Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila
- Péter Lőw†1,
- Ágnes Varga†1,
- Karolina Pircs1,
- Péter Nagy1,
- Zsuzsanna Szatmári1,
- Miklós Sass1 and
- Gábor Juhász1Email author
© Lőw et al.; licensee BioMed Central Ltd. 2013
Received: 22 February 2013
Accepted: 21 June 2013
Published: 25 June 2013
Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized.
Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (α, β or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1α.
We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1α/sima may contribute to upregulation of autophagy by impaired proteasomal activity.
The proteasome is a multi-subunit protease complex, consisting of a 20S core particle and a 19S regulatory particle. 20S contains predominantly structural α and catalytic β subunits. 19S contains non-ATPase subunits that are involved in the recognition of polyubiquitinated proteins, and ATPase subunits responsible for unfolding substrates and maintaining protease activity. The ubiquitin-proteasome system (UPS) is considered to be the main route for the degradation of short-lived proteins. Proteasomal activity is also essential for eliminating excess or unfolded proteins and thus contributes to establishing steady-state overall protein levels. The selective and often well-timed degradation of regulatory proteins by the proteasome orchestrates and modulates multiple biological processes such as transcription, metabolism, cell cycle progression and differentiation .
During the main pathway of autophagy, initially appearing membrane cisterns called phagophores capture portions of the cytoplasm in double-membrane autophagosomes. These vesicles then deliver cargo for lysosomal degradation [2, 3]. Autophagy is thought to be mainly responsible for degrading long-lived proteins and whole organelles, and stress-induced autophagy maintains the amino acid pool and energy production necessary for biosynthetic processes during chronic starvation [1, 3].
UPS and autophagy were long considered as independent, parallel pathways. Recent observations challenged this view, suggesting interactions between these degradative routes [4, 5]. First, autophagy also plays an important role in the degradation of ubiquitinated proteins. Loss of autophagy leads to neurodegeneration accompanied by the formation of pathological protein aggregates both in mice and flies [3, 6, 7]. These aggregates contain ubiquitinated proteins and p62 (also known as Ref2P in flies), an autophagy receptor. p62 facilitates selective autophagic removal of ubiquitinated cargo by binding to both ubiquitin and Atg8 that is covalently bound to phagophore and autophagosome membranes [8, 9]. Second, protein degradation by the autophagy and UPS pathways is coordinately regulated by the evolutionarily conserved Foxo transcription factors in muscles [10, 11]. In addition, we recently showed that genes encoding proteins of the UPS are upregulated in response to starvation in autophagy mutant Drosophila larvae compared to starved control animals . Third, impairment of the UPS has been shown to enhance autophagy in various cells, but the molecular links are poorly characterized [4, 5].
In this study, we investigated the consequences of proteasome inhibition on aggregate formation and autophagic activity in somatic clones of larval fat body cells in Drosophila. We showed that genetic activation of hypoxia signaling induces autophagy, and identified HIF-1α as a novel molecular link between UPS and autophagy.
Depletion of genes encoding various proteasome subunits leads to impaired proteasomal degradation in vivo
Proteasome maturation protein (Pomp) facilitates the main steps in 20S core complex formation at the ER by coordinating the assembly process, and it is necessary for the continuous supply of newly formed proteasomes. Similar to the above results, depletion of Pomp also resulted in accumulation of GFP-CL1 (Figure 1C).
