Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function - PubMed (original) (raw)

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

Nadia Jaber et al. Proc Natl Acad Sci U S A. 2012.

Abstract

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34(f/f) mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34(f/f) mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34(f/f) mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Ablation of Vps34 leads to defective autophagosome formation and endocytic protein turnover in MEFs. (A) PCR analysis with the Pik3c3-5′ and 3′ arm primers (

Fig. S1

) using genomic DNA isolated from MEFs generated from Vps34+/f breeding pair. (B) MEFs were observed by phase/contrast microscopy upon control or Cre infection for 6 d. Representative images are shown. (C) MEFs infected with vector control or Cre were left in full medium or serum starved for 6 h and observed by EM. Large-sized empty vacuoles are observed in Vps34-null MEFs representing single-membraned swollen endosomes/lysosomes. Serum starvation induced the appearance of autophagosomes in wild-type, but not Vps34-null MEFs. Quantification of the number of autophagosomes was obtained by counting 10–20 cells and the averages ± SEM are shown. (D and E) Endocytic EGFR degradation but not transferrin recycling is blocked in Vps34-null cells. (D) Vector control or Cre-infected MEFs were serum starved for 4 h and incubated with biotinylated-transferrin, then stripped and chased with unlabeled transferrin for the indicated times. Cells and culture media were collected and analyzed for biotinylated-transferrin levels. The relative amounts of intracellular transferrin at each time point was quantified by densitometry and normalized to that of time 0. (E) Vector control or Cre-infected MEFs were serum-starved overnight and stimulated with EGF (100 ng/mL) for the indicated times. EGFR protein level is quantified relative to Lamin B1.

Fig. 2.

Fig. 2.

Autophagy flux is impaired in Vps34-null MEFs. (A) Vps34f/f MEFs were infected with control or Cre virus. Cell lysates were collected at indicated time points after infection and probed for Vps34, LC3, and p62. The fluctuation of the levels of LC3 and p62 in control cells may be due to the change of confluency during passaging. (B) Vps34f/f MEFs were infected with vector control or Cre. At 7 d after infection, cells were left untreated or serum deprived for 6 h, with or without the addition of lysosomal protease inhibitors E64D (10 μg/mL) and PepA (10 μg/mL). (C) MEFs were labeled with 14C-valine for 24 h and left untreated or cultured in serum-free medium for 6 h. Degradation of long-lived proteins was measured. Average of three independent experiments ± SEM is shown. *P < 0.05. (D) Vps34f/f MEFs infected with vector or Cre virus were cultured in serum-free medium for 6 h. Cells were stained for LC3 for immunofluorescence analysis. Note that although serum deprivation induces LC3 puncta in vector control cells, LC3 forms large-sized structures upon Cre infection, with no further increase upon serum starvation. (E) Vector and Cre-infected Vps34f/f MEFs were transfected with mCherry-GFP-LC3. Forty-eight hours after transfection, cells were starved in Hanks buffer or serum-free medium. Cells were observed under a deconvolution microscope. Representative images are shown. The average numbers of yellow or red puncta were obtained from three countings. Note although nutrient starvation induces both autophagosomes (yellow) and autolysosomes (red) in wild-type cells, both inductions are inhibited in Vps34-null cells.

Fig. 3.

Fig. 3.

Localized production of PI(3)P, autophagosome formation, and mTOR signaling are impaired in the absence of Vps34. (A) Ablation of Vps34 leads to diminished PI(3)P production. Vps34f/f MEFs stably expressing GFP-FYVE were infected with vector control or Cre. Three representative cells from each group are shown. Note the lack of FYVE puncta in Cre-infected MEFs, indicating the absence of PI(3)P. (B) Serum starvation fails to induce DFCP1 and Atg12 puncta in Vps34-null cells. Vps34f/f MEFs were infected with vector control or Cre virus for 5 d. Cells were transiently transfected with GFP-DFCP1. Forty-eight hours after transfection, cells were fixed and immunofluorescence for Atg12 was performed. Representative images are shown. The average numbers of yellow or red puncta were determined from eight countings and are shown with SEM. Note that upon serum withdrawal, vector control cells exhibit colocalization of DFCP1 and Atg12 puncta. In contrast, no puncta formation was observed for either DFCP1 or Atg12 in the Vps34-null cells. (C) Amino acid-stimulated mTOR activation is compromised in the absence of Vps34. Vps34f/f MEFs infected with vector or Cre were starved and stimulated for 30 min with 2× MEM amino acids. mTOR signaling was measured by the levels of phospho-S6 and phospho-4EBP1. (D and E) Steady-state mTOR signaling is not affected by the loss of Vps34. Whole liver lysates from wild-type and Vps34f/f;Alb-Cre+ mice (D) and lysates from cardiomyocytes isolated from wild-type and Vps34f/f;Mck-Cre+ mice (E) were probed for phospho-S6 and total S6.

Fig. 4.

Fig. 4.

Ablation of Vps34 in the liver leads to hepatomegaly and steatosis. (A) Lysates from primary hepatocytes with indicated genotypes were probed with indicated antibodies. Note the accumulation of p62, LC3-I/II, and polyubiquitinated proteins in Vps34-null hepatocytes, indicative of blocked autophagic protein degradation. (B) Gross anatomical views of representative mice and livers with indicated genotypes. (C) Livers isolated from mice with indicated genotypes were stained with hematoxylin and eosin (H&E), Oil Red O, or Periodic Acid Schiff (PAS). Representative images are shown. (D) The mass of livers with indicated genotypes was measured and expressed relative to body mass (Left). Total protein content of wild-type and Vps34-null livers was quantified and expressed relative to the body weight (Right). (E) Wild-type and Vps34f/f;Alb-Cre+ mice were fasted for 24 h. Liver/body mass ratio was determined and expressed as normalized to that of respective fed animals. Note the difference between fed and fasted Vps34-null livers is statistically insignificant. (F) Electron micrographic images of livers from fed and 24 h fasted Vps34f/f;Alb-Cre− and Vps34f/f;Alb-Cre+ mice. Higher magnification views of boxed areas are numbered and shown in Lower. Gly, glycogen area; arrowheads point to autophagosomes; asterisks denote lipid droplets. Note the lack of glycogen deposition, accumulation of lipid droplets, and smaller mitochondria in fed Vps34-null livers. Starved Vps34-null livers are devoid of autophagosomes but are noted to have swollen mitochondria.

Fig. 5.

Fig. 5.

Cardiac dysfunction in Mck-Cre;Vps34f/f mice. (A) Hearts isolated from 6-wk-old Mck-Cre;Vps34+/f and Mck-Cre;Vps34f/f mice were photographed. (B) Hearts were weighed, and the heart mass is normalized to body mass. (C) Representative B-mode (left ventricle labeled) and M-mode echocardiographic images of control and Mck-Cre;Vps34f/f mice. (D) Kaplan–Meier survival curve of Mck-Cre;Vps34f/f mice. (E) Representative EM images of control and Mck-Cre;Vps34f/f hearts. Note that wild-type heart section shows characteristic alignment of mitochondria and organization of Z-lines, whereas the Vps34-null tissue shows disorganized mitochondria and Z-lines. (F) Cardiomyocytes were isolated from control and Mck-Cre;Vps34f/f hearts. Cell lysates were probed with indicated antibodies.

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