疫反应性磷酸酪氨酸含量增加。酪氨酸激酶抑制剂赫曲霉素A（5微摩尔）可阻止血栓素刺激的酪氨酸磷酸化，但不能阻止血栓素刺激的肥大，这表明酪氨酸磷酸化并非血栓素刺激血管平滑肌生长所必需。这些结果表明，内源性bFGF表达和释放的增加介导了血管平滑肌对血栓素的肥大反应，而不是直接的酪氨酸磷酸化。