We have also tested the level of p62, a well-characterized intracellular receptor for ubiquitinated proteins. As ubiquitin-containing proteins were shown to be captured into aggregates of p62 [9, 14, 15], inactivation of the proteasome was expected to increase p62 aggregation due to a block in ubiquitinated protein degradation. Note that in the various staining experiments in genetic mosaic animals, GFP-positive RNAi cells are always surrounded by control cells in the very same tissue, which serve as an internal control to directly compare phenotypes to (Figure 1D). Immunostainings indeed revealed large ubiquitin- and p62-positive aggregates in the cytoplasm of proteasome subunit RNAi cells compared to control non-GFP cells (Figure 1E, F, see also Additional file 2: Figure S2 for further images). Importantly, aggregates never colocalized with the lysosome marker Lamp1-GFP in these cells, suggesting that aggregated proteins are not accumulated inside lysosomes. Quantification of immunostainings revealed that 20 out of the 27 RNAi lines (targeting 26 different proteasome subunits and Pomp) led to large-scale accumulation of endogenous p62 aggregates (Figure 1G). Notably, exactly the same seven lines failed to trigger a statistically significant elevation of both p62 and GFP-CL1 aggregates. This may be due to that these RNAi lines did not work properly in fat body cells, although it cannot be excluded that some of the proteasome subunits targeted by these seven RNAi lines are dispensable for proteasome function. The transcription of p62 is also regulated during starvation and stress, both in Drosophila and mammalian cells [12, 16, 17]. RT-PCR experiments revealed that p62 transcription in proteasome depleted animals was also increased relative to controls (see Additional file 3: Figure S3).
It was obvious in these experiments that smaller fat body cells had higher GFP-CL1 levels (see Additional file 4: Figure S4). This suggested that proteasomal degradation is necessary for proper growth and endomitosis of larval fat body cells, and a more severe block of proteasome function likely produces smaller cells.
We selected five proteasome subunit RNAi lines for further experiments that all worked very well in both in vivo proteasome activity assays (that is, GFP-CL1 and p62 aggregation). These represent different subunit types: 20S core particle subunits Prosα1, Prosα5, Prosβ2, and 19S regulatory particle subunits Rpt1, Rpn2 (Figure 1C).
Proteasome impairment enhances starvation-induced and basal autophagy
These genetic manipulations also activated autophagy in a cell-autonomous manner in well-fed larvae, based on increased LTR and autophagosome-associated punctate anti-Atg8a immunostainings (Figure 2B-E, see also Additional file 7: Figure S7 for further images). Similarly, knockdown of genes encoding proteasome subunits led to elevated levels of autophagosome-associated, lipidated Atg8a-II in western blots of well-fed larvae (Figure 2F). Levels of the ubiquitin-binding selective autophagy cargo p62 also showed a marked increase in these samples (Figure 2F).
We found that upon proteasome RNAi, smaller cells always had increased normalized LTR dot number (see also Additional file 8: Figure S8). Thus, more severe proteasome inactivation seems to induce higher levels of autophagy and a stronger cell growth impairment.
Proteasome RNAi leads to accumulation of cytoplasmic aggregates and enhances autophagic flux
We then carried out electron microscopy to completely rule out the possibility that these large aggregates were inside autophagosomes. Ultrastructural images of proteasome depleted fat bodies dissected from well-fed animals revealed the presence of large cytoplasmic protein aggregates that were never surrounded by membranes (Figure 3E, F). In addition, double-membrane autophagosomes and degrading autolysosomes were also readily detected in well-fed larval samples, consistent with autophagy induction in these cells (Figure 3E, F). See also Additional file 11: Figure S11 for further ultrastructural images.
It was striking that upon proteasome RNAi, smaller cells always had increased p62 accumulation (see Additional file 12: Figure S12). These results altogether indicated that even though autophagic activity is enhanced in these cells, it fails to remove the excess protein aggregates formed due to impaired proteasomal degradation.
As expected, LTR practically never colocalized with Atg8a or p62 reporters (see Additional file 13: Figure S13). Even the size of these structures was different: LTR-positive digesting lysosomes in fat body cells had an approximate diameter of 1-3 μm, while p62 was present in the large protein aggregates (approximate diameter: 5 μm), and GFP-Atg8a labeled small autophagosomes (approximate diameter: 1 μm) in addition to being captured into the large p62 aggregates.
These data altogether suggest that p62 accumulation is due to both transcriptional and post-transcriptional mechanisms, that is, increased rate of transcription, and reduced protein turnover by autophagy due to large-scale aggregate formation.
