Oxidative stress-induced inhibition of Sirt1 by caveolin-1 promotes p53-dependent premature senescence and stimulates the secretion of interleukin 6 (IL-6).

Oxidative stress can induce premature cellular senescence. Senescent cells secrete various growth factors and cytokines, such as IL-6, that can signal to the tumor microenvironment and promote cancer cell growth. Sirtuin 1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including senescence. We found that caveolin-1, a structural protein component of caveolar membranes, is a direct binding partner of Sirt1, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain of Sirt1 (amino acids 310-317). Our data show that oxidative stress promotes the sequestration of Sirt1 into caveolar membranes and the interaction of Sirt1 with caveolin-1, which lead to inhibition of Sirt1 activity. Reactive oxygen species stimulation promotes acetylation of p53 and premature senescence in wild-type but not caveolin-1 null mouse embryonic fibroblasts (MEFs). Either down-regulation of Sirt1 expression or re-expression of caveolin-1 in caveolin-1 null MEFs restores reactive oxygen species-induced acetylation of p53 and premature senescence. In addition, overexpression of caveolin-1 induces stress induced premature senescence in p53 wild-type but not p53 knockout MEFs. Phosphorylation of caveolin-1 on tyrosine 14 promotes the sequestration of Sirt1 into caveolar membranes and activates p53/senescence signaling. We also identified IL-6 as a caveolin-1-specific cytokine that is secreted by senescent fibroblasts following the caveolin-1-mediated inhibition of Sirt1. The caveolin-1-mediated secretion of IL-6 by senescent fibroblasts stimulates the growth of cancer cells. Therefore, by inhibiting Sirt1, caveolin-1 links free radicals to the activation of the p53/senescence pathway and the protumorigenic properties of IL-6.

Oxidative stress induces premature senescence by stimulating caveolin-1 gene transcription through p38 mitogen-activated protein kinase/Sp1-mediated activation of two GC-rich promoter elements.

Cellular senescence is believed to represent a natural tumor suppressor mechanism. We have previously shown that up-regulation of caveolin-1 was required for oxidative stress-induced premature senescence in fibroblasts. However, the molecular mechanisms underlying caveolin-1 up-regulation in senescent cells remain unknown. Here, we show that subcytotoxic oxidative stress generated by hydrogen peroxide application promotes premature senescence and stimulates the activity of a (-1,296) caveolin-1 promoter reporter gene construct in fibroblasts. Functional deletion analysis mapped the oxidative stress response elements of the mouse caveolin-1 promoter to the sequences -244/-222 and -124/-101. The hydrogen peroxide-mediated activation of both Cav-1 (-244/-222) and Cav-1 (-124/-101) was prevented by the antioxidant quercetin. Combination of electrophoretic mobility shift studies, chromatin immunoprecipitation analysis, Sp1 overexpression experiments, as well as promoter mutagenesis identifies enhanced Sp1 binding to two GC-boxes at -238/-231 and -118/-106 as the core mechanism of oxidative stress-triggered caveolin-1 transactivation. In addition, signaling studies show p38 mitogen-activated protein kinase (MAPK) as the upstream regulator of Sp1-mediated activation of the caveolin-1 promoter following oxidative stress. Inhibition of p38 MAPK prevents the oxidant-induced Sp1-mediated up-regulation of caveolin-1 protein expression and development of premature senescence. Finally, we show that oxidative stress induces p38-mediated up-regulation of caveolin-1 and premature senescence in normal human mammary epithelial cells but not in MCF-7 breast cancer cells, which do not express caveolin-1 and undergo apoptosis. This study delineates for the first time the molecular mechanisms that modulate caveolin-1 gene transcription upon oxidative stress and brings new insights into the redox control of cellular senescence in both normal and cancer cells.

Mapping of oxidative stress response elements of the caveolin-1 promoter.

According to the "free radical theory" of aging, normal aging occurs as the result of tissue damages inflicted by reactive oxygen species (ROS). ROS are known to induce cellular senescence, and senescent cells are believed to contribute to organismal aging. The molecular mechanisms that mediate the cellular response to oxidants remain to be fully identified. We have shown that oxidative stress induces cellular senescence through activation of the caveolin-1 promoter and upregulation of caveolin-1 protein expression. Here, we describe how reactive oxygen species activate the caveolin-1 promoter and how the signaling may be assayed. These approaches provide insight into the functional role of caveolin-1 and potentially allow the identification of novel ROS-regulated genes that are part of the signaling machinery regulating cellular senescence/aging.

Caveolin-1 regulates the antagonistic pleiotropic properties of cellular senescence through a novel Mdm2/p53-mediated pathway.

We show that caveolin-1 is a novel binding protein for Mdm2. After oxidative stress, caveolin-1 sequesters Mdm2 away from p53, leading to stabilization of p53 and up-regulation of p21(Waf1/Cip1) in human fibroblasts. Expression of a peptide corresponding to the Mdm2 binding domain of caveolin-1 is sufficient to up-regulate p53 and p21(Waf1/Cip1) protein expression and induce premature senescence. Oxidative stress-induced activation of the p53/p21(Waf1/Cip1) pathway and induction of premature senescence are compromised in caveolin-1 null mouse embryonic fibroblasts (MEF). We also show that reintroduction of caveolin-1 in oncogenic Ras (Ras(G12V))-transformed fibroblasts, which express residual levels of caveolin-1, is sufficient to promote cellular senescence. Moreover, caveolin-1 expression in MEFs is required for senescent fibroblast-induced stimulation of cell growth and tumorigenesis of both Ras(G12V)-transformed fibroblasts and MDA-MB-231 breast cancer epithelial cells both in vitro and in vivo. Thus, our results propose caveolin-1 as a key mediator of the antagonistic pleiotropic properties of cellular senescence.