Regulation of MCP-1 chemokine transcription by p53

Abstract

Background: Our previous studies showed that the expression of the monocyte-chemoattractant protein (MCP)-1, a chemokine, which triggers the infiltration and activation of cells of the monocyte-macrophage lineage, is abrogated in human papillomavirus (HPV)-positive premalignant and malignant cells. In silico analysis of the MCP-1 upstream region proposed a putative p53 binding side about 2.5 kb upstream of the transcriptional start. The aim of this study is to monitor a physiological role of p53 in this process.

Results: The proposed p53 binding side could be confirmed in vitro by electrophoretic-mobility-shift assays and in vivo by chromatin immunoprecipitation. Moreover, the availability of p53 is apparently important for chemokine regulation, since TNF-alpha can induce MCP-1 only in human keratinocytes expressing the viral oncoprotein E7, but not in HPV16 E6 positive cells, where p53 becomes degraded. A general physiological role of p53 in MCP-1 regulation was further substantiated in HPV-negative cells harboring a temperature-sensitive mutant of p53 and in Li-Fraumeni cells, carrying a germ-line mutation of p53. In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-alpha treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-alpha addition, directly confirming a crosstalk between p53 and MCP-1.

Conclusion: These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.

Figure 1

MCP-1 expression in HPV16 immortalized human keratinocytes and in vitro binding activity of wild-type p53 at the MCP-1 regulatory region. (A) Upper panel: RT-PCR for MCP-1 and GAPDH in HPV16 E6-, E7-, and E6/7 immortalized human foreskin keratinocytes. Cells were treated with TNF-α (250 U/ml) (+) for 6 h. (-) untreated control. The RT-PCR products were separated on 1.5% agarose gel. Lower panel: Western blot analysis of p53. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Equal loading was confirmed using an actin specific antibody. (B) RT-PCR for MCP-1, p53 and GAPDH in E7-immortalized keratinocytes after lentiviral p53 shRNA delivery. Cells were treated as described in panel A. (C) Schematic overview of the MCP-1 gene. The region -2750 to -2150 bp upstream of the MCP-1 start site harbors two NFκB and one AP-1 binding site. The in silico identified putative p53 binding site (-2422/-2391) relative to NFκB and AP-1 is indicated. Black boxes: exon I-III; white box: polyadenylation site (poly-A), (striped grey box): 3'-regulatory region. (D) Left panel: Electrophoretic mobility shift assay (EMSA): 60 ng of recombinant p53 protein was incubated with [³²P]-labeled double-stranded oligonucleotides harboring the wild-type p53-specific RGC-site, the mutated version thereof and the putative MCP-1 p53 binding site, respectively. In addition, binding was also performed in the presence of 200 ng of BSA, anti-GAPDH, DO-1, PAb1801, or PAb421. The positions of free probe, DNA in complex with p53, and DNA in complex with p53 plus supershifting antibodies are indicated by arrows. (D) Right panel: EMSA: comparative binding analysis for wild-type p53 (wtp53) using specific RGC and non-specific RGC mutated versus MCP-1 DNA. DNA binding activities of wild-type p53 (wtp53), p53pro248 and p53pro273 mutants are indicated.

Figure 2

MCP-1 induction by TNF-α after reconstitution of wild-type p53 activities. 4Bv (A) and BT2E (B) cells were cultivated at 37°C or shifted to 32°C overnight. Stimulation was done with TNF-α for 4 h or 6 h. Total cellular RNA was separated in a 1% agarose gel and transferred to a Gene screen Plus membrane. The filter was subsequently hybridized with a p21, MCP-1 and c-myc cDNA probe. The positions of the 28S and 18S ribosomal RNA are indicated.

Figure 3

Temperature shifting does not affect the cell cycle or TNF-α signaling. (A) 4Bv cells were maintained at 37°C or 32°C overnight, followed by treatment with or without TNF-α for 6 h. Flow-cytometric analysis was performed on DAPI/SR 101 stained cells to determine the percentage of 4Bv cells in different cell cycle phases (G1, S, G2/M). Mean values (columns) and standard deviations (bars) are given for three independent experiments. Steady state expression (B) and phosphorylation (C) of p38 MAP kinase was analyzed after treatment of 4Bv cells (at 37°C or 32°C) with TNF-α as indicated. Total cellular protein (50 μg) was separated in a 12% SDS-PAGE gel. After electrotransfer, the filter was consecutively incubated with antibodies as indicated and re-probed with anti-actin antibody as loading-controls. (-): untreated control cells; (15')/(30')/(60'): cells treated with 250 U/ml of TNF-α for the indicated time.

Figure 4

Expression of MCP-1 in Li Fraumeni fibroblasts and after p53 knockdown in A172 cells. (A) RT-PCR of MCP-1 and GAPDH in Li Fraumeni cells (MDAH041) in passage 8 (p:8, p53 mut/wt) and passage 160 (p:160, p53 mut/mut), respectively. Cells were treated with 250 U/ml of TNF-α for 6 h (+). Untreated control cells: (-). (B) Western blot analysis of p53 in MDAH041 cells (p:8) and (p:160) treated with TNF-α (250 U/ml) (+) for 6 h. (-): untreated control. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Actin confirms equal loading. (C) A172 cells were transiently transfected with pSUPER-p53 or with the empty pSUPER vector. After 24 h, cells were stimulated with TNF-α for additional 5 h. Cells were harvested and Western blot analysis was performed. Filters were probed with anti-p53 (DO-1) (see also panel B). (D) 4 μg of total RNA were separated in a 1% agarose gel and transferred to a Gene screen Plus membrane. The filter was subsequently hybridized with a MCP-1 cDNA probe. Hybridization of the same filter with a cDNA probe coding for the housekeeping gene β-actin confirmed equal loading. The positions of the 28S and 18S ribosomal RNA are indicated.

Figure 5

p53 binds to the enhancer region of MCP-1 in vivo. (A) DNA sequence of the 5'-regulatory region of the MCP-1 gene (-2550 to -2250) indicating MCP-1 specific primer pairs used for the chromatin immunoprecipitation (ChIP) assay by arrows. The p53 binding site is marked with bold letters. (B) 4Bv cells were maintained at 37°C or shifted to 32°C for 5 h and stimulated with TNF-α for additional 5 h. Cells were harvested for chromatin immunoprecipitation (ChIP) assay as described in Materials and Methods. Samples were subjected to immunoprecipitation without antibody (no ab), with p53 antibody or rabbit IgG (IgG); total lysate was used as a control for PCR amplification (input). (B, left panel) p53 binding was tested by using MCP-1 specific primers; (B, right panel) p21 primers were used as a positive control. Untreated control cells (-); cells treated with 250 U/ml of TNF-α for 5 h (+).