Activation of hypoxia signaling induces autophagy in Drosophila
We also carried out anti-active caspase 3 stainings to analyze cell death, but no apoptotic caspase activation was detected in cell clones undergoing proteasome or Vhl RNAi, or overexpressing sima in larval tissues (see Additional file 14: Figure S14)
Proteasome inactivation-induced autophagy requires Atg genes and sima/HIF-1α
Since genetic activation of hypoxia signaling increased autophagy, we first tested whether proteasome RNAi activates this pathway. BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3) is a well-characterized transcriptional target of HIF1 in mammalian cells . Our RT-PCR experiments revealed that transcription of Drosophila BNIP3 (CG5059) is enhanced upon silencing of Rpn2 to a similar extent as in Vhl RNAi animals (Figure 4L). Additionally, proteasome RNAi increased the expression level of an in vivo transcriptional reporter for hypoxia signaling (see Additional file 15: Figure S15).
These results suggested that hypoxia signaling may be involved in proteasome RNAi-induced autophagy. To evaluate the genetic pathways mediating compensatory autophagy upon proteasome impairment, we tested the potential role of sima, core Atg genes required for starvation-induced autophagy, and p62.
As a final confirmation of our results, we also tested two additional proteasome RNAi lines for hypoxia and p62 signaling. Depletion of sima, but not p62, also reduced the number of LTR puncta in Prosα1 and Prosα5 RNAi cells (Figure 5H).
Autophagy and UPS coordinately sustain cellular homeostasis through protein quality control and clearance. A wide range of neurodegenerative diseases (often described as proteinopathies) are characterized by accumulation of aggregate-prone proteins that are not efficiently removed by proteasome. Compensatory autophagy may alleviate disease pathology in these cases, as numerous studies showed that substrates of autophagy and UPS overlap [1, 4, 5]. Indeed, genetic or pharmacological activation of autophagy facilitates aggregate clearance and neuronal function in disease models [1, 5]. Chronic block of proteasomal degradation also leads to toxicity, ER stress and cell death in various mammalian and Drosophila cells, and proteasome inhibitors are in clinical use to trigger apoptosis of cancer cells in multiple myeloma patients [31–34]. While we found no evidence of cell death in larval Drosophila fat body upon silencing of genes encoding essential subunits of the proteasome or genetic activation of hypoxia signaling, these genetic manipulations strongly enhanced both basal and starvation-induced autophagy in these cells, and this effect was dependent on canonical autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12.
An important aspect of our study is that overexpressed tagged Atg8 is not ideal for analyzing autophagy in proteasome RNAi cells, as excess p62-containing aggregates incorporate these reporter molecules, likely due to the physical interaction of p62 and Atg8. This artefact may then lead to false conclusions regarding autophagic activity, similar to the case of co-overexpressed p62 and Atg8 reporters .
How does proteasome impairment lead to compensatory autophagy? Multiple scenarios are possible, and these are not mutually exclusive. One hypothesis would be that accumulation of excess cargo triggers the formation of autophagosomes. However, overexpression of specific autophagy substrates p62 or blue cheese (the fly homolog of human Alfy that is also involved in the clearance of ubiquitinated proteins) did not result in an obvious increase in autophagy in Drosophila, unlike proteasome impairment [21, 35]. Moreover, p62 was found to be dispensable for both viability and starvation-induced autophagy in knockout mice . Multiple selective autophagy receptors have been identified in mammalian cells, but these are relatively uncharacterized in Drosophila, as only HDAC6 and blue cheese have been analyzed so far besides p62 [13, 35, 36]. Further studies are necessary to find out whether any of the selective adaptors is involved in autophagy induced by proteasome inactivation, or by starvation. After all, autophagy is thought to be responsible for breakdown and recycling of bulk cytoplasm upon nutrient limitation , so it is possible that none of the receptors are actually required for these large-scale autolysosomal degradation processes.
A second hypothesis is that proteasomal degradation is essential to maintain free intracellular amino acid levels and compensatory autophagy is induced by amino acid shortage, as suggested in a recent publication . In this paper, addition of exogenous amino acids was reported to attenuate proteasome inactivation-induced autophagy and partially rescue lifespan reduction in flies treated with a proteasome inhibitor . In this scenario, loss of autophagy in cells with impaired proteasome activity would further reduce amino acid levels as both recycling pathways are inhibited, presumably resulting in a stronger block of cell growth. However, blocking the function of core autophagy genes did not further decrease the growth of proteasome RNAi cells in fat body clones, indicating that reduced amino acid levels are likely not the only link between these processes in this setting.
