Transcription factor Sp1 is involved in expressional regulation of coxsackie and adenovirus receptor in cancer cells

Abstract

Coxsackie and adenovirus receptor (CAR) was first known as a virus receptor. Recently, it is also known to have tumor suppressive activity such as inhibition of cell proliferation, migration, and invasion. It is important to understand how CAR expression can be regulated in cancers. Based on an existence of putative Sp1 binding site within CAR promoter, we investigated whether indeed Sp1 is involved in the regulation of CAR expression. We observed that deletion or mutation of Sp1 binding motif (-503/-498) prominently impaired the Sp1 binding affinity and activity of CAR promoter. Histone deacetylase inhibitor (TSA) treatment enhanced recruitment of Sp1 to the CAR promoter in ChIP assay. Meanwhile, Sp1 binding inhibitor suppressed the recruitment. Exogenous expression of wild-type Sp1 increased CAR expression in CAR-negative cells; meanwhile, dominant negative Sp1 decreased the CAR expression in CAR-positive cells. These results indicate that Sp1 is involved in regulation of CAR expression.

Figure 1

Comparative analysis of CAR promoter activity. (a) Schematic description of CAR gene promoter containing specific transcription factor binding sites and its deletion constructs. The luciferase reporter gene constructs were cloned into pGL3 vector. (b, c) Each CAR promoter-luciferase reporter construct was cotransfected into two different CAR-positive cells with β-galactosidase gene. *P = 0.0003, **P < 0.001 relative to other constructs, not pGL3. Bars represent SD. (d) Either Wt or mut of the core promoter (−585/−400) was cotransfected into four different CAR-positive cells with β-galactosidase gene. From total cell extracts, luciferase activities were examined and normalized by β-galactosidase activity. The relative luciferase activities were determined by statistical analysis of three independent experiments. ^#P < 0.05 relative to each of wild-type. Bars represent the mean ± SD.

Figure 2

Effect of the mutation in Sp1 binding motif or Sp1 on CAR promoter activity. (a, b) For the examination of the effect of Sp1 on the promoter activity, either Sp1 or its dominant negative form (Sp1 DN) was cotransfected into MCF-7 (a) and PC-3 cells (b) with reporter construct of core promoter and β-galactosidase gene in the presence or absence of mithramycin. *P < 0.05, ***P > 0.05 relative to pCMV vector control, **P < 0.05 relative to Sp1 control. (c, d) For the examination of the effect of Sp1 binding site on the core promoter activity in response to ectopic expression of Sp1, either wild type or mutant type of core promoter was cotransfected with Sp1 and β-galactosidase into MCF-7 (c) and PC-3 (d), and the following procedures were identical to above case. From total cell extracts, luciferase activities were examined and normalized by β-galactosidase activity. The relative luciferase activities were determined by statistical analysis of three independent experiments. ^#P = 0.0003, ^{# # #}P < 0.05 relative to pGL3 vector control, ^{# #}P < 0.05 relative to wild-type core promoter (−585/−400) DMSO. Bars represent the mean ± SD.

Figure 3

Sp1 binds to its putative binding motif within the core promoter. (a) Labeled probe and cold wild or mutant type probes used for competition assay were used as described in Section 2. The closed triangle indicates the Sp1-DNA probe complex. (b) Labeled probe was preincubated with either MTM or DMSO (vehicle) for 1 h at 4°C before incubation with the nuclear proteins. (c, d) Chromatin immunoprecipitation assay. Each of cell lysates was extracted from MCF-7 cells or HepG2 (e) cells after treatment either by TSA or MTM. Chromatin immunoprecipitation was performed with anti-Sp1, anti-acetyl histone H3, and then PCR was carried out with primers flanking Sp1 binding site.

Figure 4

Effects of MTM on CAR promoter activity and mRNA expressions. HCT116 (a), HeLa (c), and HepG2 (e) were treated with either MTM or DMSO (vehicle) for 24 h at the concentration indicated. The mRNA expressions of CAR and GAPDH used as an internal control were examined by Semiquantitative RT-PCR. Core promoter reporter construct was cotransfected with β-galactosidase into the three different cells and followed by treatment with either MTM or DMSO for 24 h at the concentration indicated (b, d, and f). The relative luciferase activity was determined by statistical analysis of three independent experiments. Bars represent the mean ± SD.

Figure 5

Effects of Sp1 or MTM on TSA-induced CAR promoter activity and mRNA expression. HepG2 (a, b) and MCF-7 (c) cells were pretreated with either MTM or DMSO for 30 min and followed by TSA treatment for 24 h. The mRNA expressions of CAR and GAPDH were examined by Semiquantitative RT-PCR. The extent of histone3 acetylation with actin used as a loading control was analyzed by western blotting. In contrary to (a), CAR mRNA expression was examined depending on TSA concentration with fixed concentration of MTM (c). The mRNA levels of lane1 were set to 1.0. (d) MCF-7 cells were transfected with the core promoter reporter construct and β-galactosidase and treated as in (c). The relative luciferase activity was determined. Bars represent S. D. Effects of WT-Sp1 and DN-Sp1 overexpression on CAR expression. (e) MCF-7 cells were transfected with WT-Sp1 (0.1, 0.25, 0.5, and 1 μg), treated with TSA (50 ng/mL) for 24 h, and examined for CAR mRNA expression by Semiquantitative RT-PCR. Relative expression of CAR mRNA to GAPDH was calculated based on the band intensity. (f) Similarly, HCT116 cells were transfected with DN-Sp1 (0.1, 0.25, 0.5, and 1 μg) in the absence of TSA, and relative expression of CAR mRNA was determined. The mRNA levels of lane1 were set to 1.0.

Figure 6

Effect of Sp1 binding on infectivity of adenoviral containing β-gal. (a) MCF-7 cells were treated by TSA with or without pretreatment of mithramycin and infected by Ad-β-gal (50 MOI). Photographs of the cells stained for β-galactosidase activity. (a) represents whole cells and some part of the whole cells under a higher magnification, respectively, scale bar: 50 μm. (b) Quantitation of β-galactosidase-positive cells after the virus infection. β-galactosidase-positive cells were counted from three nonoverlapping fields of the cells treated as indicated. Bars represent the mean ± SD.