Hif-2α promotes degradation of mammalian peroxisomes by selective autophagy.

Cell Metabolism

Article Hif-2a Promotes Degradation of Mammalian Peroxisomes by Selective Autophagy Katharina M. Walter,1,2,10 Miriam J. Scho¨nenberger,1,2,10 Martin Tro¨tzmu¨ller,3 Michael Horn,1 Hans-Peter Elsa¨sser,4 Ann B. Moser,5 Miriam S. Lucas,6 Tobias Schwarz,6 Philipp A. Gerber,7 Phyllis L. Faust,8 Holger Moch,9 Harald C. Ko¨feler,3 Wilhelm Krek,1,2,* and Werner J. Kovacs1,2,* 1Institute

of Molecular Health Sciences Center for Systems Physiology and Metabolic Diseases ETH Zurich, CH-8093 Zurich, Switzerland 3Core Facility for Mass Spectrometry, Center for Medical Research, Medical University of Graz, A-8010 Graz, Austria 4Department of Cytobiology, Philipps-University Marburg, D-35037 Marburg, Germany 5Kennedy Krieger Institute, Baltimore, MD 21205, USA 6ScopeM – Scientific Center for Optical and Electron Microscopy, ETH Zurich, CH-8093 Zurich, Switzerland 7Division of Endocrinology and Diabetes, University Hospital Zurich, CH-8091 Zurich, Switzerland 8Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA 9Institute of Surgical Pathology, University Hospital Zurich, CH-8091 Zurich, Switzerland 10Co-first Authors *Correspondence: [email protected] (W.K.), [email protected] (W.J.K.) http://dx.doi.org/10.1016/j.cmet.2014.09.017 2Competence

SUMMARY

Peroxisomes play a central role in lipid metabolism, and their function depends on molecular oxygen. Low oxygen tension or von Hippel-Lindau (Vhl) tumor suppressor loss is known to stabilize hypoxia-inducible factors alpha (Hif-1a and Hif-2a) to mediate adaptive responses, but it remains unknown if peroxisome homeostasis and metabolism are interconnected with Hif-a signaling. By studying liver-specific Vhl, Vhl/Hif1a, and Vhl/Hif2a knockout mice, we demonstrate a regulatory function of Hif-2a signaling on peroxisomes. Hif-2a activation augments peroxisome turnover by selective autophagy (pexophagy) and thereby changes lipid composition reminiscent of peroxisomal disorders. The autophagy receptor Nbr1 localizes to peroxisomes and is likewise degraded by Hif-2a-mediated pexophagy. Furthermore, we demonstrate that peroxisome abundance is reduced in VHL-deficient human clear cell renal cell carcinomas with high HIF-2a levels. These results establish Hif-2a as a negative regulator of peroxisome abundance and metabolism and suggest a mechanism by which cells attune peroxisomal function with oxygen availability.

INTRODUCTION Oxygen (O2) is an essential substrate in cellular metabolism and signaling and, as such, is inseparably linked to survival and normal function of all metazoan. Central to the molecular mechanisms underlying O2 homeostasis are hypoxia-inducible factors (HIFs) that function as master regulators of the adaptive response to hypoxia. HIFs are heterodimers composed of a constitutively

expressed HIF-1b subunit and O2-regulated a subunits (HIF-1a and HIF-2a) (Keith et al., 2012). Under normoxia, HIF-a subunits are hydroxylated and targeted for proteasomal degradation by an ubiquitin ligase complex containing the von Hippel-Lindau (VHL) tumor suppressor protein pVHL. Under hypoxia, HIF-a hydroxylation is inhibited, thereby stabilizing HIF-a protein. HIF-a dimerizes with HIF-1b to activate target genes. HIF-1a and HIF-2a have both common and distinct target genes and are differentially regulated in various physiological and pathophysiological conditions, such as cancer and ischemic diseases (Semenza, 2012; Keith et al., 2012). HIF-induced genes promote angiogenesis, erythropoiesis, metastasis, and metabolic reprogramming, such as shifting metabolism from oxidative phosphorylation to glycolysis. Activation of HIFs also reprograms lipid metabolism leading to lipid accumulation (Kucejova et al., 2011; Qu et al., 2011; Kim et al., 2006; Rankin et al., 2009; Krishnan et al., 2009), but it remains controversial how they promote lipid accumulation. Abundant evidence suggests key roles for HIF-1a in the regulation of mitochondrial metabolism (Semenza, 2011). However, so far there is no evidence linking HIF signaling to peroxisomes, whose function depends highly on molecular O2. In fact, peroxisomes may be responsible for as much as 20% of O2 consumption and 35% of H2O2 production in tissues such as the liver (Fransen et al., 2012). Peroxisomes are extremely versatile and dynamic organelles whose number, size, and function are dependent on cell type and metabolic needs. They play key roles in the degradation of fatty acids (i.e., very long-chain fatty acids [VLCFAs] and polyunsaturated fatty acids [PUFAs]), ether lipid synthesis, cholesterol and bile acid synthesis, and metabolism of reactive oxygen species (Van Veldhoven, 2010; Faust and Kovacs, 2014; Fransen et al., 2012). The importance of peroxisomal metabolism is illustrated by peroxisome biogenesis disorders in which functional peroxisomes are absent or disorders caused by single peroxisomal enzyme deficiencies (Raymond et al., 2009). Lack of peroxisomal metabolism leads to extensive cellular metabolic disarray, and patients with peroxisomal disorders,

882 Cell Metabolism 22, 882–897, November 4, 2014 ª2014 Elsevier Inc.

Cell Metabolism Hif-2a Promotes Mammalian Pexophagy

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Cell Metabolism 22, 882–897, November 4, 2014 ª2014 Elsevier Inc. 883


as well as peroxisome-deficient mice, display severe hepatic dysfunction with fatty liver, cholestasis, and eventually cirrhosis (Raymond et al., 2009; Baes and Van Veldhoven, 2012; Faust and Kovacs, 2014). Since peroxisomal function depends on O2, we hypothesized that to minimize O2 consumption under hypoxic conditions, HIF-a signaling may inhibit O2-dependent peroxisomal metabolism and/or decrease the number of peroxisomes. In addition, HIF-mediated reduction in peroxisomal activity could promote metabolic reprogramming causing changes in lipid metabolism. To test this hypothesis, we chose the mouse liver as model system because peroxisomes are highly abundant in the liver, and liver-specific loss of pVhl causes severe lipid accumulation. In this study, we systematically investigated peroxisome homeostasis and metabolism in wild-type and liver-specific Vhl, Vhl/ Hif1a, and Vhl/Hif2a knockout mice and explored the role of Hif-1a and Hif-2a in this context. RESULTS

To characterize the peroxisomal population in more detail, we isolated peroxisomes from control and Vhl/ livers (Figure S2A). Catalase activity was significantly decreased in postnuclear supernatants from Vhl/ livers (Figure S2B). Peroxisomal protein levels were significantly decreased in subcellular fractions from Vhl/ livers, whereas levels of the mitochondrial proteins Tomm20, Trap1, and Vdac were similar to controls (Figure S2C). Immunoblots of gradient fractions from control livers showed that peroxisomes were enriched in fractions 17–20 (density range of 1.144–1.169 g/ml), whereas peroxisomal protein levels were significantly decreased in gradient fractions from Vhl/ livers (Figure S2D). The distribution pattern of catalase activity displayed a major peak coincident with peroxisomal proteins in control livers, and catalase activity was significantly decreased in Vhl/ livers (Figure S2E). These results argue for the presence of intact peroxisomes in Vhl/ livers, but the distribution pattern of peroxisomal proteins suggests a heterogeneous peroxisome population. In summary, hepatic loss of Vhl function is associated with a reduction in peroxisome abundance.

