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

Abstract

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.

Fig. 29.1

Oxidative stress activates the caveolin-1 promoter by acting through two GC-rich boxes. (a) Luciferase assay. Caveolin-1 promoter deletion mutant constructs were transiently transfected in NIH 3 T3 cells. pTA-luc alone was used as a control. Twenty-four hours after transfection, cells were treated with or without 150 μM H₂O₂ for 2 h. Cells were collected 48 h after oxidative stress and luciferase activity measured. Values represent means ±SEM. *^/#P < 0.001. (b) Luciferase assay. The caveolin-1 promoter −244/−222 and −124/−101 regions, both containing a GC-rich box, were cloned upstream of the luciferase gene in the pTA-luc vector. These constructs were transiently transfected in NIH 3 T3 cells. pTA-luc alone was used as a control. Twenty-four hours after transfection, cells were treated with or without 150 μM H₂O₂ for 2 h. Cells were collected 48 h after oxidative stress and luciferase activity measured. Values represent means ±SEM. *P < 0.001. Figure adapted from ref. .

Fig. 29.2

Oxidative stress stimulates the binding of nuclear proteins to Sp1 consensus elements within the caveolin-1 promoter. (a) and (b) EMSA studies. Electrophoretic mobility shift assays were performed with nuclear extracts from untreated and H₂O₂-treated (150 μM for 2 h) NIH 3 T3 cells 48 h after oxidative stress. Nuclear extracts were incubated with either Cav-1 (−244/−222) (a) or Cav-1 (−124/−101) (b) biotin-labeled oligonucleotides. Lack of nuclear extract was used as a negative control. Note that two nucleoprotein complexes were identified in (a) (Complex I and III) and two in (b) (Complex II and IV). Incubation with excess unlabeled Sp1 consensus oligonucleotides was performed to show specificity of complex I and II. Figure adapted from ref. .

Fig. 29.3

Oxidative stress promotes the binding of Sp1 to GC-rich elements within the caveolin-1 promoter. ChIP assay. Chromatin immunoprecipitation assay was done on chromatin derived from untreated or hydrogen peroxide-treated (150 μM for 2 h) NIH 3 T3 cells 48 h after oxidative stress using an antibody probe specific for Sp1. PCR was performed using primers surrounding the region of the caveolin-1 promoter containing the two GC-rich boxes. Amplification of input DNA from both untreated and H₂O₂-treated cells was performed before immunoprecipitation. A vector containing the entire caveolin-1 promoter sequence was used as a positive control for PCR. Figure adapted from ref. (30).