The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced aut

1. The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells J Li, M Ni, B Lee, E Barron, DR Hinton and AS Lee Cell Death and Differentiation (2008) 15, 1460-1471

2. Endoplasmic Reticulum (ER) ER stress Inducers: Thapsigargin (TG); Tunicamycin (Tun); Brefeldin A (BFA) http://en.wikipedia.org/wiki/Unfolded_protein_response

3. Unfolded Protein Response (UPR) Grp94 PDI http://en.wikipedia.org/wiki/Unfolded_protein_response

4. Autophagy: a defensive mechanism ER Stress GRP78/BiP UPR 3-Methyladenine (3-MA); Wortmannin (WM) ? ? ? http://www.nature.com/cdd/journal/v14/n9/full/4402200a.html

5. Methods • Cell culture: HEK293 and Hela cells • Western blot analysis • Co-immunoprecipitation assay • Transfection and RNA interference • Fluorescence and electron microscopy • RT-PCR • Clonogenic survival assay

6. Compare ER stress-induced autophagy formation and UPR activation • ER stress inducers: • TG: a SERCA inhibitor that blocks Ca2+ reuptake to the ER • Tun: an inhibitor of N glycosylation • BFA: a blocker of protein transportation from ER to Golgi • Autophagy: • Inducer: Nutrient Starvation (NS) • LC3 conversion from LC3-I (18 kDa) to LC3-II (16 kDa) • Fluorescence microscopy to check autophagosome formation • Electron microscopy to check autophagosome formation • UPR activation:Induction of CHOP and GRP78

7. Results Fig1a&b&S1 ER stress induces autophagy formation and UPR activation HEK293 cells were treated with TG (2μM), Tun (3μM), BFA (2.5μg/ml). Cell lysates were analyzed by western blot for LC3 conversion, CHOP and GRP78 induction and b-actin (a). Fluorescent images showed GFP-LC3 punctate dot (autophagosome) formation (S1), which were quantified and expressed as the mean with the indicated S.D.(b).

8. Results Fig1c&d ER stress induces autophagosome formation (c) Representative electron micrographs of HEK293 cells treated with different conditions. Autophagosomes are indicated by black arrow heads, ER by white arrow heads; N, nucleus; (d) Enlarged autophagic vacuoles.

9. 2. The dependence of UPR activation on autophagy formation • ER stress inducers: TG, and Tun • Autophagy inducer: Nutrient Starvation (NS) • Autophagy inhibitors: • 3-methyladenine: 3-MA, a inhibitor of endogenous lysosomal protein degradation that specifically targets PI3KC3 • Wortmannin: WM, another PI3K inhibitor • UPR activation: • Induction of CHOP and GRP78, ATF4 • Activation of eIF2a and JNK1 • Splicing of Xbp-1 (X-box-binding protein-1) mRNA

10. Results Fig2a Autophagy suppression by 3-MA inhibits ER stress-induced CHOP induction HEK293 cells were either non-treated (NT) or pretreated with 3-MA (10mM) or WM (100nM) for 1h. The cells were then either subjected to NS for 2h, TG or Tun treatment for 4h, either with or without 3-MA or WM. Cell lysates were analyzed forlevels of LC3-I conversion to LC3-II, CHOP, GRP78 and b-actin expression by western blot.

11. Results Fig2b&c Autophagy suppression by 3-MA inhibits ER stress-induced ATF4 induction HEK293 cells or Hela cells were either non-treated (NT) or pretreated with 3-MA (10mM) or WM (100nM) for 1h. The cells were then either subjected to NS for 2h, TG or Tun treatment for 4h, either with or without 3-MA (b) or WM (c). Cell lysates were analyzed for CHOP, ATF4, GRP78 and b-actin expression by western blot.

12. Results Fig2d&e 3-MA inhibits Tun-induced eIF2a and JNK1 activation, Xbp-1 mRNA splicing HEK293 cells were non-treated (0h) or pretreated with 3-MA (d) or WM (e) for 1h prior to Tun treatment for the indicated time. Cell lysates were analyzed for levels of phospho-JNK1, JNK1, phospho-eIF2a and Eif2a (d). Total RN was extracted and analyzed for expression of Xbp-1, spliced Xbp-1 and b-actin by RT-PCR (d and e).

13. 3. The role of Beclin1 in UPR activation • ER stress inducers: TG, and Tun • Beclin1 siRNA:Beclin1 is required for NS-induced autophagy and is an interactive protein of PI3KC3, a putative target of 3-MA • Autophagy activation: LC3 conversion • UPR activation: • Induction of CHOP • Activation of JNK1 • Splicing of Xbp-1 (X-box-binding protein-1) mRNA

14. Results Fig3a&b Beclin1 knockdown suppresses ER stress-induced LC conversion, but not CHOP induction HEK293 cells were transfected with either control siRNA (siCtrl) or siRNA against Beclin1 (siBec). The cells were non-treated (NT) or treated with TG or Tun for 5h. Cell lysates were analyzed for levels of LC3 conversion (a) and CHOP induction (b) as well as Beclin1 expression and b-actin (as a loading control).

15. Results Fig3c&d Beclin1 knockdown has no effect on Tun-inducedJNK1 activation and Xbp-1 splicing SiRNA-transfected HEK293 cells were treated with Tun. Cell lysates were analyzed for levels of p-JNK1 and JNK1 by western blot (c). Total RNA was extracted and analyzed for expression of Xbp-1, Xbp-1s and b-actin by RT-PCR (c). Quantification of the p-JNK1 level (d) is shown after normalization with total JNK1 level during the time course of Tun treatment in transfected cells.

