The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

Abstract

In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3'-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

Figure 1

ER stress induces endogenous LC3 conversion and autophagosome formation. (a) HEK293 cells were treated with thapsigargin (TG, 2 μM) or tunicamycin (Tun, 3 μM) for the indicated time. Cell lysates were analyzed by Western blot for LC3 conversion (LC3-I, 18 kDa; LC3-II, 16 kDa), CHOP and GRP78 induction and β-actin (as a loading control) using antibodies sequentially. (b) Quantitation of autophagosomes per cell. HeLa cells transiently transfected with GFP-LC3 were either non-treated (NT), nutrient starved (NS) for 2 h or treated with TG (2 μM), Tun (3 μM) or BFA (2.5 μg/ml) for 4 h. For each condition, 15 to 35 cells were examined. The experiments were repeated 3 times. The results were summarized and expressed as the mean with the indicated standard deviation. (c) Representative electron micrographs of HEK293 cells either non-treated (NT), NS for 2 h or treated with Tun (3 μM) for 5 h. Autophagosomes are indicated by black arrow heads, ER by white arrow heads and N=nucleus; Bar=1 μm. (d) Enlarged images of autophagic vacuoles shown in (c) from NS and Tun-treated cells; Bar=0.2 μm.

Figure 2

Suppression of ER stress-induced autophagy by 3-MA inhibits the UPR. (a) HEK293 cells were either non-treated (NT) or pretreated with 3-methyladenine (3-MA, 10 mM) or wortmannin (WM, 100 nM) for 1 h. The cells were then either subjected to NS for 2 h, TG (2 μM) or Tun (3 μM) treatment for 4 h, either in the absence or continuous presence of 3-MA or WM as indicated. Cell lysates were analyzed for levels of LC3-I conversion to LC3-II, CHOP, GRP78 and β-actin by Western blot using antibodies sequentially. (b) HEK293 or HeLa cells were treated the same as in (a). Cell lysates were analyzed for CHOP, ATF4, GRP78 and β-actin expression by Western blot using antibodies sequentially. (c) Same as (b) except the cells were pretreated with wortmannin. (d) HEK293 cells were non-treated (0 h) or pretreated with 3-MA (10 mM) for 1 h, then treated with Tun (3 μM), either in the absence or continuous presence of 3-MA for the indicated time. Cell lysates were analyzed for levels of phospho-JNK1 (p-JNK1), JNK1, phospho-eIF2α (p-eIF2α) and eIF2α Further, total RNA were extracted and analyzed for expression of Xbp-1, spliced Xbp-1 (Xbp-1s) and β-actin (as a control) by RT-PCR. (e) HEK293 cells were non-treated (0 h) or pre-treated with wortmannin (100 nM) prior to Tun treatment and assayed for Xbp-1 splicing as described in (d). For all panels, the experiments were repeated 2 to 3 times.

Figure 3

Beclin1 is required for ER-stress induced autophagy but not for UPR activation. HEK293 cells were transfected with either control siRNA (siCtrl) or siRNA against Beclin1 (siBec). The cells were non-treated (NT) or treated with TG (2 μM) or Tun (3 μM) for 5 h. (a) Cell lysates were analyzed for levels of LC3-I conversion to LC3-II, Beclin1 and β-actin (as a loading control), or (b) CHOP, Beclin1 and β-actin by Western blot using antibodies sequentially. (c) SiRNA transfected HEK293 cells were treated with Tun (3 μM) for indicated time. Cell lysates were analyzed for levels p-JNK1 and JNK1 by Western blot using antibodies sequentially. Total RNA were extracted and analyzed for expression of Xbp-1, Xbp-1s and β-actin (as a control) by RT-PCR. (d) Quantitation of the p-JNK1 level shown in (c) after normalization with total JNK1 level during the time course of Tun treatment in cells transfected with either siCtrl or siBec. (e) Time course of TG induction of CHOP and Xbp-1 splicing in cells transfected with either siCtrl or siBec. A lower concentration of TG (0.3 μM) was used in this experiment. The levels of Beclin1 and β-actin were monitored in parallel.

