NorUDCA promotes degradation of α1-antitrypsin mutant Z protein by inducing autophagy through AMPK/ULK1 pathway.

Alpha-1 Antitrypsin (α1AT) Deficiency is a genetic disease in which accumulation of α1AT mutant Z (α1ATZ) protein in the ER of hepatocytes causes chronic liver injury, liver fibrosis, and hepatocellular carcinoma. No effective medical therapy is currently available for the disease. We previously found that norUDCA improves the α1AT deficiency associated liver disease by promoting autophagic degradation of α1ATZ protein in liver in a mouse model of the disease. The current study unravels the novel underlying cellular mechanism by which norUDCA modulates autophagy. HTOZ cells, modified from HeLa Tet-Off cells by transfection with the resulting pTRE1-ATZ plasmid and expressing mutant Z proteins, were studied in these experiments. The role of norUDCA in inducing autophagy, autophagy-mediated degradation of α1ATZ and the role of AMPK in norUDCA-induced autophagy were examined in the current report. NorUDCA promoted disposal of α1ATZ via autophagy-mediated degradation of α1ATZ in HTOZ cells. Activation of AMPK was required for norUDCA-induced autophagy and α1ATZ degradation. Moreover, mTOR/ULK1 was involved in norUDCA-induced AMPK activation and autophagy in HTOZ cells. Our results provide novel mechanistic insights into the therapeutic action of norUDCA in promoting the clearance of α1ATZ in vitro and suggest a novel therapeutic approach for the treatment of α1ATZ deficiency disease and its associated liver diseases

Activation of ULK1 is required for norUDCA-induced AMPK activation and reduction of polymers of α1ATZ.

<p><b>A.</b> Western blotting analysis for phosphorylation of ULK1 (Ser555) in HTOZ cells treated with norUDCA at different concentrations for 1 hour. The lower panel is the densitometry of phospho-ULK1 Ser555 after normalization with total ULK1. Data is expressed as mean ± SD, #<0.05 vs untreated cells. AICAR is used as positive control. <b>B.</b> Western blotting analysis for phosphorylation of ULK1 Ser317, Ser757 and Ser777 in HTOZ cells treated with norUDCA at 200 μM for 1 hour. The left panels are the densitometry of p-ULK1 after normalization with total ULK1, #<0.05 vs untreated cells. <b>C</b>. Western blotting analysis for phosphorylation of ULK1 S555 in HTOZ cells pretreated with compound C (10 μM) for 1 hour and followed by absence or presence of norUDCA at 200 μM for an additional 1 hour. The lower panel is the densitometry of p-ULK1 Ser555 after normalization with total ULK1. Data is expressed as mean ± SD, #<0.05 vs untreated cells and §<0.05 vs norUDCA alone treated cells. Compound C is used as negative control. For A, B and C, total ULK1 is used for normalization and GAPDH is used as a loading control. <b>D</b>. Polymers and monomers of α1ATZ were determined by western blotting analysis in HTOZ cells pretreated with SBI-0206965 (10mM), inhibitor of ULK1, for 1 hour prior to addition of norUDCA at 200 μM for additional 24 hours. The lower panel is the densitometry of α1ATZ after normalization with GAPDH. Data is expressed as mean ± SD, #<0.05 vs untreated cells, §<0.05 vs norUDCA alone treated cells. GAPDH is used as loading control and for normalization.</p

NorUDCA modulates mTOR via AMPK in HTOZ cells.