16. Results Fig3e Beclin1 knockdown has no effect on TG-induced CHOP induction and Xbp-1 splicing Time course of TG induction of CHOP and Xbp-1 splicing in cells transfected with either siCtrl or siBec. A lower concentration of TG (0.3μM) was used in this experiment.

17. The role of GRP78 in ER stress-induced autophagy formation • The consequence of GRP78 knockdown • The expression of other ER chaperones: GRP94, CHOP, Calreticulin(CRT), Protein Disulphide Isomerase (PDI) • Cell viability: clonogenic survival assay • UPR activation: JNK1 activation; Xbp-1 splicing • The effect of GRP78 knockdown on ER stress-induced autophagy formation • Autophagosome formation • LC3 conversion • PI3KC3-Beclin1 association • XBP-1 associated: ER membrane expansion

18. Results Fig4a&b GRP78 knockdown affects other chaperones’ expression and cell viability HEK293 cells were transfected with 40nM siCtrl or siGrp78 FOR 48h. Cell lysates were analyzed for GRP78, GRP94, calreticulin (CRT), CHOP, PDI and b-actin by western blot (a). Or cells were subjected to clonogenic survival assay. The survival fraction of siCtrl-transfected cells was set as 100%. S.D. was shown, *P<0.05.

19. Results Fig4c&d GRP78 knockdown spontaneously activates and enhances Tun-induced UPR activation HEK293 cells were transfected with 40nM siCtrl or siGrp78 FOR 48h. The cells were then treated with Tun for indicated time. Cell lysates were analyzed for levels of p-JNK1 and JNK1 or GRP78 and b-actin by western blot (c). Total RNA was extracted and analyzed for Xbp-1 splicing by RT-PCR (c). The levels of p-JNK1 were quantitated, normalized against JNK1 and plotted with the relative level in non-treated (0h), siCtrl-transfected cells set as 1.

20. Results Fig5a&b&c GRP78 knockdown suppresses Tun-induced autophagosome formation Hela cells were transiently transfected with GFP-LC3 alone (-) or in combination with 40nM of siCtrl or siGrp78. the cells were then either non-treated (NT) or treated with Tun for 4h. Representative images of GFP-LC3 puntate dot formation in live cells captured by fluorescence microscopy are shown (a). (b) shows the quantitation of qutophagosome formation in treated cells. Western blot analysis of GRP78, GRP94 and b-actin protein levels (c).

21. Results Fig5d&e&f GRP78 knockdown suppresses NS-induced autophagosome formation Hela cells were transiently transfected with GFP-LC3 alone (-) or in combination with 40nM of siCtrl or siGrp78. the cells were then either non-treated (NT) or treated with NS for 2h. Representative images of GFP-LC3 puntate dot formation in live cells captured by fluorescence microscopy are shown (d). (e) shows the quantitation of qutophagosome formation in treated cells. Western blot analysis of GRP78, GRP94 and b-actin protein levels (f).

22. Results Fig5g GRP78 knockdown has no effect on LC3 conversion Hela cells were transfected with siCtrl or siGrp78 and subjected to NS treatment for 2h, TG or Tun treatment for 4h in the absence or presence of zVAD-fmk (40µM). The levels of LC3-I and II, CHOP and b-actin were detected by western blot.

23. Results Fig6a&b GRP78 has no effect on PI3KC3-Beclin1 association (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. Part of the cell lysates was also analyzed for GRP78, PI3KC3 and Beclin1 expression. (b) Same as (a), but here GRP78 was overexpressed in HEK293 cells.

24. Results Fig6c&d GRP78 knockdown disrupts ER integrity (c) Representative electron micrograph of cells transfected with siCtrl showing sparse ER structure (white arrow heads) or siGrp78 showing increased ER structures with expanded lumens (white arrow heads). N, nucleus. (d) Quantitation of the ER area in the cytoplasm in cells transfected with either siCtrl or siGrp78. S.D. is shown, *P<0.0001

25. Results Fig6e XBP-1 mediates the effect of GRP78 knockdown on autophagosome formation 1: siCtrl 2: siGrp78 3: siXbp-1 4: siGrp78 + siXbp-1 (e) Knockdown of XBP-1 recovered normal levels of stress-induced autophagosome formation inhibited by GRP78 knockdown. The indicated siRNA(s) was transiently cotransfected with GFP-LC3 into Hela cells which then subjected different treatments. The formation of autophagosome was assayed by fluorescence microscopy and quantitated. The inset shows western blot analysis of GRP78, XBP-1 and b-actin levels.

26. Fig7 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 PI3K3. 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 cell with wortmannin or knockdown of Beclin1 blocks both NS and ER stress-induced LC3 conversion, but has no effect on UPR activation. JNK activation and eIF2a 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.

27. Conclusion The unfolded protein response regulator GRP78/BiP is required for ER integrity and ER stress-induced autophagy in mammalian cells The specific autophagy inhibitor, 3-MA, is a novel inhibitor of UPR activation Beclin1 is not the link between ER stress-induced autophagy and UPR activation GRP78 knockdown spontaneously activates UPR GRP78 knockdown blocks ER stress and NS-induced autophagy formation XBP-1 is responsible for the suppression of GRP78 knockdown on autophagy

28. PROs and CONs • PROs • The organization is clear • Strong background introduction and results discussion • An inspiring article • CONs • How to tell UPR is activated and autophagy is formed • The specificity of 3-MA: any side-effect? • Some experimental resultsare not convincing (Fig2d p-eIF2a; Fig4a, loading control b-actin; Fig4c p-JNK1; Fig5g LC3 conversion; ) • The results go too detailed: negative results from Beclin1 knockdown; the effect of GRP78 knockdown on autophagosome formation • The effect of GRP78 overexpression on autophagy formation