Peroxisome Abundance Is Reduced in Vhl–/– Livers To investigate whether peroxisomal abundance and/or metabolism are regulated by Hif-a, we inactivated the Vhl gene in the liver. As previously reported (Kucejova et al., 2011; Rankin et al., 2009), 4-week-old (P28) liver-specific Vhl/ mice were significantly smaller than control mice but had an increased liver to bodyweight ratio, displayed severe steatosis, were hypoglycemic, and Hif target gene expression was increased (Figures S1A–S1G available online). Intriguingly, the protein levels of the peroxisomal matrix proteins catalase, acyl-CoA oxidase (Acox1), multifunctional protein 1 (Mfp1), urate oxidase (Uox), as well as the peroxisomal membrane proteins Pex14p and Pex3p were significantly decreased in livers of P28 Vhl/ mice (Figure 1A), a phenomenon already evident at 2 weeks of age (Figure S1H). The effects of pVhl loss on peroxisomal protein levels were highly selective, as representative ER and mitochondrial protein levels were similar in control and Vhl/ mice (Figures 1A and S1H). Only the mitochondrial outer membrane protein Vdac was moderately reduced in P28 Vhl/ mice. Analysis of the number of peroxisomes by cytochemical localization of catalase and immunofluorescence of Pex14p and Acox1 revealed a strong reduction in peroxisome abundance in P14– P28 Vhl/ livers (Figures 1B and S1I). Catalase activity was decreased 60%–70% in Vhl/ livers compared with control livers (Figures 1C and S1J). Electron microscopic comparisons of control and Vhl/ livers revealed that peroxisomes are similar in size and shape (Figure S1K).

Changes in Lipid Composition in Vhl–/– Livers Are Reminiscent of Peroxisomal Disorders Since peroxisomes play important roles in the degradation of VLCFAs and VLC-PUFAs, as well as in the synthesis of polyunsaturated FAs, we investigated whether the decrease in peroxisome abundance had consequences on the lipid composition in Vhl/ livers. We quantified the major lipid classes and the distribution of fatty acids within these classes in P28 control and Vhl/ livers and observed profound lipid alterations in Vhl/ livers. The profile of triacylglycerol (TAG) species showed a disproportionate increase of TAGs with VLCFAs and VLC-PUFAs in Vhl/ livers (Figures 1D and 1E; Table S1). C62 to C64 species were hardly detectable in control livers but reached concentrations between 10–100 nmol/g liver tissue in Vhl/ livers. Most TAG species showed an accumulation of 10-fold in Vhl/ livers, but TAG species with a higher number of double bonds were disproportionately increased 30- to 100-fold (Figures 1D and 1E). Next, we determined the distribution of fatty acids in the liver. The hepatic levels of docosahexaenoic (DHA, C22:6n3) and arachidonic acid (AA, C20:4n6) were significantly decreased in Vhl/ mice, whereas the parent fatty acids, linolenic (C18:3n3) and linoleic acid (C18:2n6), respectively, as well as docosapentaenoic acid (C22:5n3) upstream to DHA synthesis were significantly increased (Figure 1F; Table S2). Blood levels of VLCFA C26:0 and C24:0 lyso-phosphatidyl cholines were increased in Vhl/ mice (Figure S1L). Hence, VLCFAs and VLC-PUFAs accumulations as well as DHA and AA deficiencies

Figure 1. Peroxisome Abundance Is Decreased in Vhl–/– Livers Peroxisomes were analyzed in P28 Vhlf/f (control) and liver-specific Vhl/ mice. (A) Immunoblots of liver lysates with antibodies against peroxisomal matrix (Catalase, Acox1, Mfp1, and Uox) and membrane proteins (Pex14p and Pex3p), mitochondrial (Vdac), and ER (ERp57 and Grp94) proteins. (B) Cytochemical localization of catalase and immunofluorescence of Pex14p and Acox1 in livers. (C) Catalase activity in liver lysates. (D) Concentration spectrum of TAG species. TAGs are designated numerically as carbon and double bond number (after the colon) in acyl chains. (See Table S1 for detailed quantitative lipid profiles.) (E) Relative abundance of the TAG lipid species in Vhl/ livers normalized to control values. (F) Fatty acid profiles in total lipid extracts from livers were determined by gas chromatography-mass spectrometry (GC-MS). Relative signal intensities were normalized to an internal standard and expressed as percentage of total fatty acids. Data are mean ± SD (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001 versus control mice.



Figure 2. Hif-2a-Dependent Decrease of Peroxisome Abundance in Vhl–/– Livers Peroxisomes were analyzed in P28 control (Cre: Vhlf/f, Vhlf/f/Hif1af/f, and Vhlf/f/Hif2af/f) and liver-specific Vhl/, Vhl//Hif1a/, and Vhl//Hif2a/ mice. (A) Immunoblots of peroxisomal proteins, Hif-2a, and pVhl in liver lysates. (B) Immunofluorescence of Pex14p in livers. (C) Catalase activity in liver lysates. (legend continued on next page)



are characteristic features of hepatic Vhl loss of function. These changes in lipid composition are reminiscent of alterations due to defective peroxisomal b-oxidation in peroxisome-deficient mice and patients with peroxisomal disorders (Baes and Van Veldhoven, 2012; Raymond et al., 2009; Van Veldhoven, 2010). Hif-2a but Not Hif-1a Activation Mediates the Decrease of Peroxisome Abundance in Vhl–/– Livers To examine if the reduction of peroxisome number upon pVhl loss is mediated by Hif-1a or Hif-2a, we analyzed peroxisome abundance in liver-specific Vhl/Hif1a and Vhl/Hif2a double knockout mice (Figures 2 and S3). P28–P42 Vhl//Hif1a/ mice were significantly smaller than control mice, they were hypoglycemic, and their liver was enlarged and revealed severe steatosis similar to Vhl/ mice (Figures S3A–S3C). In contrast, Vhl//Hif2a/ mice were phenotypically similar to control mice (Figure S3A). With respect to the peroxisomal phenotype, we observed a striking rescue in Vhl//Hif2a/ but not Vhl//Hif1a/ mice. Peroxisomal protein levels were significantly decreased in Vhl//Hif1a/ livers (Figures 2A and S3D), whereas their levels were similar in control and Vhl// Hif2a/ livers (Figures 2A and S3E). Immunofluorescence of Pex14p revealed a strong reduction of peroxisome number in Vhl//Hif1a/ but not Vhl//Hif2a/ livers (Figure 2B). Catalase activity was similarly decreased in P28 Vhl/ and Vhl// Hif1a/ livers compared to controls, whereas catalase activity was similar in control and Vhl//Hif2a/ livers (Figures 2C and S3F). Hepatic TAG levels were significantly increased in Vhl/ and Vhl//Hif1a/ livers and only marginally increased in Vhl//Hif2a/ livers compared to controls (Figure 2D). The expression of Hif target genes was, with the exception of Epo, similarly increased in P28–P42 Vhl/, Vhl//Hif1a/, and Vhl//Hif2a/ livers (Figures 2E and S3G). Thus, both Hif-1a and Hif-2a were stabilized in Vhl/ livers, but the reduction in peroxisome number depends exclusively on Hif-2a activity. Liver-specific loss of Hif-1a or Hif-2a alone did not affect peroxisomal protein levels (Figure S3H). Next, we investigated the composition of hepatic TAGs in Vhl//Hif1a/ and Vhl//Hif2a/ livers. TAG species with VLCFAs and VLC-PUFAs increased in Vhl//Hif1a/ livers (Figure 2F; Table S1) but changed only moderately in Vhl// Hif2a/ livers (Figure S3I; Table S1). Changes in the fatty acid profile of Vhl//Hif1a/ livers were similar as in Vhl/ livers (Figure 2G; Table S2). Importantly, fatty acid profiles from Vhl/ and Vhl//Hif1a/ livers were comparable to P13 peroxisomedeficient Pex2/ livers that also displayed steatosis (Figure S4A) (Keane et al., 2007; Kovacs et al., 2012). Bile acids were analyzed in plasma from our mouse models. The plasma of Vhl/ and Vhl//Hif1a/ mice was yellowish with increased levels of cholic acid (CA) and chenodeoxycholic acid (CDCA) (Figures S4B and S4C), which is a sign of cholestasis. Importantly, the levels of the C27-bile acids 3a,7a-dihy-