<p><b>A</b>. Western blotting analysis for phosphorylation of mTOR (Ser2448) in HTOZ cells after treatment with norUDCA for 1 hour. <b>B</b>. Western blotting analysis for phosphorylation of mTOR (Ser2448) in HTOZ cells after pretreatment with AICAR or compound C for 1 hour and then in the presence or absence of norUDCA for an additional 1 hour. For A and B, the lower panels are the densitometry of phospho-mTOR (Ser2448) after normalization with total mTOR. Data is expressed as mean ±SD, #<0.05 vs untreated cells, §<0.05 vs norUDCA treated cells. Total mTOR is used for normalization and GAPDH is used as a loading control. Representative is from three independent experiments. <b>C & D</b>. Western blotting analysis for phosphorylation of AKT in HTOZ dox+ or HTOZ dox- cells after treatment with norUDCA for 1 hour. The lower panels are the densitometry of phosphorylation of AKT after normalization with total AKT. Data is expressed as mean ± SD, #<0.05 vs no Z expression (C) or untreated cells (D). GAPDH is used as a loading control. SS is used as negative control.</p

NorUDCA induces autophagy in vitro.

<p><b>A.</b> Western blotting analysis of LC3 in HTOZ cell line treated with norUDCA at 200 μM for different time points. <b>B.</b> LC3 puncta analysis, the left panel: LC3 puncta was shown in pLC3-EGFP plasmid transfected HTOZ cells followed by treatment with/out norUDCA for 16 hours or 24 hours (original magnification 40X); the right panel: percentage of LC3 puncta positive cells was out of total LC3-EGFP positive cells. Data is expressed as mean ± SD. Representative is from three independent experiments. <b>C.</b> Real-time PCR analysis of ATG5 mRNA levels in HTOZ or HTOM cell line treated with norUDCA at different concentrations for 24 hours, data is present as fold changes comparing to untreated cells and expressed as mean ± SD, #<0.05 vs control group, n = 3. SS was used as positive control. Representative is from three independent experiments. <b>D.</b> Western blotting analysis of ATG5-ATG12 complex and p62 in HTOZ cell line treated with norUDCA at different concentrations for 24 hours. Representative comes from three independent experiments. For <b>A</b> and <b>D,</b> the adjacent panels show the respective densitometry analysis. Data is expressed as mean ± SD, #<0.05 vs untreated cells. GAPDH was used as a loading control and normalization. For <b>A, C</b> and <b>D</b>, SS is used as positive control.</p

norUDCA reduces the steady-state protein levels, but does not change the mRNA levels of α1ATZ in HTOZ cell line.

<p><b>A.</b> Z protein expression in HTOZ or M expression at 24h in HTOM cells is turned off by doxycycline (DOX, final concentration 40ng/ml), which are called HTOZ dox+ or HTOM dox+ cells. <b>B.</b> Western blotting analysis of α1ATZ in HTOZ cells after 24h expression at time 0 treated with norUDCA at 200 μM as indicated time points. <b>C.</b> Western blotting analysis after 24h expression of α1ATZ in HTOZ cells treated with norUDCA at different doses for 24 hours. <b>D & E.</b> Real-time PCR analysis of α1ATZ mRNA levels, 24h expression at time 0, in HTOZ cells treated with norUDCA at 200 μM for indicated time points (D) or at different doses for 24 hours (E). Values are expressed as mean ± SD, n = 3. Representative is from three independent experiments. <b>F.</b> Western blotting analysis of insoluble and soluble fractions of α1ATZ at 24h expression in HTOZ cells treated with norUDCA at different doses for 24 hours. <b>G.</b> Western blotting analysis of insoluble and soluble fractions of α1ATZ after 72h expression in HTOZ cells passed and seeded at lower density to allow for time course, treated with norUDCA at different doses for 1–3 days. For B, C, F, and G, lower panel showed the densitometric analysis. Values are expressed as mean ± SD after normalization over GAPDH, #<0.05 vs untreated cells, §<0.05 vs d1 (50μM). GAPDH was used as equal loading control. Representative is from three independent experiments.</p

AMPK mediates norUDCA-induced α1ATZ polymer reduction and autophagy in HTOZ cells.

