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. 2014 Jun 20:5:4233.
doi: 10.1038/ncomms5233.

Hepatoprotective role of Sestrin2 against chronic ER stress

Hwan-Woo Park  1 , Haeli Park  1 , Seung-Hyun Ro  1 , Insook Jang  1 , Ian A Semple  1 , David N Kim  1 , Myungjin Kim  1 , Myeongjin Nam  2 , Deqiang Zhang  1 , Lei Yin  1 , Jun Hee Lee  1
Affiliations

Hepatoprotective role of Sestrin2 against chronic ER stress

Hwan-Woo Park et al. Nat Commun. .

Abstract

Upon prolonged endoplasmic reticulum (ER) stress, cells attenuate protein translation to prevent accumulation of unfolded proteins. Here we show that Sestrin2 is critical for this process. Sestrin2 expression is induced by an ER stress-activated transcription factor CCAAT-enhancer-binding protein beta (c/EBPβ). Once induced, Sestrin2 halts protein synthesis by inhibiting mammalian target of rapamycin complex 1 (mTORC1). As Sestrin2-deficient cells continue to translate a large amount of proteins during ER stress, they are highly susceptible to ER stress-associated cell death. Accordingly, dietary or genetically induced obesity, which does not lead to any pathological indication other than simple fat accumulation in the liver of wild-type (WT) mice, can provoke Sestrin2-deficient mice to develop severe ER stress-associated liver pathologies such as extensive liver damage, steatohepatitis and fibrosis. These pathologies are suppressed by liver-specific Sestrin2 reconstitution, mTORC1 inhibition or chemical chaperone administration. The Sestrin2-mediated unfolded protein response (UPR) may be a general protective mechanism against ER stress-associated diseases.