droxycholestanoic acid (DHCA) and 3a,7a,12a-trihydroxycholestanoic acid (THCA) were significantly increased in the plasma of these mice (Figures S4B and S4C). C27-bile acids accumulate in plasma of patients and mice with peroxisome deficiency disorders; they are toxic and have been suggested to contribute to liver disease associated with peroxisomal disorders (Keane et al., 2007; Ferdinandusse et al., 2009; Hiebler et al., 2014). CA and CDCA levels were similar in the plasma of control and Vhl//Hif2a/ mice, and DHCA and THCA levels were decreased in the plasma of Vhl//Hif2a/ mice compared to controls (Figure S4D). Peroxisome Proliferation Can Be Induced in Vhl–/– Livers Peroxisome homeostasis is achieved by balancing biogenesis and degradation of peroxisomes. To explore potential mechanisms underlying the pronounced reduction of peroxisome abundance in Vhl/ livers, we assessed the expression of Pex genes, which encode peroxins essential for peroxisome biogenesis (Smith and Aitchison, 2013). The expression of Pex genes was similar in P14–P28 control and Vhl/ livers (Figures 3A and S1M). Pex11a was the only peroxin that showed significant transcriptional upregulation in Vhl/ (Figures 3A and S1M) and Vhl//Hif1a/ (Figure 3B) livers, whereas Pex11a was similarly expressed in control and Vhl//Hif2a/ livers (Figure 3B). Since overexpression of Pex11a promotes peroxisome division and induces peroxisome abundance (Schrader et al., 2012), this result may reflect a feedback loop in which Vhl/ and Vhl//Hif1a/ hepatocytes sense the reduction of peroxisomes and attempt to counterbalance their lack by inducing a positive regulator of peroxisome biogenesis. Given the fact that the expression of the peroxisome biogenesis machinery was not impaired in Vhl/ livers and that peroxisome biogenesis proceeds, at least in part, through division of pre-existing peroxisomes, we tested whether pharmacological induction of peroxisome proliferation was possible in Vhl/ livers. We treated Vhl/ mice either with WY-14,643 (WY), which is a peroxisome proliferator-activated receptor a (Ppara) ligand, or 4-phenylbutyrate (PBA), which acts independently of Ppara. Treatment of mice for 3 days with PBA resulted in a strong induction of catalase activity (Figure 3C) and peroxisomal protein levels (Figure 3E) in control and Vhl/ livers. However, PBA treatment was stopped after 3 days due to a negative effect on the health state of Vhl/ mice. WY treatment for 7 days strongly increased catalase activity (Figure 3D) and peroxisomal protein levels in control livers and to a lesser extent in Vhl/ livers (Figure 3F). Cytochemical staining of catalase activity revealed that peroxisome number was significantly increased both in control and Vhl/ livers upon WY treatment (Figure 3G), but in Vhl/ livers, peroxisome abundance was still lower than in untreated controls. Electron microscopy demonstrated that peroxisomes from Vhl/ livers were similarly shaped as those from control mice, although generally smaller in size (Figure 3G).

(D) Hepatic TAG content. (E) Expression of Hif target genes in P28 livers. Each value represents the amount of mRNA relative to that in control mice, which was arbitrarily defined as 1. (F) Concentration spectrum of TAG species in livers. Data are mean ± SD (n = 6; see Table S1 for detailed quantitative lipid profiles). (G) Fatty acid profiles in total lipid extracts from livers were determined by GC-MS (n = 6 mice for each genotype). Relative signal intensities were normalized to an internal standard and expressed as percentage of total fatty acids. Data are mean ± SD (n = 7 for Vhlf/f, n = 5 for Vhl/, n = 3 for Vhlf/f/Hif1af/f, n = 4 for Vhl// Hif1a/, n = 4 for Vhlf/f/Hif2af/f, n = 5 for Vhl//Hif2a/ mice). *p < 0.05; **p < 0.01; ***p < 0.001 versus control mice.



Figure 3. WY and PBA Induce Peroxisome Proliferation in Vhl–/– Mice (A) Expression of Pex genes in P28 livers (n = 5–7 mice). (B) Expression of Pex11a in P28–P42 livers (n = 10 mice). (C and D) Catalase activity in liver lysates. (legend continued on next page)



The short-term induction of peroxisomes in Vhl/ livers upon PBA or WY treatment was insufficient to reduce hepatic TAG levels and rescue the massive steatosis in Vhl/ mice (Figures 3H and 3I). These results argue for a functional peroxisome biogenesis machinery in Vhl/ livers. Since peroxisome proliferation in Vhl/ mice was only moderately increased in response to WY treatment and accumulation of VLCFAs and VLC-PUFAs is known to induce Ppara signaling and peroxisome proliferation, we determined the mRNA levels of Ppara target genes in WY-treated mice (Figure 3J). The mRNA levels of Ppara and its target genes were significantly increased both in control and Vhl/ mice upon WY treatment (Figure 3J), but WY-mediated transcriptional induction of Ppara target genes was much weaker in Vhl/ mice (Figure 3J). Except for Pex11a, Ppara target gene expression was similar in vehicletreated control and Vhl/ mice (Figure 3J). Likewise, induction of Ppara target genes was impaired upon fasting in Vhl/ mice, whereas their expression was similar in ad-libitum-fed control and Vhl/ mice (Figure 3K). Thus, Hif-2a signaling has a repressive effect on ligand-dependent Ppara transcriptional activity and by extension on stimulated peroxisome biogenesis. Autophagy Is Induced in Vhl–/– Mice Since peroxisome biogenesis and proliferation are functional, we addressed if increased degradation of peroxisomes underlies the pronounced reduction of peroxisome abundance in Vhl/ livers. Three mechanisms for peroxisome degradation have been described in mammalian cells, which include selective autophagy (pexophagy), proteolysis by Lon protease 2 (Lonp2), and 15-lipoxygenase-1 (Alox15)-mediated autolysis (Till et al., 2012). To quantify autophagic flux in vivo, control and Vhl/ mice were infected with an adenovirus expressing GFP-tagged LC3 (Ad-GFP-LC3). The number of GFP-LC3-positive vesicles was increased 3-fold in Vhl/ livers compared to control mice (Figure 4A), indicating increased autophagic flux. Conversion of nonlipidated LC3-I into the phosphatidylethanolamine-conjugated LC3-II form was significantly increased and protein levels of the autophagic substrate p62 were decreased in Vhl/ livers (Figure 4B). In summary, these results show that autophagic flux is increased in Vhl/ livers. Immunofluorescence of catalase revealed that GFP-LC3-positive autophagosomes colocalized with peroxisomes in Vhl/ livers (Figures 4C, S5A, and S5B). We analyzed the autophagic sequestration of peroxisomes with superresolution microscopy capable of 3D-structured illumination (3D-SIM), which provides an increase of both axial and lateral resolution by a factor of two compared to confocal microscopy. Reconstructed 3D-SIM images show that both single and multiple peroxisomes were sequestrated in autophagosomes and surrounded by an autophagosomal membrane decorated with GFP-LC3 (Figures 4D, S5C, and S5D; Movies S1 and S2). Importantly, autophagosome-like

structures containing either single or clusters of peroxisomes were detected by electron microscopy in hepatocytes of Vhl/ but not in control mice (Figures 4E and 4F), arguing for increased pexophagy in Vhl/ livers. Lonp2 expression was similar in control and Vhl/, Vhl// Hif1a/, and Vhl//Hif2a/ livers (Figures S5F–S5H). Hepatic mRNA levels of Alox15 were very low and only moderately increased in Vhl/ and Vhl//Hif1a/ livers compared to controls (Figures S5F and S5G). Protein levels of Alox15 were similar in control and Vhl/ livers (Figure S5E). Hence, Lonp2 and Alox15 do not behave as Hif target genes whose induction could promote peroxisome turnover. The Autophagy Machinery Is Required for Hif-2a-Mediated Pexophagy To directly assess whether autophagy is responsible for the peroxisomal phenotype in Vhl/ livers, we examined the effect of constitutive Hif-2a signaling on peroxisome abundance in liver-specific, autophagy-deficient Atg7/ mice. We generated an adenovirus coexpressing GFP and HIF-2a P405/531A (hereafter called Ad-HIF-2a(mt)), a HIF-2a variant that escapes degradation by pVHL. We injected Atg7f/f and liver-specific Atg7/ mice either with Ad-HIF-2a(mt) or an adenovirus expressing GFP (Ad-GFP) as control and sacrificed mice after 6 days. Expression of HIF-2a(mt) caused hepatomegaly and severe steatosis in Atg7f/f livers, while Atg7/ livers remained unaffected (Figure 5A). Almost all hepatocytes in Atg7f/f livers expressing HIF-2a(mt) displayed macrovesicular steatosis (Figure 5A), an effect not seen in HIF-2a(mt)-expressing Atg7/ livers (Figure 5A). Peroxisome abundance and peroxisomal protein levels were significantly decreased in Atg7f/f livers expressing HIF-2a(mt) compared to controls (Figures 5B and 5C), while peroxisome homeostasis was similar in Atg7/ hepatocytes expressing HIF-2a(mt) compared to GFP-expressing Atg7/ hepatocytes. Overexpression of HIF-2a(mt) did not affect mitochondrial and ER protein levels (Figure 5C). In accord with the histological analysis revealing severe steatosis, lipid droplet protein Adfp (adipose differentiation-related protein) levels increased significantly in Atg7f/f but not Atg7/ livers expressing HIF-2a(mt) (Figure 5C). Catalase activity was decreased 60% in Atg7f/f and remained unchanged in Atg7/ livers expressing HIF-2a(mt) as compared to controls (Figure 5D). Although Atg7f/f and Atg7/ mice received identical amounts of Ad-HIF-2a(mt), HIF-2a(mt) protein levels were higher in Atg7f/f compared to Atg7/ livers (Figure 5C). A possible explanation is that autophagy is important for efficient adenoviral infection and replication (Zeng and Carlin, 2013). Hence, one could argue that the amounts of HIF-2a(mt) produced in Atg7/ liver cells are insufficient to promote pexophagy. To address this, we compared Hif-2a levels in Vhl/ and Vhl//Hif1a/ livers with HIF-2a(mt) levels in Atg7/ livers (Figure 5E). Hif-2a levels