Figure 4

GRP78 knockdown spontaneously activates the UPR and impairs cell viability. (a) HEK293 cells were either transfected with 40 nM siCtrl or siRNA against human Grp78 (siGrp78) for 48 h. Cell lysates were analyzed for GRP78, GRP94, calreticulin (CRT) and β-actin (as loading control) or CHOP, PDI and β-actin expression by Western blot using antibodies sequentially. The experiments were repeated 2 times. (b) After transfection with either 40 nM siCtrl or siGrp78 for 48 h, HEK293 cells were subjected to clonogenic survival assay. The survival fraction of siCtrl transfected cells was set as 100%. The standard deviation was shown, *, p<0.05. (c) HEK293 cells were transfected with 40 nM siCtrl or siGrp78 for 48 h. The cells were then treated with Tun (3 μM) for indicated time. Cell lysates were analyzed for levels of p-JNK1 and JNK1 or GRP78 and β-actin (as a loading control) by Western blot using antibodies respectively. Total RNA were extracted and analyzed for expression of Xbp-1, Xbp-1s and β-actin (as a control) by RT-PCR. (d) The levels of p-JNK1 as shown in (c) were quantitated, normalized against JNK1 and plotted with the relative level in non-treated (0 h), siCtrl transfected cells set as 1.

Figure 5

GRP78 knockdown blocks autophagosome formation induced by ER stress or NS. (a) HeLa cells were transiently transfected with GFP-LC3 alone (−) or in combination with 40 nM of siCtrl or siGrp78. The cells were then either non-treated (NT) or treated with Tun (3 μM) for 4 h. Representative images of GFP-LC3 punctate dot formation in live cells captured by fluorescence microcopy are shown. (b) Quantitation of autophagosome formation in cells treated as in (a). The results from 3 independent experiments were summarized and expressed as the mean with the indicated SD. (c) Western blot analysis of GRP78, GRP94 and β-actin (as loading control) protein levels in cells treated as in (a). (d–f) Same as (a–c) except the cells were either non-treated (NT) or subjected to NS. (g) HeLa cells were transfected with 40 nM of either siCtrl or siGrp78 and subjected to NS treatment for 2 h, TG or Tun treatment for 4 h in the absence or presence of zVAD-fmk (40 μM) as indicated on top. The levels of LC3-I and II, CHOP and β-actin were detected by Western blot. The experiments were repeated 2 times.

Figure 6

GRP78 knockdown disrupts ER integrity but not PI3KC3-Beclin1 complex formation. (a) Cell lysates from siRNA transfected HEK293 cells were immunoprecipitated with anti-Beclin1 antibody. The immunocomplex was analyzed for PI3KC3 and Beclin1 levels by Western blot using antibodies sequentially. Part of the cell lysates were also analyzed for GRP78, PI3KC3 and Beclin1 expression by Western blot using antibodies sequentially. (b) HEK293 cells were transfected with pcDNA3 (vector control, vec) or indicated amount of pcDNA3-His-GRP78 (His-GRP78). Immunoprecipitation, analysis of immunocomplex and His-GRP78, PI3KC3 and Beclin1 expression were done the same as in (a). (c) Representative electron micrograph of cells transfected with siCtrl (left panel) showing sparse ER structures (white arrow heads) or siGrp78 (right panel) showing increased ER structures with expanded lumens (white arrow heads). N, nucleus; Bar=1 μm. (d) Quantitation of the ER area in the cytoplasm in cells transfected with either siCtrl or siGrp78. The standard deviation is shown, *, p<0.0001. (e) Knockdown of XBP-1 recovered normal levels of stress-induced autophagosome formation inhibited by knockdown of GRP78. The indicated siRNA(s) was transiently co-transfected with GFP-LC3 into HeLa cells. The cells were either non-treated (NT), treated with Tun (3 μM) for 4 h or NS for 2 h. The formation of autophagosome under each condition was assayed by fluorescence microscopy and quantitated. The experiments were repeated 2 times. The results were summarized and expressed as the mean with the indicated standard deviation. The insert shows Western blot analysis of GRP78, XBP-1 and β-actin levels in cells transfected with siCtrl (lane 1), siGrp78 (lane 2), siXbp-1 (lane 3) and siGrp78 plus siXbp-1 (lane 4) respectively.

Figure 7

Modulation of UPR signaling and autophagy pathways by 3-MA and GRP78 in human cells. Nutrient starvation (NS) as well as ER stress leads to LC3 conversion and autophagosome formation. This step requires Beclin1, which is an interactive partner of PI3KC3. Treatment of cells with 3-MA blocks both NS and ER stress-induced LC3 conversion, as well as UPR activation by ER stress. In contrast, treatment of cells with wortmannin or knockdown of Beclin1 blocks both nutrient and ER-stress induced LC3 conversion but has no effect on UPR activation. JNK activation and eIF2α phosphorylation have been reported to induce LC3 conversion, leading to autophagy. Knockdown of GRP78 results in ER stress, activation of the UPR pathways and massive ER expansion and disorganization. In GRP78 depleted cells, the complex formation between Beclin1 and PI3KC3 is intact and LC3 conversion is slightly enhanced. However, autophagosome formation induced by both NS and ER stress is inhibited.