A third hypothesis is that stabilization of certain proteasome substrate(s) positively regulates autophagy. The levels of HIF-1α are normally kept low through ubiquitination mediated by VHL, which is followed by proteasomal degradation. Hypoxia leads to stabilization of HIF-1α, resulting in its translocation to the nucleus and upregulation of hypoxia-inducible genes that mediate survival at low oxygen concentrations . Cytoprotective autophagy is also induced by hypoxia, and hypoxia-induced autophagy is necessary for survival of cancer cells in the central, poorly vascularized regions of solid tumors . We found that genetic activation of hypoxia signaling induced autophagy in Drosophila, and silencing of the fly HIF-1α ortholog sima attenuated proteasome impairment-induced autophagy. These results suggest that autophagy is enhanced by proteasome inhibition at least in part through hypoxia signaling. A recent genome-wide association study identified mutations affecting the UPS pathway in renal cell carcinoma, which were associated with increased levels of HIF-1α in these tumors . Interestingly, cells derived from many established cancers show elevated levels of autophagy, and multiple oncogenes and tumor suppressors (such as VHL) encode enzymes of the UPS involved in ubiquitin conjugation or deconjugation . Thus, based on these and our results, activation of hypoxia signaling by UPS impairment may be responsible for increased autophagy in the absence of hypoxic conditions in a subset of established cancers.
Our work showed that genetic inactivation of proteasomal degradation induces compensatory autophagy in the widely used Drosophila fat body model, which requires core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Proteasome impairment leads to large-scale accumulation of aggregates containing ubiquitinated proteins and the selective autophagy cargo p62, but p62 itself turned out to be largely dispensable for enhanced autophagy. Finally, we showed that genetic activation of hypoxia/HIF-1α signaling induces autophagy in Drosophila, and HIF-1α/sima, a well-characterized proteasome substrate, contributes to autophagy induced by proteasome impairment.
Drosophila stocks and culture
Flies were raised at 25°C on standard cornmeal/agar media, at 50% humidity and a 12-hour light/12-hour dark daily cycle. Fly stocks used in this study were obtained from the Vienna Drosophila RNAi Center, VDRC, Vienna, Austria and Bloomington Drosophila Stock Center, Bloomington, Indiana, USA, or kindly provided by Udai Pandey (GFP-CL1)  and Pablo Wappner (LDH-GFP)  (see Additional file 16: Figure S16 for a list of stocks). Note that KK, GD and JF lines are long hairpin-based RNAi transgenics, and HMS stocks contain short microRNA-based silencing constructs.
RT-PCR, western blots and immunostainings
Total RNA was isolated from L3 stage larvae using Purelink RNA Mini Kit (Ambion), followed by cDNA synthesis with Revertaid First Strand cDNA Synthesis Kit (Fermentas) and PCR amplification at 25 cycles. The following primers were used (designed to overlap exon-exon boundaries to prevent accidental amplification from genomic DNA): TACTCCTCCCGACACAAAGC, CTGGGTCATCTTCTCACGGT (Actin5C), TGGATCGACGCTGATAAAGA, GTCTCCTGAAACGGGCAAT (p62), CTTGGATCGAACTCAGCACA, CAATGTTCCAGCTACTGGGTG (BNIP3).
Western blots were carried out as described [6, 25], using primary antibodies: rabbit anti-p62 (1:5,000) , rabbit anti-Atg8a (1:5,000)  or mouse monoclonal anti-Tubulin AA4.3 (1:1,000, DSHB), followed by alkaline phosphatase conjugated goat anti-rabbit, anti-mouse secondary antibodies (1:5,000, Millipore). Immunostainings were done as before [21, 25], using primary antibodies chicken anti-GFP (1:1,500, Invitrogen), rabbit anti-Atg8a (1:500, gift of Katja Köhler) , rabbit anti-p62 (1:2,000) , rabbit anti-ubiquitin (1:50, Dako), rabbit anti-active caspase 3 (1:500, Cell Signaling). Secondary antibodies were Alexa 488 anti-chicken, Alexa 546 anti-rabbit (1:1,500, Invitrogen).