(E and F) Immunoblots of peroxisomal proteins in liver lysates. (G) Cytochemical localization of catalase in livers. Electron micrographs of ultrathin sections are shown in the right panel. (H and I) Hepatic TAG content in livers. (J) Expression of Ppara target genes in livers. (K) Expression of Ppara target genes in livers from ad-libitum-fed and overnight-fasted P28 mice. Data are mean ± SD (Ad-libitum-fed mice: n = 5–7; 12 hr fasted mice: n = 3) (Vehicle: n = 4–8 mice; WY: n = 4–8 mice) (Vehicle: n = 3 mice; PBA: n = 5 mice). *p < 0.05; **p < 0.01; ***p < 0.001 versus vehicle-treated or ad libitumfed control mice. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus WY- or PBA-treated or fasted control mice.



Figure 4. Autophagic Flux Is Increased in Vhl–/– Livers Control and Vhl/ mice were infected with Ad-GFP-LC3, treated with CQ, and sacrificed at the age of 4 weeks. (A) Representative pictures of GFP-LC3 punctate structures in hepatocytes and quantification of GFP-LC3 punctate structures per cell. Data are mean ± SD. **p < 0.01. (B) Immunoblots of autophagy-related proteins in liver lysates. (C) Immunofluorescence analysis for the colocalization of autophagosomes with peroxisomes. Liver sections were immunostained for GFP and catalase. Arrowheads: yellow dots represent colocalized GFP-LC3-positive autophagosomes with red fluorescence-labeled peroxisomes. See also Figures S5A and S5B for analysis of confocal z stack images with the orthogonal slicer tool of the Imaris software. (legend continued on next page)



in Vhl//Hif1a/ livers were comparable with HIF-2a(mt) levels in Atg7/ livers. Since Vhl//Hif1a/ livers revealed a strong reduction in peroxisome abundance, HIF-2a(mt) levels in Atg7/ livers are likely sufficient to induce Hif-2a-mediated pexophagy, and the deficiency of autophagy prevents this outcome. To further corroborate this assertion, we asked whether lowering HIF-2a(mt) levels in Atg7f/f mice by injection of less pfus could still decrease peroxisome abundance. Injection of different pfus resulted in a dose-dependent decrease of HIF-2a(mt) levels (Figure 5F) and a mosaic infection pattern in those livers (Figure S6A). A direct comparison of peroxisome abundance in infected and uninfected hepatocytes revealed a strong reduction in peroxisome numbers in hepatocytes expressing HIF-2a(mt) compared to uninfected hepatocytes (Figures 5G and S6B). We determined the expression of Hif target genes in Atg7f/f and Atg7/ mice infected with different amounts of Ad-HIF2a(mt). The mRNA levels of Hif target genes were significantly increased in livers of Atg7f/f and Atg7/ mice infected with 3 3 109 pfu of Ad-HIF-2a(mt) compared to controls (Figure S6C), but the induction was much weaker in Atg7/ livers. However, their expression was comparable to Atg7f/f mice infected with 1 3 109 pfu of Ad-HIF-2a(mt) (Figure S6C). In mice infected with low doses of Ad-HIF-2a(mt) (1 3 108 or 5 3 108 pfus), little, if any, induction of Hif target genes was observed. These results suggest that Atg7/ livers display a mosaic infection pattern and that infected hepatocytes produce sufficient HIF-2a(mt) to induce pexophagy. Overexpression of HIF-2a(mt) in Atg7f/f mice led to similar changes in hepatic lipid composition as seen in Vhl/ and Vhl//Hif1a/ livers. Most TAG species showed an accumulation of 10-fold in HIF-2a(mt)-expressing Atg7f/f livers compared to controls, and TAGs with VLCFAs and fatty acids with a high number of double bonds were again disproportionately increased up to 160-fold (Figures 5H and S6D). In summary, these results support the view that Hif-2a-mediated pexophagy requires a functional autophagy machinery, and HIF-2a(mt) gainof-function leads to similar lipid changes as Vhl/ and Vhl// Hif1a/ livers. Pharmacological Inhibition of Autophagy Increases Peroxisome Abundance in Vhl–/– Livers Next, we tested whether pharmacological inhibition of autophagy would restore peroxisome abundance in Vhl/ livers. Treatment of Vhl/ mice with chloroquine (CQ) significantly increased peroxisomal protein levels in Vhl/ livers (Figure S7A). To inhibit autophagy early at presequestration steps (Petiot et al., 2000; Seglen and Gordon, 1982), P21 control and Vhl/ mice were treated with 3-methyladenine (3-MA) for 5 days. 3-MAtreated Vhl/ mice displayed improved tonus and activity compared to untreated Vhl/ mice. Peroxisome abundance

was significantly increased in livers of 3-MA-treated Vhl/ mice (Figures 6A and 6B), while peroxisomal protein levels were similar in untreated and 3-MA-treated control livers. Restoration of peroxisome abundance in 3-MA-treated Vhl/ mice confirmed that loss of peroxisomes in Vhl/ livers is due to enhanced pexophagy, whereas lack of peroxisome accumulation in treated control mice suggests that their turnover rate under basal conditions is low. Hif target gene expression was not affected by 3-MA treatment (Figure S7E). Adfp protein levels were decreased in 3-MA-treated Vhl/ mice compared to untreated Vhl/ mice (Figure 6A), which is consistent with attenuated hepatic steatosis (Figure S7B). 3-MA treatment did not affect ER and mitochondrial protein levels (Figure 6A). These data corroborate the functional requirement for autophagy in the decrease in peroxisome content downstream of Hif-2a stabilization in Vhl/ livers. Protein levels of the selective autophagy receptors p62 and Nbr1 (neighbor of BRCA1 gene 1) (Rogov et al., 2014) were increased in 3-MA-treated Vhl/ mice compared to untreated Vhl/ mice (Figure 6A) and were similar in untreated and 3MA-treated control mice. Nbr1 and p62 levels and peroxisome abundance were concomitantly decreased in Vhl//Hif1a/ livers, whereas their levels were similar in control and Vhl// Hif2a/ livers (Figure 6C). Loss of Atg7 resulted as expected in the accumulation of Nbr1 and p62, and Nbr1 levels were decreased in Atg7f/f livers expressing HIF-2a(mt) (Figure 6C). Overexpression of NBR1 and p62 has been shown to induce degradation of peroxisomes (Deosaran et al., 2013), but mRNA levels of Nbr1 and p62 were similar in control and Vhl/, Vhl//Hif1a/, and Vhl//Hif2a/ livers (Figures S7C and S7D). In addition, overexpression of HIF-2a in Atg7f/f livers decreased mRNA levels of Nbr1 and p62 (Figure S6C). These results demonstrate that Nbr1 and p62 are not Hif target genes whose induction could promote pexophagy. Since our data suggested that Nbr1 and p62 are involved in Hif-2a-mediated pexophagy, we determined the subcellular localization of Nbr1 and p62. The prediction would be that Nbr1 and/or p62 localize to peroxisomes in Vhl/ mice to mediate pexophagy. Immunofluorescence of catalase revealed colocalization of peroxisomes with Nbr1 and p62 in untreated Vhl/ mice (Figures 6D and S7F). Interestingly, Nbr1 already localized to peroxisomes in control livers, whereas p62 was barely detectable (Figures 6D and S7F). p62 accumulated as focal aggregates in 3-MA-treated control livers, and some of these aggregates showed colocalization with Nbr1 and catalase, but in contrast to Nbr1, most peroxisomes did not colocalize with p62 (Figure S7F). In addition to the restoration of peroxisome abundance, we observed significant clustering of Nbr1- and p62-positive peroxisomes in 3-MA-treated Vhl/ mice (Figures 6D and S7F). Moreover, immunofluorescence revealed that

