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.
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.
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