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. 2011 Oct 7;286(40):35227-35.
doi: 10.1074/jbc.M111.233502. Epub 2011 Aug 13.

Activating transcription factor-6 (ATF6) mediates apoptosis with reduction of myeloid cell leukemia sequence 1 (Mcl-1) protein via induction of WW domain binding protein 1

Nobuhiro Morishima  1 , Keiko Nakanishi, Akihiko Nakano
Affiliations

Activating transcription factor-6 (ATF6) mediates apoptosis with reduction of myeloid cell leukemia sequence 1 (Mcl-1) protein via induction of WW domain binding protein 1

Nobuhiro Morishima et al. J Biol Chem. .

Abstract

Endoplasmic reticulum (ER) stress is involved in both physiological and pathological apoptosis. ER stress triggers the unfolded protein response (UPR), which can then initiate apoptosis, when the cell fails to restore ER homeostasis. However, the mechanism employed by the UPR to lead cells into apoptosis is unknown. Among the three proximal sensors of ER stress, activating transcription factor-6 (ATF6) is specifically activated in apoptotic myoblasts during myoblast differentiation. This implies that active ATF6 has the ability to mediate apoptosis. Here, we demonstrate that overexpression of active ATF6 induced apoptosis in myoblast cells. Moreover, coexpression of a dominant negative form of ATF6 suppressed apoptosis. This suggested that apoptosis-related pathways depended on ATF6-mediated transcription activation. ATF6 caused up-regulation of the WBP1 (WW domain binding protein 1), probably via an indirect mechanism. Furthermore, WBP1 was also found to be proapoptotic. The silencing of WBP1 with small hairpin RNAs caused partial, but significant suppression of ATF6-induced apoptosis. Overexpression of active ATF6 or WBP1 caused a specific reduction in an anti-apoptotic protein, Mcl-1 (myeloid cell leukemia sequence 1). This suggested a molecular link between the UPR and an apoptosis regulator. Neither Bcl-2 nor Bcl-x(L) were reduced upon apoptosis induction in C2C12 cells that overexpressed ATF6 or WBP1. Cells treated with ER stressors underwent apoptosis concomitant with an up-regulation of WBP1 and suppression of Mcl-1. These results suggested that Mcl-1 is a determinant of cell fate, and ATF6 mediates apoptosis via specific suppression of Mcl-1 through up-regulation of WBP1.