(D) Superresolution microscopy shows that both single (left panel) or multiple (right panel) peroxisomes (shown in red) are sequestrated in autophagosomes and surrounded by an autophagosomal membrane decorated with GFP-LC3. Liver sections were stained with antibodies against GFP and catalase. See also Figures S5C and S5D and Movies S1 and S2. (E and F) Electron microscopy detection of peroxisomal autophagy in Vhl/ livers. Livers were fixed and stained with DAB for catalase, which generates an electron-dense precipitate over the organelle. (E) The left panel shows a double-membrane structure, possibly a phagophore, enclosing a peroxisome. The right panel shows a peroxisome that is sequestered within a double-membrane autophagosome. (F) A cluster of peroxisomes is contained in a single-membrane-bound compartment, representing a putative autolysosome.



(legend on next page)



GFP-LC3-positive autophagosomes colocalized with Nbr1 and catalase in Vhl/ livers (Figure S7G). Analysis of purified peroxisomes from livers of untreated and 3-MA-treated control and Vhl/ mice also confirmed the peroxisomal localization of Nbr1 (Figures S2, S7H, and S7I). 3-MA treatment significantly increased catalase activity in peroxisomal peak fractions of control and Vhl/ livers (Figure S7H). Interestingly, the density of peroxisomes was increased in 3-MA-treated mice (Figure S7H). Peroxisomal protein levels were increased in gradient fractions of 3-MA-treated Vhl/ mice compared to untreated knockouts (Figure S7I). Nbr1 was present in peroxisomal fractions from untreated control livers, whereas in untreated Vhl/ livers Nbr1 was not detected probably due to the low amount of peroxisomes (Figures S2D and S7I). 3-MA treatment increased Nbr1 and p62 protein levels and both autophagic receptors were present in peroxisomal fractions of control and Vhl/ livers (Figure S7I). Collectively, we conclude that Nbr1 and p62 are recruited to peroxisomes and participate in Hif2a-mediated pexophagy in Vhl/ livers, where they are degraded together with peroxisomes in autolysosomes. Peroxisome Abundance Is Reduced in Clear Cell Renal Cell Carcinomas with High HIF-2a Levels To explore the relevance of HIF-2a-dependent pexophagy in human disease, peroxisome abundance was analyzed in more than 200 clear cell renal cell carcinoma (ccRCC) tissue samples on a tissue microarray (TMA) and compared with HIF-2a expression and differentiation grade. This disease setting was chosen for several reasons. First, loss of VHL function occurs in up to 90% of sporadic ccRCC, the most common form of kidney cancer (Hakimi et al., 2013). Second, HIF-2a is considered to be a driver oncoprotein for ccRCC. Third, peroxisomes are highly abundant in the kidney, and kidneys are affected in peroxisome biogenesis disorder patients. Fourth, at least in the context of steatosis and angiogenesis, ccRCC and Vhl/ livers behave similarly. Peroxisome abundance varies between the different cell types of the kidney. They are highly abundant in cells of proximal tubules and less abundant in distal tubules and collecting duct (Figure 7A). Organelle abundance was classified according to the situation in normal tissue: many organelles correspond to proximal tubules, a medium number to distal tubules and few organelles describe a number below the abundance observed in normal kidney tissue (Figure 7B). In tumor specimen with strong HIF-2a staining, 42% had few peroxisomes and only 8% had many peroxisomes (Figure 7D). Among the specimens that were negative for HIF-2a only 15% displayed few peroxisomes

in contrast to 39% with medium number and 46% with many peroxisomes (Figure 7D). Interestingly, peroxisome abundance is reduced more frequently in well-differentiated grade 1 as in grade 2 or 3 tumors (Figure 7E). In summary, the negative regulatory effect of HIF-2a signaling on peroxisome abundance is not restricted to hepatocytes and may extend to other tissues and disease settings that are characterized by high levels of HIF-2a such as ccRCC. DISCUSSION The main finding of this report is that the transcription factor Hif2a, but not Hif-1a, is a negative regulator of peroxisome abundance in hepatocytes. In fact, Hif-2a promotes pexophagy since Hif-2a-mediated downregulation of peroxisome abundance was suppressed in liver-specific autophagy-deficient Atg7/ mice and after inhibition of autophagy with 3-MA in Vhl/ mice. The autophagy receptor Nbr1 localizes to peroxisomes and is degraded together with peroxisomes by Hif-2a-mediated pexophagy. Reduced number of peroxisomes and the ensuing deficiency in peroxisomal function encompass major changes in the hepatic lipid profile that are reminiscent of peroxisomal disorders and include the accumulation of VLCFAs and VLC-PUFAs. These lipids are activating ligands for the transcription factor Ppara, a promoter of peroxisome proliferation. However, Hif2a restrains ligand-induced Ppara-mediated peroxisome proliferation. Thus, by simultaneously inducing pexophagy and counteracting Ppara, Hif-2a ensures efficient depletion of the peroxisome pool (Figure 7F). Finally, we demonstrate that peroxisome abundance is significantly reduced in ccRCCs that are characterized by high HIF-2a levels, providing a link between reduced peroxisome abundance and HIF-2a expression in human disease. Peroxisomes adapt rapidly to cellular demands, and their homeostasis is achieved by the counterbalance between organelle biogenesis and degradation (Smith and Aitchison, 2013; Till et al., 2012). Peroxisomal function depends highly on molecular O2, and the identification of Hif-2a as an activator of pexophagy suggests that peroxisome homeostasis is directly coupled to O2 availability. Hence, Hif-2a-mediated pexophagy may well be part of adaptive responses to hypoxia. Hypoxia signaling is involved in the pathogenesis of various liver diseases that are associated with changes in lipid metabolism (Nath and Szabo, 2012), and Hif-2a-mediated pexophagy might contribute to those changes in lipid metabolism. Indeed, we showed that Hif-2a-mediated pexophagy associated with decreased peroxisomal metabolism resulted in the accumulation of VLCFAs and VLC-PUFAs and

Figure 5. Overexpression of HIF-2a P405/531A (HIF-2a(mt)) Reduces Peroxisome Abundance in Atg7f/f but Not in Atg7–/– Livers (A) Hematoxylin and eosin staining on liver sections. (B) Immunofluorescence of Pex14p and GFP in livers. Some Atg7/ hepatocytes displayed large peroxisomal clusters, and confocal z stack analysis with 3D reconstruction revealed that these clusters represent a network of interconnected, reticular peroxisomal structures and individual peroxisomes (see Movies S3 and S4). (C) Immunoblots of liver lysates. The asterisk indicates a nonspecific band. (D) Catalase activity in liver lysates. Data are mean ± SD (Ad-GFP: n = 3–6; Ad-HIF-2a(mt): n = 7). ***p < 0.001 versus GFP-infected mice. (E and F) Immunoblots of Hif-2a in P28 liver lysates. (G) Immunofluorescence of Pex14p and GFP in livers from Atg7f/f mouse infected with 1 3 109 pfu of Ad-HIF-2a(mt). (H) Concentration spectrum of TAG species in livers of Atg7f/f mice infected with Ad-GFP or Ad-HIF-2a(mt). Data are mean ± SD (n = 6 to 7) (see Table S1 for detailed quantitative lipid profiles).