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Figures

Figure 1
Figure 1. Obesity induces Sestrin2 through ER stress signaling
(a-c) HepG2 cells were treated with palmitic acid (PA) for indicated hr and analyzed by immunoblotting (images with black bands) and RT-PCR (images with white bands) (n = 3). P values were calculated between untreated (0 hr) and indicated groups (b,c). (d,e) At 48 hr after infection with GFP (Con)- or SERCA2b-overexpressing adenoviruses, cells were treated with BSA (0 hr) or PA for indicated hr (d) or 9 hr (e) (n = 3). (f,g,l,m) 6-month-old WT mice kept on LFD or HFD for 4 months were injected daily with vehicle (PBS) or TUDCA (500 mg per kg body weight i.p.) or transduced once with adenoviruses expressing SERCA2b, GFP (Con) or shRNA targeting c/EBPβ. After 10 days, livers were analyzed by immunoblotting (n = 3). (h,k) At 48 hr before PA treatment, cells were infected with lentiviruses expressing shRNAs targeting luciferase (Con), PERK (h) and c/EBPβ (k). After 9 hr of BSA (−) or PA treatment, cells were analyzed by immunoblotting. (i,j) Chromatin immunoprecipitation (ChIP) analysis of cells with indicated treatments (n = 3). (n) At 24 hr after infection with GFP (Con)- or c/EBPβ-overexpressing adenoviruses, cells were analyzed by immunoblotting. All data are shown as the mean ± s.e.m. P values are from Student’s t test. Molecular weight markers are indicated in kDa (immunoblots) or bp (agarose gels).
Figure 2
Figure 2. Sestrin2 suppresses mTORC1 and protein synthesis in response to ER stress
(a-e) At 48 hr after infection with shRNA lentiviruses for luciferase (Con) or Sestrin2, HepG2 cells were treated with PA for 9 hr and analyzed by immunoblotting (n = 3). AMPK signaling activity was monitored by phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). mTORC1 signaling activity was monitored by phosphorylation of p70 ribosomal protein S6 kinase (S6K), ribosomal protein S6 and eIF4E-binding protein (4E-BP). (f-h) 2-month-old WT or Sesn2−/− mice kept on LFD were injected with tunicamycin (Tm, 500 mg per kg body weight i.p.). After indicated hr, livers were analyzed by immunoblotting (n = 4). (i,j) Relative intracellular ATP levels of indicated cells (i, n = 3) and liver tissues (j, n = 4). As a positive control for ATP depletion, HepG2 cells were treated with oligomycin (OM, 5 μg ml−1) for 1 hr. (k-r) HepG2 cells transduced with sh-Con or sh-Sesn2 (k,m, n = 5) or primary hepatocytes (1°-HPC) isolated from WT or Sesn2−/− mice (l,n, n = 4) were treated with BSA (−) or PA for 9 hr. 2-month-old WT or Sesn2−/− mice were injected with Tm (o,q, n = 4) or kept on LFD or HFD for additional 2 months (p,r, n = 4). After indicated treatments, newly synthesized proteins in cells (k-n) and liver tissues (o-r) were visualized and quantified by Click-IT AHA® labeling system. The protein bands denoted by stars correspond to serum albumin (70kD), which is the most actively synthesized protein in normal hepatocytes. All data are shown as the mean ± s.e.m. P values are from Student’s t test. Molecular weight markers are indicated in kDa.
Figure 3
Figure 3. Sestrin2 deficiency exacerbates ER stress upon chemical insults or obesity
(a,b) 2-month-old WT or Sesn2−/− mice kept on LFD were injected with Tm (500 mg per kg body weight, i.p.). After indicated hr, livers were harvested from the treated mice and analyzed (n = 4). Protein phosphorylation and expression were analyzed by immunoblotting (images with black bands) (a) and quantified (b). XBP1 mRNA splicing was examined through semi-quantitative RT-PCR (images with white bands) (a). (c-i) 6-month-old WT (n = 6) and Sesn2−/− (n = 5) mice kept on LFD or HFD for 4 months (c-f) and 4-month-old Lepob/ob/Sesn2+/− (Con, n = 4) and Lepob/ob/Sesn2−/− (n = 6) mice kept on LFD (g-i) were analyzed. Protein phosphorylation and expression were analyzed by immunoblotting (c,g) and quantified by densitometry (d,h). Liver sections were stained with indicated antibodies (e). Relative mRNA expression of ER stress-inducible genes was quantified through qRT-PCR (f,i). Scale bars, 100 μm. All data are shown as the mean ± s.e.m. P values are from Student’s t test. Molecular weight markers are indicated in kDa (immunoblots) or bp (agarose gels).
Figure 4
Figure 4. Sestrin2 controls ER homeostasis through the AMPK-mTORC1 axis
