Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages

Abstract

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

Figure 1

Infection with BCG induces Notch1 upregulation in BMM. (a) BMM from C57/BL6 mice was infected with BCG at various MOIs for 28 h, and the expression of Notch1 was detected by western blot. (b) BMM from C57/BL6 mice or MyD88^−/− mice were infected with BCG at an MOI of 1∶10, and the cell lysate samples that were collected at different time points were analyzed by western blot. β-actin was used as a loading control. BCG, Bacillus Calmette-Guérin; BMM, bone marrow-derived macrophage; MOI, multiplicity of infection.

Figure 2

PPD treatment and BCG infection induces Mcl-1 expression in BMM. (a) BMM were treated with PPD (5 µg/ml) for the indicated times, and the expression of Mcl-1, Notch1 and cleaved Notch1 (Val1744) were detected by western blot. (b) BMM were infected with BCG (MOI 1∶10) for the indicated times, and the expression of Mcl-1 and Notch1 were detected by western blot. (c, d) BMM were treated with PPD (c) or infected with BCG (d) for the indicated times, and the levels of mcl-1 mRNA were detected by qPCR. Cells without any stimulation was labeled as ‘unstimulated' and cells without BCG infection was labeled ‘uninf'. Cells The results represent two independent experiments carried out in triplicate. * indicates where statistical significance (P<0.05) was observed between the levels in uninfected cells and cells infected for 10 and 28 h, respectively. BCG, Bacillus Calmette-Guérin; BMM, bone marrow-derived macrophage; Mcl-1, myeloid cell leukemia sequence-1; MOI, multiplicity of infection; PPD, purified protein derivative; qPCR, quantitative real-time PCR.

Figure 3

GSI treatment decreases Mcl-1 expression in BMM treated with PPD or infected with BCG. (a, b) BMM were pre-treated with GSI (25 µM) or a vehicle control (DMSO) for 1 h prior to stimulation with PPD (5 µg/ml). The expression of Mcl-1 and Notch1 were detected by western blot at 24 h post PPD treatment (a). The levels of mcl-1 mRNA were measured by q-PCR 1 h post PPD treatment (b). The results represent two independent experiments carried out in triplicate. * indicates where statistical significance (P<0.05) is observed. (c, b) BMM were pre-treated with GSI (25 µM) or a vehicle control (DMSO) for 1 h prior to infection with BCG (MOI 1∶10). The expression of Mcl-1 and Notch1 was detected by western blot 24 h post PPD treatment (c). The levels of mcl-1 mRNA were measured by q-PCR 10 h post infection (d). The results represent two independent experiments carried out in triplicate. * indicates where statistical significance (P<0.05) is observed. BCG, Bacillus Calmette-Guérin; BMM, bone marrow-derived macrophage; DMSO, dimethyl sulfoxide; GSI, gamma secretase inhibitor; Mcl-1, myeloid cell leukemia sequence-1; MOI, multiplicity of infection; PPD, purified protein derivative; qPCR, quantitative real-time PCR.

Figure 4

Silencing Notch1 in RAW264.7 decreases Mcl-1 expression. (a) RAW264.7 cells were treated with PPD (5 µg/ml) for the indicated times, and the expression of Mcl-1 and Notch1 were detected by western blot. (b) RAW264.7 cells were transiently transfected with the indicated plasmids for 36 h and then treated with PPD (5 µg/ml) for 12 h. Expression of Notch1 and Mcl-1 were detected by western blot. (c) RAW264.7 cells were transfected with indicated siRNA for 24 h. After the transfection, cells were treated with PPD (5 µg/ml) for 3 h. The levels of mcl-1 mRNA were measured by qPCR. * indicates where statistical significance (P<0.05) is observed. Mcl-1, myeloid cell leukemia sequence-1; PPD, purified protein derivative; qPCR, quantitative real-time PCR.

Figure 5

GSI treatment enhances apoptosis of BMM. (a) BMM were pre-treated with GSI or DMSO for 1 h and then stimulated with PPD (5 µg/ml) for 18 h. The results represent two independent experiments. (b) BMM were pre-treated with GSI or DMSO for 1 h and infected with BCG (MOI 1∶10). On day 5 post infection, apoptotic cells were measured. The results represent two independent experiments performed in triplicate. BCG, Bacillus Calmette-Guérin; BMM, bone marrow-derived macrophage; DMSO, dimethyl sulfoxide; GSI, gamma secretase inhibitor; PPD, purified protein derivative.

Figure 6

Direct association of Notch1 with mcl-1 promoter. (a) The consensus sequence of a potential CSL/RBP-Jκ binding site on the regulatory region in the human and murine mcl-1 promoter is shown. The area highlighted in grey is the conserved binding site. +1 indicates the translation start site. A potential binding site for CSL/RBP-Jκ was found between −171 and −163 bp upstream of transcription initiation site. * indicates conserved nucleotide sequences between human and murine species. (b) BMM were treated with PPD (5 µg/ml) for 24 h, and the genome of treated cells was immunoprecipitated using either an anti-RNA polymerase II or anti-Notch1 antibody. The resulting products were used to PCR amplify the promoter region of mcl-1 containing the potential CSL/RBP-Jκ binding site. Rabbit or mouse normal IgG were used as a control. The PCR reaction performed without addition of the precipitated DNA was used as a negative control, as indicated ‘Neg. control' in the figure. Input indicated the results when the PCR reaction was performed using genomic DNA for ChIP assay as a template. BMM, bone marrow-derived macrophage; ChIP, chromatin immunoprecipitation; GSI, gamma secretase inhibitor; Mcl-1, myeloid cell leukemia sequence-1; PPD, purified protein derivative.