Han Ji-Soo, Macarak Edward, Rosenbloom Joel, Chung Kwang Chul, Chaqour Brahim
University of Pennsylvania, Department of Anatomy and Cell Biology, Philadelphia, PA 19104, USA.
Eur J Biochem. 2003 Aug;270(16):3408-21. doi: 10.1046/j.1432-1033.2003.03723.x.
Cysteine-rich protein 61 (Cyr61/CCN1) is an angiogenic factor and a member of a family of growth factor-inducible immediate-early genes with functions in cell adhesion, proliferation and differentiation. We investigated the regulatory mechanisms and signaling pathways involved in Cyr61/CCN1gene activation in smooth muscle cells. Treatment of these cells with sphingosine 1-phosphate (S1P), a bioactive lysolipid, increased rapidly but transiently the expression of the Cyr61/CCN1 gene at both the mRNA and protein levels. Cyr61/CCN1 mRNA stability was not altered but the transcription rate of the Cyr61/CCN1 gene was increased fivefold in isolated nuclei from S1P-stimulated cells indicating that the level of control is primarily transcriptional. Transfection experiments showed that a 936-bp promoter fragment of the human Cyr61/CCN1 gene is functional and induces a reporter gene activity in S1P-treated cells. Using a combination of cis-element mutagenesis and expression of dominant negative inhibitors of transcription factors, we showed that both a CRE and AP-1 site and their cognate transcription factors, cAMP response element binding protein (CREB) and AP-1, were responsible for the promoter activity in S1P-stimulated cells. Furthermore, by using either pharmacological inhibitors or active forms of known signaling molecules, we showed that inducible Cyr61/CCN1 gene expression occurs through RhoA GTPase and that additional signaling through the p38 pathway is required. In particular, p38 seems to regulate Cyr61/CCN1 promoter activity through modulation of phosphorylation of CREB and the CREB kinase, MSK1. These findings demonstrate the transcriptional regulation of the Cyr61/CCN1 gene and provide clues to the signaling molecules and transcription factors involved in such regulation.
富含半胱氨酸的蛋白61（Cyr61/CCN1）是一种血管生成因子，属于生长因子诱导的即刻早期基因家族成员，在细胞黏附、增殖和分化中发挥作用。我们研究了平滑肌细胞中Cyr61/CCN1基因激活所涉及的调控机制和信号通路。用生物活性溶血磷脂鞘氨醇-1-磷酸（S1P）处理这些细胞，可使Cyr61/CCN1基因在mRNA和蛋白质水平上迅速但短暂地表达增加。Cyr61/CCN1 mRNA稳定性未改变，但在S1P刺激细胞的分离细胞核中，Cyr61/CCN1基因的转录速率增加了五倍，这表明调控水平主要在转录阶段。转染实验表明，人Cyr61/CCN1基因的一个936 bp启动子片段具有功能，并在S1P处理的细胞中诱导报告基因活性。通过顺式元件诱变和转录因子显性负抑制剂的表达相结合，我们发现一个CRE和AP-1位点及其相应的转录因子，即cAMP反应元件结合蛋白（CREB）和AP-1，负责S1P刺激细胞中的启动子活性。此外，通过使用药理学抑制剂或已知信号分子的活性形式，我们表明可诱导的Cyr61/CCN1基因表达通过RhoA GTP酶发生，并且需要通过p38途径的额外信号传导。特别是，p38似乎通过调节CREB和CREB激酶MSK1的磷酸化来调节Cyr61/CCN1启动子活性。这些发现证明了Cyr61/CCN1基因的转录调控，并为参与这种调控的信号分子和转录因子提供了线索。
