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Supplementary Figure 5B from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 2A-B from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 2A from 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

Supplementary Figure 5E-F from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 1B from 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

Supplementary Figures 4A and B from 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

Supplementary Figure 4 from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 1 Legend from 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

Supplementary Figure Legends 1-6 from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 1D-F from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 2C from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 5C-D from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 3 from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

Supplementary Figure 3A from 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

Supplementary Figure 6A-B from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

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. (Cancer Res 2006; 66(22): 10805-14)

Supplementary Figure 1A-C from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway

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.

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 p21Waf1/Cip1 in human fibroblasts. Expression of a peptide corresponding to the Mdm2 binding domain of caveolin-1 is sufficient to up-regulate p53 and p21Waf1/Cip1 protein expression and induce premature senescence. Oxidative stress–induced activation of the p53/p21Waf1/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 (RasG12V)–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 RasG12V-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. [Cancer Res 2009;69(7):2878–86]

Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.

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. (Cancer Res 2006; 66(22): 10805-14)

Extracellular nucleotides are ubiquitous signaling molecules. ATP signals through two receptor types: the ionotropic P2X receptors, and the metabotropic P2Y receptors. ATP acts as a chemorepellent in Tetrahymena thermophila, where it causes a distinct avoidance response. The intracellular mechanisms by which ATP causes avoidance in this organism, however, are unknown. In this study, we use in vivo pharmacological assays along with enzyme immuno-assays to obtain information about the ATP chemorepellent pathway and its associated second messenger systems. Our data show strong similarities between the presumed ATP receptor of T. thermophila and members of the P2Y family of receptors. The ATP response of T. thermophila appears to be coupled to phospholipase C, a defining characteristic of the P2Y receptor family. In addition, the ATP chemoresponse appears to be linked to a G(i/o) protein, nitric oxide synthase, and adenylyl cyclase, all of which are characteristic of some P2Y receptors. This is an important first step in describing the pathways involved in ATP chemoresponse of this organism.

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.