Cellular senescence is believed to represent a natural tumor suppressor mechanism. We have previously shown that upregulation of caveolin-1 was required for oxidative stressinduced 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 (À1,296) 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 À244/À222 and À124/À101. The hydrogen peroxide-mediated activation of both Cav-1 (À244/ À222) and Cav-1 (À124/À101) 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 À238/ À231 and À118/À106 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.

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 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.

Background: Oxidative stress promotes premature senescence in eukaryotic cells. Results: Inhibition of Sirt1 by caveolin-1 is induced by free radicals and promotes cellular senescence and secretion of tumorigenic IL-6 in fibroblasts. Conclusion:The caveolin-1/Sirt1/IL-6 signaling pathway contributes to explain the ability of senescent cells to stimulate cancer cell growth. Significance: This novel signaling cascade brings new insights into how senescent cells regulate the tissue microenvironment.

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.

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.