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Figures

FIGURE 1.
FIGURE 1.
Overexpression of active ATF6 induced apoptosis in myoblast cells. A, apoptosis induced by GFP-ATF6(1–360) was assessed by cell morphology. Bars represent an average of three independent experiments. B, cell morphology 24 h after cell transfection with GFP-ATF6(1–360), shows nuclei stained with Hoechst 33342 dye. Scale bar, 50 μm. C, merged image of GFP-ATF6(1–360) and corresponding phase contrast images. Arrow and arrowhead indicate live and dead cells, respectively. Scale bar, 50 μm. D, active ATF6 induced apoptosis in different cell lines.
FIGURE 2.
FIGURE 2.
Active ATF6 induced caspase activation. A, C2C12 cells were electroporated with GFP vector or GFP-ATF6(1–360). After 24 h, dead and live cells were separated and subjected to Western blot analysis. Arrow indicates GFP-ATF6(1–360). α-Tubulin was the loading control. B, the same blot shown in A was sequentially probed with anti-caspase-12, anti-caspase-9, anti-caspase-3, and anti-BiP antibodies. Black arrows show caspase precursors; gray arrows show cleaved caspase products. IB, immunoblot.
FIGURE 3.
FIGURE 3.
Dominant negative form of ATF6 suppressed apoptosis induced by active ATF6. A, C2C12 cells grown in six-well plates were transiently co-transfected with GFP or GFP-ATF6(1–360) (0.3 μg) and either the pDsRed vector (control) or DsRed-ATF6(158–360) (dominant negative) (1.8 μg). Apoptosis was assessed by cell morphology. B, CHOP did not induce apoptosis in C2C12 cells. C2C12 cells were transiently transfected with GFP or GFP-tagged CHOP. The apoptotic efficiencies of GFP-ATF6(1–360) and the dominant negative GFP-ATF6(158–360) are included in the bar graph.
FIGURE 4.
FIGURE 4.
WBP1 induced apoptosis in C2C12 cells. A, transfection of WBP1-GFP caused apoptosis in C2C12 cells. B, Western blot shows WBP1 expression in apoptotic cells after electroporation with WBP1-GFP. High molecular mass complexes of WBP-GFP (asterisk) that were resistant to denaturation were reproducibly detected. C, activation of caspases in apoptotic cells. The same blot in B was sequentially probed with anti-caspase-12, anti-active caspase-9, and anti-caspase-3. Black arrows show caspase precursors; gray arrows show cleaved caspase products. D, co-localization of WBP1-GFP with Sar1. C2C12 cells were transfected with WBP1-GFP, fixed, and immunostained with anti-Sar1 antibody. Scale bar, 10 μm. IB, immunoblot.
FIGURE 5.
FIGURE 5.
WBP1 was induced in apoptotic cells. A, real-time quantitative PCR analysis. Fold changes in WBP1 and BiP expression were determined by comparing the GAPDH-normalized ratios of mRNAs from active ATF6-induced samples versus vector-transfected samples. Bars represent an average of three independent experiments. B, active ATF6 induced WBP1 expression in apoptotic cells. C2C12 cells were electroporated with GFP-ATF6(1–360). After 24 h, dead and live cells were separated and subjected to Western blot analysis. C, WBP1 was induced in apoptotic cells treated with ER stressors tunicamycin (TUN) and thapsigargin (TG). UT, untreated; IB, immunoblot.
FIGURE 6.
FIGURE 6.
WBP1 knockdown suppressed apoptosis induced by active ATF6. A, C2C12 cells were electroporated with GFP-ATF6(1–360) and WBP1 shRNA, version A, at a ratio of 1:3 (w/w). After 24 h, apoptosis was assessed by cell morphology. B, WBP1 levels were analyzed by Western blot (IB). UT, untreated.
FIGURE 7.
FIGURE 7.
Analysis of Bcl-2 family proteins in apoptotic cells. A, specific reduction of Mcl-1 expression in apoptotic cells transfected with active ATF6 or WBP1. B, co-expression of Bcl-xL suppressed apoptosis induced by WBP1. C2C12 cells were grown in six-well plates and transiently co-transfected with GFP or WBP1-GFP (0.3 μg) and pcDNA3.1 vector or Bcl-xL (1.8 μg). C, down-regulation of Mcl-1 in cells that overexpressed Bcl-xL. C2C12 cells were electroporated with WBP1-GFP and Bcl-xL at a 1:1 ratio (w/w). Mcl-1 was analyzed by Western blot. The band intensity was normalized with α-tubulin as a standard. Representative data from three independent experiments are shown. D, specific reduction of Mcl-1 in apoptotic cells treated with ER stressors tunicamycin (TUN) and thapsigargin (TG). UT, untreated; IB, immunoblot.
FIGURE 8.
FIGURE 8.
Mcl-1 was a cell fate determinant in C2C12 cells. A, Mcl-1 knockdown caused apoptosis. C2C12 cells were transfected with Mcl-1 shRNA; dead cells were counted after 24 h. Mcl-1 was analyzed by Western blot (lower panel). B and C, coexpression of Mcl-1 caused suppression of the apoptosis induced by active ATF6 (B) or WBP1 (C). C2C12 cells were grown in six-well plates and transiently co-transfected with GFP, GFP-ATF6(1–360), or WBP1-GFP (0.3 μg) and pcDNA3.1 vector or Mcl-1 (1.8 μg).
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