Zhou Limei, Goldsmith Adam M, Bentley J Kelley, Jia Yue, Rodriguez Michael L, Abe Mark K, Fingar Diane C, Hershenson Marc B
Department of Pediatrics and Communicable Diseases, University of Michigan, 1150 W. Medical Center Dr., Room 3570, MSRBII, Box 0688, Ann Arbor, MI 48109-0688, USA.
Am J Respir Cell Mol Biol. 2005 Aug;33(2):195-202. doi: 10.1165/rcmb.2004-0411OC. Epub 2005 May 18.
The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.
气道平滑肌肥大是重度哮喘的一个特征，其分子机制目前仍知之甚少。我们之前利用一种温度敏感型SV40大T抗原建立了一种条件永生化的人支气管平滑肌细胞系。温度变化和大T抗原的缺失会导致G1期细胞周期停滞，同时伴有气道平滑肌细胞大小增加。在本研究中，我们假设真核起始因子-4E（eIF4E）结合蛋白（4E-BP）的磷酸化是气道平滑肌肥大所必需的，这种磷酸化随后会释放eIF4E并启动帽依赖性mRNA翻译。用PI 3激酶和雷帕霉素哺乳动物靶点的化学抑制剂处理细胞会阻断蛋白质合成和细胞生长，同时降低4E-BP的磷酸化水平并增加4E-BP与eIF4E的结合，这与肥大需要4E-BP1磷酸化和eIF4E功能的观点一致。为了直接验证这一点，我们用编码一种能显性抑制eIF4E的4E-BP1磷酸化位点突变体（AA-4E-BP-1）的逆转录病毒感染细胞。温度变化后，感染AA-4E-BP-1的细胞而非空载体对照细胞未能发生肥大生长。我们得出结论，4E-BP的磷酸化、eIF4E的释放以及帽依赖性蛋白质合成是人气道平滑肌细胞肥大所必需的。
