A caspase-dependent pathway is involved in Wnt/β-catenin signaling promoted apoptosis in Bacillus Calmette-Guerin infected RAW264.7 macrophages

Abstract

Apoptosis of alveolar macrophages following Mycobacterium tuberculosis infection have been demonstrated to play a central role in the pathogenesis of tuberculosis. In the present study, we found that Wnt/β-catenin signaling possesses the potential to promote macrophage apoptosis in response to mycobacterial infection. In agreement with other findings, an activation Wnt/β-catenin signaling was observed in murine macrophage RAW264.7 cells upon Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection at a multiple-of-infection of 10, which was accompanied with up-regulation of pro-inflammatory cytokines TNF-α and IL-6 production. However, the BCG-induced TNF-α and IL-6 secretion could be significantly reduced when the cells were exposed to a canonical Wnt signaling ligand, Wnt3a. Importantly, the activation of Wnt/β-catenin signaling was able to further promote apoptosis in BCG-infected RAW264.7 cells in part by a mitochondria-dependent apoptosis pathway. Immunoblotting analysis further demonstrated that Wnt/β-catenin signaling-induced cell apoptosis partly through a caspase-dependent apoptosis mechanism by down-regulation of anti-apoptotic protein Mcl-1, and up-regulation of pro-apoptotic proteins Bax and cleaved-caspase-3, as well as enhancement of caspase-3 activity in BCG-infected RAW264.7 cells. These data may imply an underlying mechanism of alveolar macrophages in response to mycobacterial infection, by which the pathogen induces Wnt/β-catenin signaling activation, which in turn represses mycobacterium-trigged inflammatory responses and promotes mycobacteria-infected cell apoptosis.

Figure 1.

The impacts of Wnt/β-catenin signaling in RAW264.7 cells in response to BCG infection. (A) The impact of BCG infection on macrophages was first ascertained by evaluating the relative firefly luciferase activity and the expression of Wnt target gene Cyclin D1 in RAW264.4 cells that transfected with β-catenin/TCF reporter TOPflash and treated with indicated conditions. In the absent of BCG stimulation, RAW264.7 cells showed a weak baseline Wnt signaling activity. However, the Wnt signaling activity could be dramatically elevated by Wnt3a and the BCG infection, and repressed by a Wnt signaling inhibitor, DKK1; (B) Immunoblots showed the expressions of Wnt target gene Cyclin D1, nuclear β-catenin and. P65 subunit of NFκB; (C) An ELISA analysis of the supernatants of RAW264.7 cells treated with indicated conditions showed the impacts of Wnt3a signaling on the productions of IL-6 and TNF-α in macrophages. Compared to a naïve control, * p < 0.05; ** p < 0.01. Data represented the mean ± SD from three independent triplicated experiments (n = 15).

Figure 2.

The activation of Wnt/β-catenin signaling promotes BCG-infected cell apoptosis. (A) An MTT assay determined the cell viability of RAW264.7 cells treated with indicated conditions for 24 h; (B) Representatives of dot plot from five independent experiments of flow cytometry analysis of the apoptotic fraction of RAW264.7 cells treated with indicated conditions for 24 h; (C) A time-dependent apoptotic cell death fraction of RAW264.7 cells treated with indicated condition for different time points. Compared to a naïve control, * p < 0.05; ** p < 0.01. Data represented the mean ± SD from five independent triplicated experiments (n = 15 in A; n = 9 in B and C).

Figure 3.

Morphological analysis of the impact of Wnt3a on BCG-infected RAW264.7 cells apoptosis. RAW264.7 cells exposed to Wnt3a-CM or control-CM, followed by infection of BCG at MOI of 10 for 24 h. (A) Representative images of SEM (top panel) and TEM (bottom panel) of healthy RAM264.7 cells (left panel) and apoptotic cells (right panel); (B) Percentages of cells with an apoptotic phenotype as determined by morphology using EM images. Compared to a naïve control, ** p < 0.01. Data represented the mean ± SD from three independent triplicated experiments (n = 9). Bar in SEM images = 2 μm; Bar in TEM images = 10 μm. Arrows in A indicated apoptotic bodies.

Figure 4.

Impact of Wnt/β-catenin signaling on mitochondrial membrane potential (ΔΨm) of RAW264.7 cells. (A) Representatives of dot plot of flow cytometry analysis for ΔΨm of cells treated with indicated condition for 12 h; (B) fractions of cells with low ΔΨm treated with indicated conditions for 12 h; (C) Immunoblots of apoptosis-related proteins in RAW264.7 cells treated with indicated conditions for 24 h. Compared to a naïve control, * p < 0.05; ** p < 0.01. Data represented the mean ± SD from three independent duplicate experiments (n = 9).

Figure 5.

Wnt/β-catenin promotes BCG-infected RAW264.7 cell apoptosis is in part through a caspase-dependent pathway. A caspase-dependent apoptotic pathway was examined in the RAW264.7 cells treated with Wnt3a, BCG, pan caspase inhibitor zVAD, and/or Wnt signal inhibitor DKK1. (A) Cell viability determined by an MTT assay for RAW264.7 cells treated with indicated conditions for 6 h; (B) Apoptotic cell fraction determined by a flow cytometric assay for RAW264.7 cells treated with indicated conditions for 24 h; (C) Relative caspase activity of RAW264.7 cells treated with indicated conditions containing pan caspase inhibitor zVAD for 24 h; (D) Relative caspase activity of RAW264.7 cells treated with indicated conditions containing Wnt signal inhibitor DKK1 for 24 h. Compared to a naïve control, * p < 0.05; ** p < 0.01; compared to the cells treated with zVAD (C) or DKK1 (D) alone, ^# p < 0.05; ^## p < 0.01. Data represented the mean ± SD from three independent triplicate experiments (n = 15); and (E) A proposed mechanism whereby the regulatory role of Wnt/β-catenin signaling in the apoptosis macrophages upon mycobacterial infection. In this model, upon invasion of mycobacteria, the pathogen is first recognized by TLRs of the cells, which in turn triggers the TLR signaling cascade to initiate inflammatory response and activates Wnt/β-catenin signaling that plays a negative feedback inhibitory role in modulation of inflammatory responses. Meanwhile, the activation of the Wnt/β-catenin pathway subsequently promotes apoptotic cell death in the mycobacteria-infected macrophages, in part through a caspase-dependent apoptosis pathway.