<p><b>A & B</b>. phosphorylation of AMPK (Thr172) in HTOZ cells treated with norUDCA at 200 μM for indicated time points (A) or at different doses for 1 hour (B) was determined by western blotting analyses. Compound C (20 μM) or AICAR (1.0 mM) was used as negative or positive control. Representative is from three independent experiments. <b>C</b>. Phosphorylation of AMPK (Thr172) in HTOZ cells pretreated with compound C at different concentrations (0–20 μM) for 1 hour prior to the presence of norUDCA at 200 μM for additional 1 hour was determined by western blotting analyses. AICAR (1.0 mM) was used as positive control. For A, B and C, the lower panels are the densitometry of phospho-AMPK after normalization with total AMPK. GAPDH is used as a loading control. Data is expressed as mean ± SD, #<0.05 vs untreated cells, and §<0.05 vs norUDCA treated cells alone. Representative is from three independent experiments. <b>D</b>. LC3 in HTOZ cells treated with AICAR at different concentrations (0–1.0 mM) for 24 hours was determined by western blotting analyses. <b>E</b>. mRNA levels of ATG5 and p62 were determined by real-time PCR in HTOZ cells treated without/with AICAR at 1.0 mM for 24 hours. Data is expressed as mean ± SD, # < 0.05 vs control, n = 3. <b>F</b>. LC3 in HTOZ cells treated with AICAR at 1.0 mM or norUDCA at 200 μM with/out compound C at 20 μM for 24 hours was determined by western blotting analyses. For D and F, the lower panels are the ratio of LC3-II/LC3-I. Data is expressed as mean ± SD, #<0.05 vs untreated cells, and §<0.05 vs norUDCA treated cells alone. Representative is from three independent experiments. <b>G</b>. A1ATZ polymers and monomers were isolated and determined by western blotting analyses in HTOZ cells treated with AICAR at 1.0 mM or norUDCA at 200 μM with/out compound C at 20 μM for 24 hours. The lower panel is the densitometry of α1ATZ after normalization with GAPDH. Data is expressed as mean ± SD, #<0.05 vs untreated cells, and §<0.05 vs norUDCA treated cells alone. GAPDH is used as an equal loading control. Representative is from three independent experiments.</p

NorUDCA reduces insoluble α1ATZ via inducing autophagy in HTOZ cell line.

<p><b>A.</b> Western blotting analysis for soluble and insoluble fraction of α1ATZ in HTOZ cells pretreated with chloroquine (CQ), commonly used pharmacological autophagy inhibitor, at different concentrations (0, 5, 10 μM) for 1 hour before addition of norUDCA at 200 μM for an additional 24 hours. p62 was detected to show that autophagy was blocked by CQ. <b>B.</b> Western blotting analyzes the efficacy of ATG5 knock-down. The specific band showed the ATG5-ATG12 complex (56 kD). <b>C.</b> Western blotting analysis for soluble and insoluble fraction of α1ATZ in ATG5 expressing (ATG5-WT) or ATG5 knock-down (ATG5-KD) HTOZ cells. <b>D</b>. Western blotting analysis for soluble and insoluble fraction of α1ATZ in ATG5-WT or ATG5-KD HTOZ cells treated with/out norUDCA at 200 μM for 24 hours. <b>E.</b> Western blotting analysis for soluble and insoluble fraction of α1ATZ in HTOZ cells pretreated with MG132 (30 μM) for 1 hour and subsequently incubated with norUDCA (200 μM) for an additional 24 hours. The cells that were incubated with MG132 alone served as control to validate that the proteasome was inhibited. For A, B, C, D and E, the lower panels show the densitometry of specific bands normalized with GAPDH. Data are expressed as mean ± SD, #<0.05 vs control or ATG5 expressing cells (C), §<0.05 vs norUDCA treated cells. Representatives come from three independent experiments. GAPDH was used as an equal loading control.</p

Schema of proposed mechanisms by which norUDCA promotes degradation of α1ATZ.

<p>NorUDCA activates AMPK and inhibits mTOR. AMPK phosphorylates ULK1 at Ser317, Ser555 and Ser777, and inhibits Ser757, and subsequently initiates autophagy, resulting in degradation of polymers of α1ATZ and reduction of accumulation of α1ATZ in hepatocytes.</p