(a-c) HepG2 cells stably transduced with Sestrin2 shRNA (sh-Sesn2) were treated with PA for indicated hr (a) or 6 hr (b). PBS (Con), AICAR (1 mM), Rapamycin (Rap, 100 nM), PP242 (1 μM), cycloheximide (CHX, 180 μM), BHA (100 μM) and NAC (10 mM) were applied 1 hr before treating with PA. AICAR is an AMPK activator, PP242 is an mTOR inhibitor, and CHX is a protein translation inhibitor. Protein phosphorylation and expression were examined (a,b) and quantified (c) (n = 3). P values were calculated between PA+PBS (Con) and indicated groups. (d,e) Sestrin2-silenced HepG2 cells were transduced with shRNA lentiviruses for luciferase (Con) or Raptor (sh-Raptor). After 48 hr, cells were treated with BSA (−) or PA for 6 hr and analyzed by immunoblotting (n = 3). (f,g) At 48 hr after infection with shRNA lentiviruses for luciferase (Con) or TSC2 (sh-TSC2), HepG2 cells were treated with BSA (−) or PA for 6 hr and analyzed by immunoblotting (n = 3). (h-k) 5-month-old Sesn2−/− mice kept on HFD for 3 months were transduced once with adenoviruses expressing GFP (Con, n = 5) or constitutive active AMPK (AMPKCA, n = 6) (h,i) or were injected daily with vehicle (PBS, n = 6) or AICAR (250 mg per kg body weight per day i.p., n = 5) (j,k). After 10 days, livers were harvested, and protein phosphorylation and expression were examined (h,j) and quantified (i,k). All data are shown as the mean ± s.e.m. P values are from Student’s t test. Molecular weight markers are indicated in kDa.
Figure 5
Figure 5. Sestrin2 prevents liver damage during obesity
(a-c) Livers and sera were collected from 6-month-old WT (n = 6) and Sesn2−/− (n = 5) mice kept on HFD for 4 months (HFD panels) or from 4-month-old Lepob/ob/Sesn2+/− (Con, n = 4) and Lepob/ob/Sesn2−/− (n = 6) mice kept on LFD (Lepob panels). Liver sections were subjected to TUNEL (red) and DAPI (blue) staining (a). TUNEL-positive cells were quantified (b). Serum ALT levels were quantified from indicated groups of mice (c). (d-f) Livers from obese Sesn2−/− mice transduced with AAV-Con (n = 4) or AAV-Sesn2 (n = 3) were subjected to TUNEL (red) and DAPI (blue) staining (d). TUNEL-positive cells in livers (e) and serum ALT levels (f) were quantified. (g-i) 5-month-old Sesn2−/− mice kept on HFD for 3 months were daily injected with vehicle (PBS, n = 4) or TUDCA (500 mg per kg body weight per day i.p., n = 5). After 10 days of the treatment, mice were sacrificed, and livers and sera were collected. Livers were subjected to TUNEL (red) and DAPI (blue) staining (g). TUNEL-positive cells in livers (h) and serum ALT levels (i) were quantified. Scale bars, 100 μm. All data are shown as the mean ± s.e.m. P values are from Student’s t test.
Figure 6
Figure 6. Sestrin2 is an endogenous attenuator of steatohepatitis progression
(a-b) Livers from WT and Sesn2−/− mice kept on HFD (HFD panels) or Lepob/ob/Sesn2+/− (Con) and Lepob/ob/Sesn2−/− mice kept on LFD (Lepob panels) were subjected to anti-F4/80 staining to visualize macrophage infiltration (a). F4/80-positive areas were quantified (b). (c-h) Livers from obese Sesn2−/− mice transduced with AAV-Con (n = 4) or AAV-Sesn2 (n = 3) (c,d) or injected with PBS (n = 4) or TUDCA (n = 5) (e,f) or PBS (n = 6) or AICAR (n = 5) (g,h) were analyzed by anti-F4/80 staining (c,e,g). F4/80-positive areas were quantified (d,f,h). Nuclei were visualized by hematoxylin (a,c,e,g). (i,j) Working model of how Sestrin2-mediated UPR attenuates NAFLD progression. In WT liver, Sestrin2 is induced during obesity through the PERK-c/EBPβ pathway to attenuate protein translation and thereby relieve ER stress (i). In Sesn2−/− liver, persistent mTORC1 activity elevates protein synthesis and subsequently aggravates ER stress, leading to facilitated NAFLD progression (j). Scale bars, 200 μm. All data are shown as the mean ± s.e.m. P values are from Student’s t test.
Figure 7
Figure 7. Sestrin2 controls liver metabolism through regulating ER homeostasis
(a-d) 6-month-old WT and Sesn2−/− mice were kept on HFD for 4 months. Livers were analyzed by Oil Red O (ORO) staining (a). ORO densities were quantified (b) (n = 4). Mice were tested for insulin resistance (ITT) (c) (n = 8) and for glucose tolerance (GTT) (d) (n = 8). Area-under-the-curve data for ITT (c) and GTT (d) were calculated. (e-h) 5-month-old Sesn2−/− mice kept on HFD for 3 months were transduced with AAV-Con (n = 4) or AAV-Sesn2 (n = 3). After 10 days, livers were harvested from the treated mice and analyzed by ORO staining (e). ORO densities were quantified (f). Mice were tested for insulin resistance at 5 days (g) and for glucose tolerance at 7 days (h) after AAV transduction. Area-under-the-curve data for ITT (g) and GTT (h) were calculated. (i-l) 5-month-old Sesn2−/− mice kept on HFD for 3 months were daily injected with vehicle (PBS, n = 4) or TUDCA (500 mg per kg body weight per day i.p., n = 5). After 10 days of treatment, livers were harvested from the treated mice and analyzed by ORO staining (i). ORO densities were quantified (j). Mice were tested for insulin resistance at 5 days (k) and for glucose tolerance at 7 days (l) after initiation of TUDCA treatment. Area-under-the-curve data for ITT (k) and GTT (l) were calculated. Scale bars, 200 μm; 10 μm (insets). All data are shown as the mean ± s.e.m. P values are from Student’s t test.
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