Figure 6. Inhibition of Autophagy with 3-MA Increases Peroxisome Abundance in Vhl–/– Livers (A) Immunoblots of liver lysates. (B) Immunofluorescence of Pex14p in livers. (legend continued on next page)



depletion of DHA and AA. Such lipid changes are characteristic features of peroxisomal disorders (Van Veldhoven, 2010; Raymond et al., 2009; Baes and Van Veldhoven, 2012). Decreased DHA and AA levels have also been observed in patients with nonalcoholic fatty liver disease and steatohepatitis (Puri et al., 2007, 2009). Since substrates for peroxisomal b-oxidation also include branched-chain and dicarboxylic FAs, C27-bile acids, and eicosanoids (Van Veldhoven, 2010), Hif-2a-mediated pexophagy likely contributes to additional lipid alterations. Moreover, alterations in lipid metabolism may derive also from indirect consequences of reduced peroxisome function like activation of ER stress pathways and mitochondrial dysfunction (Baes and Van Veldhoven, 2012; Faust and Kovacs, 2014). Autophagy is an evolutionary conserved catabolic process for degradation of macromolecules and organelles. Both nonselective ‘‘bulk’’ autophagy and selective autophagy of specific proteins or organelles have been described (Mizushima et al., 2011; Schreiber and Peter, 2014). The key factor in selective autophagy is specific cargo-recognizing receptors that connect the cargo to the core autophagic machinery. Two pexophagy receptors, Atg30 and Atg36, have been identified in yeast (Farre´ et al., 2008; Motley et al., 2012), and their overexpression stimulates pexophagy even under peroxisome-inducing conditions. However, there are no orthologous genes in mammals, and no pexophagy receptors have been identified. Previous studies showed that hypoxia-induced macroautophagy or mitophagy are Hif-1a dependent (Semenza, 2011), but a specific Hif-2a function in the context of autophagy has not been reported. While it remains an open question how Hif-2a induces pexophagy at the detailed molecular level, several possibilities exist. Hif-2a could induce the expression of a pexophagy receptor. Overexpression of NBR1 and p62, which are receptors for selective autophagy of ubiquitinated targets, induces clustering and degradation of peroxisomes in cell lines (Deosaran et al., 2013), but Nbr1 and p62 are not induced by Hif-2a. Artificial monoubiquitination of peroxisomal membrane proteins in mammalian cells is sufficient to cause NBR1- and p62-dependent pexophagy (Kim et al., 2008), but it is unknown if a peroxisomal membrane protein is ubiquitinated under pexophagyinducing conditions and whether subsequent interaction with Nbr1 and/or p62 links ubiquitinated peroxisomes to the autophagic machinery. One possibility is that Hif-2a induces an E3 ubiquitin ligase that mediates ubiquitination of a peroxisomal membrane protein. However, Hif-2a did not induce the expression of peroxisomal E3 ligases (Pex2, Pex10, and Pex12). Alternatively, Nbr1 could be recruited to peroxisomes independently of ubiquitin, because Nbr1 (but not p62) contains a membraneinteracting domain capable of binding to peroxisomes (Deosaran et al., 2013), and Nbr1 localized to peroxisomes even in control livers. In addition, p62 is not required for pexophagy when Nbr1 is in excess, but its binding to Nbr1 increases the efficiency of Nbr1-mediated pexophagy (Deosaran et al., 2013). Since 3-MA treatment of Vhl/ mice induced significant clustering of Nbr1- and p62-positive peroxisomes, we hypothesize that

Hif-2a signaling increases the rate of Nbr1 interaction with peroxisomes. The accumulation of Nbr1 on peroxisomes likely recruits p62 to achieve a critical mass required for pexophagy. This may concentrate sufficient ubiquitin-like modifiers (e.g., LC3 and Gabaraps) in close proximity to peroxisomes to prime phagophore assembly. Hif-2a could also promote a posttranslational modification of a peroxisomal membrane protein or Nbr1 that induces pexophagy. For example, hypoxia-induced dephosphorylation of FUNDC1, a mitochondrial outer membrane protein, enhances its interaction with the core autophagic machinery and promotes mitophagy (Liu et al., 2012). Nbr1 phosphorylation by glycogen synthase kinase 3 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation (Nicot et al., 2014). Hif-2a could activate mRNA translation of a pexophagy receptor or a protein-modifying gene as part of its recently identified transcription-independent role in the regulation of translation (Uniacke et al., 2012). The identification of Hif-2a as a major inducer of pexophagy provides now a path toward delineating the underlying mechanism. Low numbers of peroxisomes have been observed in various tumor cells (Frederiks et al., 2010). Our analysis of more than 200 ccRCC tissue samples revealed a striking correlation between high HIF-2a levels and loss of peroxisomes, further supporting a direct association between activation of HIF-2a and pexophagy. Of particular interest in this regard is the relationship between peroxisome abundance and the histological tumor differentiation grade, since peroxisome abundance is reduced more frequently in well-differentiated tumors. The majority of ccRCC is associated with constitutive upregulation of HIF-a due to loss of pVHL function. HIF-2a is considered to be a core oncogenic driver in ccRCC and is upregulated in the vast majority of solid tumors (Franovic et al., 2009; Qing and Simon, 2009). Accumulation of neutral lipids and glycogen are characteristic features of ccRCCs. Thus, alterations in lipid metabolism as a result of deregulated peroxisome homeostasis play likely an important but unappreciated role in ccRCC development (Currie et al., 2013; Santos and Schulze, 2012). Fatty acids support cancer growth by providing substrates for energy production or by generating building blocks for membranes and signaling lipids in proliferating cells. VLC-PUFAs could be converted to eicosanoids, biologically active lipids involved in inflammation and carcinogenesis. Eicosanoids are degraded in peroxisomes, and dysbalance of peroxisome homeostasis will affect eicosanoid signaling. Peroxisomes are essential for the synthesis of ether lipids, and they are increased in aggressive cancers (Lodhi and Semenkovich, 2014). Changes in VLCFAs, ether lipids, and DHA levels affect membrane properties, thereby influencing membrane microdomain organization and trafficking pathways (Lodhi and Semenkovich, 2014; Obara et al., 2013). In summary, the findings presented here provide now an impetus to further characterize the functional consequences of HIF-2a-driven pexophagy and associated lipid alterations in cancer and to exploit this knowledge for therapy.

(C) Immunoblots of p62 and Nbr1 in liver lysates. (D) Immunofluorescence of catalase, Nbr1, and p62 (shown in Figure S7F). The right panel shows enlarged images of the boxed regions in the overlay panels. Arrowheads represent Nbr1-positive aggregates that colocalize with catalase. Arrows represent peroxisome clusters in livers of 3-MA-treated Vhl/ mice. Insets in the bottom panel show higher magnification of the colocalization of peroxisome clusters with Nbr1 in livers of 3-MA-treated Vhl/ mice.



Figure 7. Strong HIF-2a Staining Correlates with a Reduction of Peroxisome Abundance in ccRCCs (A) Characterization of peroxisome distribution and abundance in healthy kidney. Sequential sections of a biopsy from a healthy kidney were stained with specific markers for proximal tubules (p), glomeruli (g), distal tubules (d), collecting duct (c), and with catalase as marker for peroxisomes. (B) Immunofluorescence staining of a RCC TMA for catalase. Representative examples of ccRCC biopsies with varying abundance of peroxisomes are shown. (C) Immunohistochemical staining of a RCC TMA for HIF-2a. Shown are representative images of biopsies that were classified as HIF-2a negative, weak, and strong and correspond to biopsies that were deemed to contain many, medium, and few peroxisomes, respectively. (D) Correlation of peroxisome abundance with HIF-2a staining in ccRCC tumor biopsies in a TMA. (legend continued on next page)



