Chronic exposure to 12-O-tetradecanoylphorbol-13-acetate represses sod2 induction in vivo: the negative role of p50

Abstract

It is well documented that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can activate manganese superoxide dismutase (MnSOD) expression. However, it is unclear how repeated exposure to TPA following a single application of tumor initiator 7,12-dimethylbenz-(a)-anthracene causes tumor development. We generated transgenic mice expressing human MnSOD promoter- and enhancer-driven luciferase reporter gene and used a non-invasive imaging system to investigate the effects of TPA on MnSOD expression in vivo. Our data indicate that TPA initially activates MnSOD expression, but this positive effect declines after repeated applications. Changes in MnSOD expression in vivo were verified by measuring MnSOD mRNA and protein levels. Using chromatin immunoprecipitation coupled to Western analysis of the transcription factors known to be essential for the constitutive and TPA-induced transcription of MnSOD, we found that TPA treatment leads to both activation and inactivation of MnSOD gene transcription. During the activation phase, the levels of p50, p65, specificity protein 1 (Sp1) and nucleophosmin (NPM) increase after TPA treatments. Sustained treatments with TPA lead to further increase of p50 but not p65, Sp1 or NPM, suggesting that excess p50 may have inhibitory effects leading to the suppression of MnSOD. Alteration of p50 levels by expressing p50 cDNA or p50 small interfering RNA in mouse epithelial (JB6) cells confirms that p50 is inhibitory to MnSOD transcription. These findings identify p50 as having a negative effect on MnSOD induction upon repeated applications of TPA and provide an insight into a cause for the reduction of MnSOD expression during early stages of skin carcinogenesis.

Fig. 1. Characterization of transgene and determination of MnSOD transcription in vivo

(A) MnSOD promoter–enhancer-driven luciferase expression construct for the generation of transgenic mice. The 2.7 kb human MnSOD basal promoter- and enhancer-driven luciferase gene construct was used for microinjection to generate transgenic mice. (B) a, Detection of human MnSOD gene in transgenic mice. Kpn1 and BglII restriction-digested genomic DNAs from mouse tails were electrophoresed in 1% agarose gel, transferred onto nylon membrane and then hybridized with MnSOD intronic element. Transgenic mice showed ExI₂E band at ~2.7 kb. Endogenous genes were detected in both transgenic and non-transgenic animals; b, the luciferase activity was determined from the skin tissue extracts in transgenic mice and their non-transgenic littermates using luciferase assay kit (Promega); c, MnSOD protein and d, activity levels in transgenic mice and their nontransgenic littermates. (C) Bioluminescence images of mice treated with TPA (4 µg/mouse/day) were acquired by CCD camera. (D) Bioluminescent signals were quantified in terms of photon count which in turn represents the reporter gene activity in living mice. Bioluminescent images were gated to an area (1.54 cm²) where signals are generated after the application of substrate d-luciferin. Photon counts were estimated within that defined gated area. (E) The same animals were humanely euthanized and skin tissues were harvested. The luciferase activity was determined from the skin tissue extracts using luciferase assay kit (Promega). (F) MnSOD activity (U/mg protein) in controls and TPA-treated skin tissues. All data are representative of three independent sets of experiments. Significantly different from control; *P < 0.05 and **P < 0.01; significantly different from 4-day repeated treatment groups; #P < 0.01.

Fig. 2. MnSOD mRNA and protein levels in vivo and alteration of transcription factor

(A) Mice were repeatedly treated with TPA (4 µg/mouse/day) at indicated times and skin tissues were collected for RNA isolation. RNAs were isolated and purified by TRIzol method according to the manufacturer’s instructions. cDNAs were prepared and RT–PCR was carried out using mouse-specific MnSOD primer as described under Materials and methods. For quantification of mRNA, PCR bands were densitometrically scanned and normalized with β-actin. (B) Western blotting was used to determine the MnSOD protein level in skin epithelial tissue homogenate by using rabbit polyclonal MnSOD antibody. (C) Equal amounts of nuclear protein were subjected to 10% SDS–polyacrylamide gel electrophoresis and western blotting was performed using antibodies specific to p50 and p65. (D) The same membrane was reprobed with Sp1 or NPM antibody. Protein bands were densitometrically scanned and normalized with GAPDH as loading control. All data sets consist of three representative experiments. Significantly different from control; *P < 0.05 and **P < 0.01.

Fig. 3. Association of transcription factors to MnSOD promoter and enhancer element

The binding of transcription factor to the promoter or enhancer region of MnSOD gene was evaluated by ChIP assay in isolated skin tissues as described in Materials and methods. (A) Chromatins were immunoprecipitated with p50, Sp1 or NPM antibodies. Immunoprecipitated DNA was amplified by PCR using primers targeted to the enhancer element (1742–2083) and the promoter element (−154 to +6) of the MnSOD gene. (B) The immunoprecipitated proteins were detected by western analysis.

Fig. 4. Transcription factor binding with DNA

(A) Isolated skin cells were cross-linked with formaldehyde. The binding of proteins with chromatins was evaluated by ChIP assay. The product of the ChIP experiment using the p50 antibody was subjected to SDS–polyacrylamide gel electrophoresis for protein analysis. Proteins were detected by western blotting using antibodies specific to p65, p50, Sp1 or normal IgG. (B) Electrophoretic mobility shift assay was performed using purified nuclear extract from skin tissue as described in Materials and methods. For super-shift experiment, electrophoretic mobility shift assay reaction mixture was incubated with 1 µg of antibody specific to p50. The arrows point to the protein–DNA complex and super-shift protein–antibody complex. To verify the specificity for NF-κB binding, a 100-fold excess of non-radiolabeled NF-κB (cold) oligonucleotide was used to compete with the radiolabeled NF-κB probe in nuclear extracts (designated as self). The specificity of NF-κB bindings was also demonstrated by an addition of 100-fold mutant NF-κB (cold) DNA to nuclear extracts (non-self) along with the radiolabeled NF-κB probes.

Fig. 5. Suppression of MnSOD gene transcription by p50

JB6 cells were co-transfected with p50 expression vector (pcDNA3.1/p50) along with MnSOD reporter vector (pGL3/I₂E) or p50 (pcDNA3.1/p50) and Sp1 expression vector (pcDNA3.1/Sp1) with MnSOD reporter vector. Separately, p50 siRNA or control siRNAwas co-transfected with Sp1 expression vector in JB6 cells. (A) After transfection, cells were collected and luciferase activity was measured as a determinant of MnSOD gene transcription. (B) The over-expressed proteins were evaluated by western blotting using the same cellular extract. (C) The siRNA effect on p50 protein levels was detected by western blotting. (D) p50 siRNA increases MnSOD gene transcription mediated by Sp1 are shown. All data sets consist of three representative experiments. Statistically significant from control; *P < 0.05 and **P < 0.01; #P < 0.01.

Fig. 6. Suppression of MnSOD mRNA by p50

JB6 cells were co-transfected with either p50 expression vector (pcDNA3.1/p50) or empty vector (pcDNA3.1) individually or in combination with Sp1 expression vector (pcDNA3.1/Sp1) along with MnSOD reporter gene. Twenty-four hours after co-transfection, cells were treated with TPA (100 nM) for 12 h. Cells were then collected and RNA was isolated and purified. (A) Over-expressed p50 and Sp1 are shown by western blotting of cell homogenates. (B) RT–PCR of RNA isolated from control and TPA-treated cells was carried out using primers specific to mouse MnSOD gene and β-actin gene used as loading control. A representative of duplicate experiments with identical results is shown.