Role of Matrix Metalloproteinase-1 in Endothelial Cell Senescence

描述（由申请人提供）：在体外由癌基因和氧化应激诱导的细胞过早衰老概括了复制衰老和体内细胞衰老的许多方面。随着最近的研究发现衰老细胞在体内具有肿瘤抑制作用，细胞衰老的建立成为一种生存适应，旨在限制应激和受损细胞的增殖。然而，细胞衰老建立和维持的因素和分子机制仍然知之甚少。锂通常用于治疗双相情感障碍，并通过抑制糖原合成酶激酶(GSK)-32激活致癌的2-catenin信号通路。我们之前的研究表明，锂在原代内皮细胞中诱导细胞周期阻滞，这与肿瘤抑制因子p53和p21cip级联的激活以及以SA- 2-半乳糖苷酶标记为特征的细胞衰老表型的发展有关。锂离子诱导的衰老表型还伴随着肿瘤抑制因子FOXO1的激活，FOXO1参与寿命和细胞衰老的控制，并通过上调纤溶酶原激活物抑制剂(PAI)-1和基质金属蛋白酶(MMP)-1这两种细胞衰老和年龄相关疾病的分泌标志物。除了降解胶原蛋白外，MMP-1还可以通过裂解和激活蛋白酶激活受体(PAR)-1来传递信号。内皮细胞中PAR1的激活触发了促炎状态，这也是衰老状态的标志。此外，锂对MMP1的上调是一个独立于炎症细胞因子、GSK32抑制和2-catenin稳定的早期事件，表明了一种新的锂依赖级联。我们提出验证一种新的锂依赖级联诱导MMP-1表达的假设，MMP-1反过来通过激活PAR-1参与细胞衰老的建立和/或维持。在Aim 1中，我们将确定MMP-1在锂诱导的细胞衰老表型中的作用及其作用机制：i)分别使用特异性抑制剂和小RNA干扰监测MMP-1存在和缺乏MMP1活性和表达时衰老标志物的表达，ii)通过分析PAR-1对锂的响应及其对MMP-1表达和活性的依赖性。在Aim 2中，我们将通过以下方法确定锂介导的内皮细胞中MMP-1上调的机制：1)描述MMP-1启动子中的锂响应元件，2)识别锂调节的转录因子，以及3)分析使用特定抑制剂和激活剂以及通过小RNA干扰敲低其表达的信号级联。公共卫生相关性：癌症和动脉粥样硬化的发生随着年龄的增长而增加，这些与年龄有关的疾病具有共同的特征，如病变内部或周围出现衰老细胞。虽然细胞衰老是对压力的生存反应，但这些衰老的细胞释放因子，如基质金属蛋白酶-1，将反过来维持和传播细胞功能失调状态。因此，了解这些因子是如何产生的，以及它们如何作用于邻近细胞，对于开发新的靶向治疗策略来预防或延缓与衰老相关的细胞功能障碍的传播至关重要。

DESCRIPTION (provided by applicant): Premature cell senescence induced by oncogenes and oxidative stress in vitro recapitulates many facets of replicative senescence and in vivo cellular aging. With the recent findings of a tumor suppressor role for senescent cells in vivo, the establishment of cell senescence emerges as a survival adaptation to stress aimed at limiting the proliferation of stressed and damaged cells. However, the factors and molecular mechanisms by which cell senescence is established and maintained remain poorly understood. Lithium is commonly used to treat bipolar disorder as well as to activate the oncogenic 2-catenin signaling pathway via inhibition of glycogen synthase kinase (GSK)-32. We have previously shown that lithium induces a cell cycle arrest in primary endothelial cells associated with the activation of the tumor suppressor p53 and p21cip cascade and the development of a cell senescent phenotype characterized by the SA- 2-galactosidase marker. The lithium-induced senescent phenotype was also accompanied with the activation of the tumor suppressor FOXO1, which is involved in the control of lifespan and cell senescence and by the up-regulation of plasminogen activator inhibitor (PAI)-1 and matrix metalloproteinase (MMP)-1, two secreted markers of cell senescence and age-related diseases. MMP-1, in addition to degrade collagen, can signals via cleavage and activation of the proteinase-activated-receptor (PAR)-1. Activation of PAR1 in endothelial cells triggers a pro-inflammatory state, which is also a hallmark of a senescent state. Moreover, MMP1 up-regulation by lithium is an early event that appears independent of inflammatory cytokines, GSK32 inhibition and 2-catenin stabilization indicative of a novel lithium-dependent cascade. We propose to test the hypothesis that a novel lithium-dependent cascade induces MMP-1 expression, which in turn participates in the establishment and/or maintenance of cell senescence via activation of PAR-1. In Aim 1, we will determine the role of MMP-1 in the establishment of lithium-induced cell senescent phenotype and its mechanism of action by i) monitoring the expression of senescence markers in presence of MMP-1 and in absence of MMP1 activity and expression using specific inhibitors and small RNA interference respectively, and ii) by analyzing the activation of PAR-1 in response to lithium and its dependency on MMP-1 expression and activity. In Aim 2, we will identify the mechanisms of lithium-mediated up-regulation of MMP-1 in endothelial cells by i) delineating the lithium- responsive element in MMP-1 promoter, ii) identifying the transcription factors regulated by lithium and iii) analyzing the signaling cascade involved using specific inhibitors and activators as well as by knock in down their expression by small RNA interference. PUBLIC HEALTH RELEVANCE: The occurrence of cancer and atherosclerosis increase with age and these age-related diseases share common features such as the appearance of senescent cells within or around the lesions. Though cell senescence is a survival response to stress, these senescent cells release factors, like the matrix metalloproteinase-1, that will in turn maintain and propagate cellular dysfunctional states. Thus understanding how these factors are produced and how they act on neighboring cells is crucial for developing novel and targeted therapeutic strategies to prevent or delay the propagation of cellular dysfunctions associated with aging.