EXPERIMENTAL PROCEDURES Mice Albumin-Cre (B6.Cg-Tg(Alb-cre)21Mgn/J), Vhlf/f (C;129S-Vhl < tm1Jae > /J), Hif1af/f (B6.129-Hif1a < tm3Rsjo > /J), and Hif2af/f (B6.Epas1 < tm1Mcs > /J) mice were purchased from the Jackson Laboratory. Atg7f/f mice (B6.CgAtg7 < tm1Tchi >) were obtained from the RIKEN Bio Resource Center (Ibaraki). Liver-specific inactivation of Vhl, Hif1a, Hif2a, and Atg7 was achieved by mating with Albumin-Cre mice. Cre-negative littermates were used as controls. WY (50 mg/kg body weight/day) or PBA (1 g/kg/day) was administered by gavage. CQ (10 mg/kg/day) or 3-MA (2 mg/kg/day) was administered by intraperitoneal injection. All protocols for animal use and experiments were approved by the Veterinary Office of Zurich (Switzerland). Lipid Measurements by Liquid Chromatography-Tandem Mass Spectrometry Lipids from livers were extracted with the MTBE method, and data acquisition was performed as described previously (Fauland et al., 2011). Adenovirus Adenovirus expressing HIF-2a P405/531A was generated by using the Rapid Adenovirus Production System (Viraquest). The HA-HIF-2a P405/531A cDNA was excised from pcDNA3.0-HA-HIF2a P405A/P531A (Addgene). The virus expresses GFP from an independent promoter in addition to HIF-2a P405/ 531A. Virus expressing only GFP served as control. Analysis of Autophagy by Microscopy The GFP-LC3-expressing adenovirus was delivered to P21 Vhlf/f and Vhl/ mice by tail-vein injection. After 7 days, mice received CQ (10 mg/kg/day) by intraperitoneal injection for 2 days. Livers were harvested and immunostained on paraffin sections against GFP. Punctuated GFP-LC3 structures were quantified using the particle analysis tool of Imaris software. Thirty to fifty single cells were evaluated in three Vhlf/f and Vhl/ mice. Quantitative RT-PCR and Biochemistry Measurements Analyses were performed as described in Kovacs et al. (2012). Antibodies are listed in Table S3. Histology and Immunohistochemistry Experiments were performed as described in Kovacs et al. (2012). Antibodies are listed in Table S3. Images were taken with a Leica SP2-AOBS confocal laser scanning microscope. Fluorescent dyes were imaged sequentially in frame interlace mode to eliminate crosstalk between the channels. For 3D visualization (movie generation, orthogonal slicer tool), Imaris software was used. 3D-SIM experiments were realized on a DeltaVision OMX V4 Blaze (GE Healthcare). 3D-SIM data were acquired with a fixed z distance of 125 nm, 15 images per z stack (three different illumination angles at 60 interval and five different phases/angle). The superresolution stack was reconstructed in softWoRx 6.0 (GE Healthcare) with channel-specific measured optical transfer functions. As comparison, wide-field images were acquired (same z range and z distance for 3D-SIM dates) and deconvolved with softWoRx 6.0. Image registration for the different acquired channels was performed in softWoRx 6.0 with alignment parameters obtained from a TetraSpeck bead preparate (bead diameter 0.2 mm). Reconstructed and registered images were 3D rendered in Imaris. From the fluorescence information, surfaces were created (surface area detail level: 0.08 mm). Renal Tumor TMA The construction of a renal tumor TMA and the tumors included have been described previously (Mertz et al., 2008). Sequential sections of a healthy kid-

ney were stained against catalase and marker proteins for proximal tubules and glomeruli, distal tubules, and collecting ducts, according to the standard protocol at the pathology of the University Hospital Zu¨rich. Statistical Analyses Data are expressed as mean ± SD. Statistical significance was evaluated by an unpaired Student’s t test. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures, three tables, four movies, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.cmet.2014.09.017. AUTHOR CONTRIBUTIONS W.J.K., K.M.W., and W.K. initiated the project. K.M.W., M.J.S., and W.J.K. designed experiments. K.M.W., M.J.S., P.A.G., and W.J.K. carried out experiments. K.M.W., M.J.S., W.K., and W.J.K. analyzed and interpreted the data. M.H., M.S.L., and H.-P.E. carried out electron microscopy analysis. A.B.M. carried out plasma lipid and bile acid analysis. M.T. and H.C.K. carried out lipidomics analysis. T.S. assisted in superresolution microscopy. P.L.F. provided lipid data from Pex2 mice. H.M. provided and analyzed human renal cancer TMAs. W.J.K., M.J.S., K.M.W., and W.K. wrote the manuscript and all authors commented on it. ACKNOWLEDGMENTS Imaging and electron microscopy was performed on instruments of ScopeM, the Scientific Center for Optical and Electron Microscopy of ETH Zu¨rich. The excellent technical assistance of J. Hehl, M. Gu¨nthert, and F. Lucas from ScopeM is gratefully acknowledged. Ad-GFP-LC3 was a generous gift from A.M. Tolkovsky. We would like to thank M. Stoffel for help with mouse work. We thank J.P. Theurillat and S.M. Mu¨hl for help with TMA analysis. We thank H. Stangl for helpful discussions. This work was supported by the Swiss National Science Foundation (SNF) grant 31003A_132982 to W.J.K. and by grants from the Gebert Ruef Foundation and the Swiss National Science Foundation (310030B_138669) to W.K. Received: July 30, 2014 Revised: August 26, 2014 Accepted: September 24, 2014 Published: November 4, 2014 REFERENCES Baes, M., and Van Veldhoven, P.P. (2012). Mouse models for peroxisome biogenesis defects and b-oxidation enzyme deficiencies. Biochim. Biophys. Acta 1822, 1489–1500. Currie, E., Schulze, A., Zechner, R., Walther, T.C., and Farese, R.V., Jr. (2013). Cellular fatty acid metabolism and cancer. Cell Metab. 18, 153–161. Deosaran, E., Larsen, K.B., Hua, R., Sargent, G., Wang, Y., Kim, S., Lamark, T., Jauregui, M., Law, K., Lippincott-Schwartz, J., et al. (2013). NBR1 acts as an autophagy receptor for peroxisomes. J. Cell Sci. 126, 939–952. Farre´, J.-C., Manjithaya, R., Mathewson, R.D., and Subramani, S. (2008). PpAtg30 tags peroxisomes for turnover by selective autophagy. Dev. Cell 14, 365–376. Fauland, A., Ko¨feler, H., Tro¨tzmu¨ller, M., Knopf, A., Hartler, J., Eberl, A., Chitraju, C., Lankmayr, E., and Spener, F. (2011). A comprehensive method

(E) Correlation of peroxisome abundance with histological differentiation grade in ccRCC. (F) Model for Hif-2a-mediated decrease in peroxisome abundance. Hypoxia or loss of pVhl results in stabilization of Hif-a. Hif-2a signaling promotes pexophagy, whereas it does not affect basal peroxisome biogenesis. Consequently, the reduced number of peroxisomes and hence decreased peroxisomal metabolic activity leads to significant changes in the lipid profile, such as accumulation of VLCFAs and VLC-PUFAs. These lipids are potent activators of Ppara, a nuclear receptor that induces peroxisome proliferation. Hif-2a signaling represses this ligand-dependent activation of Ppara and by this means prevents Ppara-mediated peroxisome proliferation and consequential restoration of peroxisome homeostasis. In summary, Hif-2a is a negative regulator of peroxisome abundance and metabolic activity. P, peroxisome.



for lipid profiling by liquid chromatography-ion cyclotron resonance mass spectrometry. J. Lipid Res. 52, 2314–2322.

Nath, B., and Szabo, G. (2012). Hypoxia and hypoxia inducible factors: diverse roles in liver diseases. Hepatology 55, 622–633.

Faust, P.L., and Kovacs, W.J. (2014). Cholesterol biosynthesis and ER stress in peroxisome deficiency. Biochimie 98, 75–85.

Nicot, A.-S., Lo Verso, F., Ratti, F., Pilot-Storck, F., Streichenberger, N., Sandri, M., Schaeffer, L., and Goillot, E. (2014). Phosphorylation of NBR1 by GSK3 modulates protein aggregation. Autophagy 10, 1036–1053.