Please see Additional file 17: Figure S17 for original images of RT-PCR gels and western blots.
Genetics and treatments
Clonal analysis in larval fat bodies was done as exhaustively described previously [6, 18, 19, 21–25]. Briefly, cell clones were spontaneously generated by Flp-mediated excision of the FRT (Flp Recombination Target, >) cassette from Act > CD2 > Gal4 to activate UAS-dependent transcription of overexpression, dominant-negative and RNAi transgenes, together with the GFP reporters indicated in figure legends. Note that cell clones arose in embryos, and all of our analyses were carried out in L3 stage larvae at 84-90 hours after egg deposition. We also generated stable lines of hsFlp, Act > CD2 > Gal4, UAS-mCD8-GFP (all recombined together on chromosome X); UAS-Rpn2 or UAS-Rpt1 or UAS-Prosβ2 RNAi (located on autosomes). These lines were crossed to various Atg, p62 and sima RNAi and dominant-negative lines in genetic interaction tests. RNAi was mediated in whole fat bodies by cg-Gal4 for electron microscopy. As systemic knockdown of proteasomal genes resulted in early stage lethality, hsFlp, Act > CD2 > Gal4, UAS-RNAi stocks were heat shocked for 1 hour at 37 degrees at 48 hours after egg deposition to induce gene silencing in polyploid cells for western blots. Larvae of the age 60-66 hours after egg deposition were transferred to fresh medium containing 3 mg/ml chloroquine (Sigma) for a 24-hour treatment in autophagic flux experiments.
Histology and imaging
Lysotracker stainings were carried out as described [19, 21–25]. Briefly, larvae were allowed to feed on food supplemented with plenty of yeast (fed) or floated in 20% sucrose solution for 3 hours (starved). Fat bodies were dissected, stained with Lysotracker Red (Invitrogen) and DAPI in PBS for 5 minutes, and photographed live. A Zeiss Axioimager M2 microscope equipped with an Apotome2 unit and a Plan-NeoFluar 40× 0.75 NA objective was used to capture images with Axiovision software. Primary images were processed in Adobe Photoshop to produce final figures. Tissues were processed for transmission electron microscopy analysis as described previously [6, 18, 25]. Images were captured on a Jeol JEM-1011 microscope using an Olympus Morada 11 megapixel camera and iTEM software (Olympus).
Autophagic structures in ultrastructural images, Lysotracker Red (LTR) and mCherry-Atg8a puncta were manually counted in Photoshop. Statistical evaluation of GFP-CL1, p62 and anti-Atg8a images was carried out using ImageJ (NIH) as described [21, 25]. As proteasome RNAi and sima overexpressing cells were usually smaller than control cells, the numbers of dots per GFP-positive clone area or control tissue area were counted. To avoid errors owing to cell size differences, dot numbers shown in figures are normalized to control cells, that is, if the average size of GFP-positive cells was 10% of control cells and contained 5 dots on average, then the normalized number of dots per cell is 50 .
Images taken from multiple individual larvae per genotype (n) as indicated in figure legends were evaluated. Data were analyzed in PASW Statistics 18 (IBM). After testing for normality of data distribution, p values were calculated by Student’s t tests or Mann-Whitney u tests in pairwise comparisons for normal or non-normal distribution data, respectively. We used ANOVA for multiple comparisons of normal distribution data and Kruskal-Wallis test for non-normal distribution data. Bar charts show average values and standard deviations. Regression analyses were also done in SPSS.
LDH-GFP fluorescence was detemined with ImageJ (NIH) software. Individual larvae were selected in the greyscale GFP channel, and the mean gray value was measured. Backgrund fluorescence from near each larva was subtracted from the mean gray value.
We would like to thank Zsófia Kováts and Sarolta Pálfia for technical assistance, and Pablo Wappner, Katja Köhler and public stock centers for providing reagents. This work was supported by grants from the Hungarian Scientific Research Fund (OTKA NK78012 for MS), and the Wellcome Trust (087518/Z/08/Z for GJ).
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