DRAM1 Regulates Autophagy Flux and Apoptosis through Lysosomes
|Keywords||DRAM1 autophagy 3-NP lysosome acidification lysosomal enzymes V-ATPase BAX|
Aim: Previous studies demonstrated that intrastiatal administration of 3-nitropropionic acid induced death of striatal neurons acopmnied by activation of autophagy and apoptosis. The p53-dependent induction of damage-regulated autophagy modulator 1 (DRAM1) played an important role of in autophagy activation and cell death. This study was designed to investgate the molecular mechanisms by which DRAM1 regulates autophagy and apoptosis.Methods: (1) To evaluate activation of autophagy and apoptotic signaling in a cellular model of mitochondrial dysfunction-induced cellular toxicity, A549 cells were treated with 3-nitropropionic Acid (3-NP) for various length of time. The protein levels of DRAM1, BAX, p62, LC3 and release of cytochrome c were determined with Western blot analysis. Expression of GFP-LC3 was determined with fluorescence. (2) To evaluate if an autophagic and apoptotic mechanism contributes to 3-NP-induced cell death, the effects of knockdown of autophagy gene Atg5 and pan-caspase inhibitor Z-VAD-FMK on cell viability determined with WST - 1 method. (3) To understand the role of DRAM1 in the regulation of autophagy, the induction of protein levels of p62 and LC3 induced by 3-NP were determined with Western blot analysis after sirencing DRAM1. Expression of GFP-LC3 induced by 3-NP after DRAM1 knockdown was determined with fluorescence. (4) To study the mechanisms of DRAM1 in regulating autophagy, the lysosomal localization of DRAM1 was examined with LysoTracker. Clearance of autophagosomes is a measure of autophagy flux, and the present study employed double immunofluorescence of cathepsin D and LysoTracker to explore the role of DRAM1 in lysosomal function. To assess lysosomal acidification, we used LysoSensor DND-167 and measured lysosomal pH in quantization.Then we examined the ATP-dependent lysosomal acidification using the pH sensitive dye FITC-dextran. (5) The lysosomal inhibitors E64d (10 ?M) and chloroquine (20 ?M) were used to evaluate if inhibition of lysosomal functions produces effects similar to knockdown of DRAM1. (6) To understand the role of DRAM1 in the regulation of apoptosis, the induction of protein levels of BAX and activation of lysosomal enzyme cathepsin D and release of cyt-c following 3-NP were determined with Western blot analysis after sirencing DRAM1.(7)Next, we analyzed if DRAM1 affects apoptosis through up-regulation of BAX. The connections between BAX and activation of lysosomal enzyme cathepsin D and release of cyt-c following 3-NP treatment were examined with sirecing BAX. The effects of knockdown of BAX on cell viability were determined with WST - 1 method.Results: (1) The results showed that 3-NP-induced a significant increase in the protein levels of DRAM1 and LC3-II from 24-72 h, and 3-NP induced a time-dependent increase in GFP-LC3 in A549 cells, while p62 decreased 24-72 h after 3-NP treatment. 3-NP induced expression of BAX 24 h after treatment. Western blot analysis showed that cyt-c levels decreased in the mitochondrial fractions but increased in the cytosolic fractions starting 24 h after treatment with 3-NP and persisted to 72 h. These results indicate that autophagic and apoptotic proteins were induced by 3-NP. (2) Western blot analysis showed that ATG5 siRNAs caused a down-regulation of ATG5 proteins and a reduction in 3-NP-induced cell death. Z-Vad-FMK (20-40 ?M) had no effect on cell survival under normal conditions, whereas inhibition of caspase activation effectively inhibited the cell death induced by 3-NP. These results suggest that both autophagy contribute to 3-NP-induced cell death. (3) Following treatment of cells with DRAM1 siRNA, the induction of LC3-II, GFP-LC3 puncta by 3-NP was markedly reduced. p62 levels increased in DRAM1 siRNA-treated cells. These lines of evidence support an important role of DRAM1 in autophagy activation. (4) Marked co-localization of DRAM1 and LysoTracker was seen, suggesting that DRAM1 predominantly localizes to lysosomes. In control cells, acute autophagy induction with rapamycin elevated LC3-II levels as revealed by immunoblotting. After removing rapamycin from the medium for 6 h, LC3-II returned towards baseline levels.While in DRAM1 siRNA-treated cells, LC3-II remained elevated 6 h after removing rapamycin. These suggest that the clearance of autophagic vacuoles is impaired in DRAM1 siRNA-treated A549 cells. 3-NP treatment increased the expression of Cathepsin D and the number of LysoTraker labeled lysosomes. The results of immunoblotting showed that knockdown of DRAM1 significantly inhibited 3-NP-induced production of the active form of Cathepsin D. In control cells, the fluorescence of LysoSensor was enhanced from 24 to 72 h after 3-NP exposure. By contrast, in DRAM1 siRNA-treated cells, the fluorescence was lower than that in the control cells. WT cells exhibited an intralysosomal pH of 4.75, and lysosomal pH decreased following 3-NP treatment. In contrast, the lysosomal pH values in DRAM1 siRNA-treated cells were 5.23 and decreased to a lesser extent following 3-NP treatment. These results suggest that there is a defective lysosomal acidification in DRAM1 siRNA-treated cells. In DRAM1 siRNA-treated cells, ATP addition-induced drop in fluorescence emission was reduced, reflecting a reduction in internal lysosomal acidification. Thus, the impairment of acidification in DRAM1 siRNA-treated cells might be due to decreased V-ATPase activity. (5) Compared with cells treated with 3-NP alone, LC3 in E64d and chloroquine-treated cells accumulated more Lamp2-positive vacuoles. LC3-II accumulated after E64d or chloroquine treatment. Western blot analysis showed that cyt-c levels decreased in the cytosolic fractions and increased in the mitochondrial fractions 24 h after treatment of E64d or chloroquine in the presence 3-NP (48 h). Although E64d and chloroquine had no effect on cell survival under normal conditions, they significantly reduced 3-NP-induced cell death 48 h after 3-NP treatment. These results suggest a defective clearance of autophagic vacuoles and a reduction of 3-NP-induced cell death in E64d- and chloroquine-treated cells. (6) Following treatment of cells with DRAM1 siRNA, the induction of BAX、release of cyt-c and activation of caspase-3 by 3-NP was markedly reduced. (7) The efficiency of over-expression and knock-down of BAX was validated by the Western blot analysis. Knock-down of BAX significantly reduced the activation of Cathepsin D. Western blot analysis showed that 3-NP-induced release of cyt-c release from mitochondria and activation of caspase-3 was significantly attenuated after knock-down of BAX with siRNA. The results showed that over-expression of BAX increased 3-NP-induced cell death, while silencing of BAX had no significant effect on cell survival under normal conditions, but significantly reduced 3-NP–induced cell death. It is indicated that DRAM1 affects apoptosis, at least, partially through up-regulation of BAX. Conclusions: The present study demonstrates that DRAM1 regulates autophagy flux through promoting lysosomal acidification and activation of lysosomal enzymes. DRAM1 also has a regulatory role in mitochondrial apoptotic signaling pathway through up-regulating BAX.Under going works: The fusion of autophagosomes with lysosomes is an important step for autophagic degradation. For a full understand of a role of DRAM1 in autophagy flux, the effects of DRAM1 on the fusion process between autophagosomes and lysosomes are currently under investigation. The cross-talk to mitochondrial apoptotic pathway through BAX is interesting. How does DRAM1 regulate the BAX expression is also under investigation.