Ferdinandusse, S., Denis, S., Faust, P.L., and Wanders, R.J.A. (2009). Bile acids: the role of peroxisomes. J. Lipid Res. 50, 2139–2147. Franovic, A., Holterman, C.E., Payette, J., and Lee, S. (2009). Human cancers converge at the HIF-2a oncogenic axis. Proc. Natl. Acad. Sci. USA 106, 21306–21311. Fransen, M., Nordgren, M., Wang, B., and Apanasets, O. (2012). Role of peroxisomes in ROS/RNS-metabolism: implications for human disease. Biochim. Biophys. Acta 1822, 1363–1373. Frederiks, W.M., Bosch, K.S., Hoeben, K.A., van Marle, J., and Langbein, S. (2010). Renal cell carcinoma and oxidative stress: The lack of peroxisomes. Acta Histochem. 112, 364–371. Hakimi, A.A., Pham, C.G., and Hsieh, J.J. (2013). A clear picture of renal cell carcinoma. Nat. Genet. 45, 849–850. Hiebler, S., Masuda, T., Hacia, J.G., Moser, A.B., Faust, P.L., Liu, A., Chowdhury, N., Huang, N., Lauer, A., Bennett, J., et al. (2014). The Pex1G844D mouse: a model for mild human Zellweger spectrum disorder. Mol. Genet. Metab. 111, 522–532. Keane, M.H., Overmars, H., Wikander, T.M., Ferdinandusse, S., Duran, M., Wanders, R.J.A., and Faust, P.L. (2007). Bile acid treatment alters hepatic disease and bile acid transport in peroxisome-deficient PEX2 Zellweger mice. Hepatology 45, 982–997. Keith, B., Johnson, R.S., and Simon, M.C. (2012). HIF1a and HIF2a: sibling rivalry in hypoxic tumour growth and progression. Nat. Rev. Cancer 12, 9–22. Kim, W.Y., Safran, M., Buckley, M.R.M., Ebert, B.L., Glickman, J., Bosenberg, M., Regan, M., and Kaelin, W.G., Jr. (2006). Failure to prolyl hydroxylate hypoxia-inducible factor alpha phenocopies VHL inactivation in vivo. EMBO J. 25, 4650–4662. Kim, P.K., Hailey, D.W., Mullen, R.T., and Lippincott-Schwartz, J. (2008). Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. Proc. Natl. Acad. Sci. USA 105, 20567–20574. Kovacs, W.J., Charles, K.N., Walter, K.M., Shackelford, J.E., Wikander, T.M., Richards, M.J., Fliesler, S.J., Krisans, S.K., and Faust, P.L. (2012). Peroxisome deficiency-induced ER stress and SREBP-2 pathway activation in the liver of newborn PEX2 knock-out mice. Biochim. Biophys. Acta 1821, 895–907. Krishnan, J., Suter, M., Windak, R., Krebs, T., Felley, A., Montessuit, C., Tokarska-Schlattner, M., Aasum, E., Bogdanova, A., Perriard, E., et al. (2009). Activation of a HIF1a-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy. Cell Metab. 9, 512–524. Kucejova, B., Sunny, N.E., Nguyen, A.D., Hallac, R., Fu, X., Pen˜a-Llopis, S., Mason, R.P., Deberardinis, R.J., Xie, X.J., Debose-Boyd, R., et al. (2011). Uncoupling hypoxia signaling from oxygen sensing in the liver results in hypoketotic hypoglycemic death. Oncogene 30, 2147–2160. Liu, L., Feng, D., Chen, G., Chen, M., Zheng, Q., Song, P., Ma, Q., Zhu, C., Wang, R., Qi, W., et al. (2012). Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat. Cell Biol. 14, 177–185. Lodhi, I.J., and Semenkovich, C.F. (2014). Peroxisomes: a nexus for lipid metabolism and cellular signaling. Cell Metab. 19, 380–392. Mertz, K.D., Demichelis, F., Sboner, A., Hirsch, M.S., Dal Cin, P., Struckmann, K., Storz, M., Scherrer, S., Schmid, D.M., Strebel, R.T., et al. (2008). Association of cytokeratin 7 and 19 expression with genomic stability and favorable prognosis in clear cell renal cell cancer. Int. J. Cancer 123, 569–576. Mizushima, N., Yoshimori, T., and Ohsumi, Y. (2011). The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132. Motley, A.M., Nuttall, J.M., and Hettema, E.H. (2012). Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae. EMBO J. 31, 2852–2868.

Obara, K., Kojima, R., and Kihara, A. (2013). Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids. J. Lipid Res. 54, 831–842. Petiot, A., Ogier-Denis, E., Blommaart, E.F.C., Meijer, A.J., and Codogno, P. (2000). Distinct classes of phosphatidylinositol 30 -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 275, 992–998. Puri, P., Baillie, R.A., Wiest, M.M., Mirshahi, F., Choudhury, J., Cheung, O., Sargeant, C., Contos, M.J., and Sanyal, A.J. (2007). A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology 46, 1081–1090. Puri, P., Wiest, M.M., Cheung, O., Mirshahi, F., Sargeant, C., Min, H.-K., Contos, M.J., Sterling, R.K., Fuchs, M., Zhou, H., et al. (2009). The plasma lipidomic signature of nonalcoholic steatohepatitis. Hepatology 50, 1827–1838. Qing, G., and Simon, M.C. (2009). Hypoxia inducible factor-2a: a critical mediator of aggressive tumor phenotypes. Curr. Opin. Genet. Dev. 19, 60–66. Qu, A., Taylor, M., Xue, X., Matsubara, T., Metzger, D., Chambon, P., Gonzalez, F.J., and Shah, Y.M. (2011). Hypoxia-inducible transcription factor 2a promotes steatohepatitis through augmenting lipid accumulation, inflammation, and fibrosis. Hepatology 54, 472–483. Rankin, E.B., Rha, J., Selak, M.A., Unger, T.L., Keith, B., Liu, Q., and Haase, V.H. (2009). Hypoxia-inducible factor 2 regulates hepatic lipid metabolism. Mol. Cell. Biol. 29, 4527–4538. Raymond, G.V., Watkins, P., Steinberg, S., and Powers, J. (2009). Peroxisomal disorders. In Handbook of Neurochemistry and Molecular Neurobiology: Neural Lipids, A. Lajtha, G. Tettamanti, and G. Goracci, eds. (Berlin: Springer Science+Business Media), pp. 631–670. Rogov, V., Do¨tsch, V., Johansen, T., and Kirkin, V. (2014). Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy. Mol. Cell 53, 167–178. Santos, C.R., and Schulze, A. (2012). Lipid metabolism in cancer. FEBS J. 279, 2610–2623. Schrader, M., Bonekamp, N.A., and Islinger, M. (2012). Fission and proliferation of peroxisomes. Biochim. Biophys. Acta 1822, 1343–1357. Schreiber, A., and Peter, M. (2014). Substrate recognition in selective autophagy and the ubiquitin-proteasome system. Biochim. Biophys. Acta 1843, 163–181. Seglen, P.O., and Gordon, P.B. (1982). 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl. Acad. Sci. USA 79, 1889–1892. Semenza, G.L. (2011). Hypoxia-inducible factor 1: regulator of mitochondrial metabolism and mediator of ischemic preconditioning. Biochim. Biophys. Acta 1813, 1263–1268. Semenza, G.L. (2012). Hypoxia-inducible factors in physiology and medicine. Cell 148, 399–408. Smith, J.J., and Aitchison, J.D. (2013). Peroxisomes take shape. Nat. Rev. Mol. Cell Biol. 14, 803–817. Till, A., Lakhani, R., Burnett, S.F., and Subramani, S. (2012). Pexophagy: the selective degradation of peroxisomes. Int. J. Cell Biol. http://dx.doi.org/10. 1155/2012/512721. Uniacke, J., Holterman, C.E., Lachance, G., Franovic, A., Jacob, M.D., Fabian, M.R., Payette, J., Holcik, M., Pause, A., and Lee, S. (2012). An oxygen-regulated switch in the protein synthesis machinery. Nature 486, 126–129. Van Veldhoven, P.P. (2010). Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabolism. J. Lipid Res. 51, 2863–2895. Zeng, X., and Carlin, C.R. (2013). Host cell autophagy modulates early stages of adenovirus infections in airway epithelial cells. J. Virol. 87, 2307–2319.


Degradation of plant peroxisomes by autophagy.

Autophagy promotes ferroptosis by degradation of ferritin.

Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.

Plant cell remodeling by autophagy: switching peroxisomes for green life.

A new role for ATM in selective autophagy of peroxisomes (pexophagy).

Peroxin Pex14p is the key component for coordinated autophagic degradation of mammalian peroxisomes by direct binding to LC3-II.

Amino-terminal arginylation as a degradation signal for selective autophagy.

Role of peroxisomes in mammalian metabolism.

DRAM1 promotes the targeting of mycobacteria to selective autophagy.

Autophagic degradation of peroxisomes in mammals.

RAD6 promotes homologous recombination repair by activating the autophagy-mediated degradation of heterochromatin protein HP1.

Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

Degradation of AF1Q by chaperone-mediated autophagy.

Selective degradation of cytosolic proteins by lysosomes.

Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.

Autophagic degradation of peroxisomes in isolated rat hepatocytes.

Organ-specific quality control of plant peroxisomes is mediated by autophagy.

Autophagy and mammalian development.

Selective degradation of PU.1 during autophagy represses the differentiation and antitumour activity of TH9 cells.

Regulation of autophagy by glucose in Mammalian cells.

Dissection of ubiquitinated protein degradation by basal autophagy.

Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria.

Hypoxia-induced oxidative stress promotes MUC4 degradation via autophagy to enhance pancreatic cancer cells survival.

NLRP3 promotes autophagy of urate crystals phagocytized by human osteoblasts.