Song Yuanda, Wu Jinhua, Oyesanya Regina A, Lee Zendra, Mukherjee Abir, Fang Xianjun
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.
Clin Cancer Res. 2009 Jan 15;15(2):492-501. doi: 10.1158/1078-0432.CCR-08-1945.
Lysophosphatidic acid (LPA), which is present in ascites of ovarian cancer patients, stimulates expression of vascular endothelial growth factor (VEGF). VEGF is essential for the development and abdominal dissemination of ovarian cancer. We examined how LPA drives VEGF expression to gain a better understanding of tumor angiogenesis under normoxic conditions.
ELISA, Northern blotting, immunoblotting, quantitative PCR, and promoter reporter analysis in combination with small interfering RNA and pharmacologic inhibitors were used to examine LPA-induced VEGF expression and the underlying mechanisms.
LPA stimulated expression of multiple VEGF variants. A 123-bp fragment proximal to the transcriptional initiation site was identified to be functional promoter region responsible for the response to LPA. The fragment harbors consensus sites for several transcription factors including c-Myc and Sp-1 but not hypoxia-inducible factor-1. Blockade of Rho, ROCK, or c-Myc reduced LPA-dependent VEGF production and promoter activation, suggesting that the G12/13-Rho-ROCK-c-Myc cascade partially contributes to VEGF induction by LPA. More significantly, the multiple Sp-1 sites within the responsive region of the VEGF promoter were essential for LPA-mediated transcription. LPA induced Sp-1 phosphorylation and DNA-binding and transcriptional activities. The silencing of Sp-1 expression with small interfering RNA or inhibition of Sp-1 with pharmacologic inhibitors blocked VEGF production induced by LPA.
LPA stimulates hypoxia-inducible factor-1-independent VEGF expression to promote tumor angiogenesis through activation of the c-Myc and Sp-1 transcription factors.
溶血磷脂酸（LPA）存在于卵巢癌患者的腹水中，可刺激血管内皮生长因子（VEGF）的表达。VEGF对于卵巢癌的发展和腹腔播散至关重要。我们研究了LPA如何驱动VEGF表达，以便更好地了解常氧条件下的肿瘤血管生成。
采用酶联免疫吸附测定（ELISA）、Northern印迹法、免疫印迹法、定量聚合酶链反应（PCR）以及启动子报告基因分析，并结合小干扰RNA和药理抑制剂，来检测LPA诱导的VEGF表达及其潜在机制。
LPA刺激多种VEGF变体的表达。转录起始位点近端的一个123 bp片段被确定为负责对LPA作出反应的功能性启动子区域。该片段含有包括c-Myc和Sp-1在内的几种转录因子的共有位点，但不含有缺氧诱导因子-1的共有位点。阻断Rho、Rho相关卷曲螺旋形成蛋白激酶（ROCK）或c-Myc可降低LPA依赖的VEGF产生和启动子激活，这表明G12/13-Rho-ROCK-c-Myc级联反应部分促成了LPA对VEGF的诱导作用。更重要的是，VEGF启动子反应区域内的多个Sp-1位点对于LPA介导的转录至关重要。LPA诱导Sp-1磷酸化以及DNA结合和转录活性。用小干扰RNA沉默Sp-1表达或用药理抑制剂抑制Sp-1可阻断LPA诱导的VEGF产生。
LPA通过激活c-Myc和Sp-1转录因子刺激不依赖缺氧诱导因子-1的VEGF表达，从而促进肿瘤血管生成。
