Regulation of cell growth by autophagy

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Jan 22, 2008 - systems acting as major growth regulatory pathways converge on autophagy genes to control cell size. Thus, autophagy may act as a.

[Autophagy 4:4, 507-509; 16 May 2008]; ©2008 Landes Bioscience

Article Addendum

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Regulation of cell growth by autophagy Tibor Vellai,1,* Bertalan Bicsák,1 Márton L. Tóth,1 Krisztina Takács-Vellai1 and Attila L. Kovács2 of Genetics and 2Department of Anatomy, Cell and Developmental Biology; Eötvös Loránd University; Budapest, Hungary

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reduced insulin/IGF-1 or TOR signaling,7,8 raising the possibility that autophagy-mediated renewal of cytosolic materials is a prerequisite of normal cell growth. The first genetic study implicating autophagy in cell growth control shows that mouse fibroblast cells defective for autophagy are unable to undergo size reduction in response to starvation.9 Nutrient limitation causes a general decrease in macromolecular synthesis and also induces autophagy to facilitate cell survival by degrading and recycling parts of the cytoplasm. When autophagic degradation is blocked, cytosolic materials are not utilized sufficiently for maintaining cellular homeostasis; thus, cells are unable to become slender. Consistently, mutations that cause feeding defects by abnormal pharyngeal neurotransmission or anatomy confer small cell size in C. elegans.10,11 These mutant nematodes display elevated levels of autophagy, indicating that long-term starvation leads to cell growth retardation via (hyper)activating autophagic degradation. In Drosophila, direct induction of autophagy by overexpressing the autophagy-specific Atg1 protein kinase also inhibits cell growth.12,13 Together, these results demonstrate that high levels of autophagy caused by prolonged starvation or genetic manipulation inhibit cell growth and lead to reduced cell size. Contrary to the normal cell size observed in Atg1 mutant flies that were maintained under normal conditions,12 we have found that inactivation of certain autophagy genes markedly reduces cell size without affecting cell number in well-fed nematodes.14 Mutant C. elegans strains deficient in UNC-51/Atg1 exhibit a small body size phenotype (Sma). The reduced body length of these animals results from a significant decrease in the size and volume of different cell types such as hypodermal and intestinal cells, which are known to primarily determine body size in this organism. Mutant animals with reduced BEC-1/Atg6 activity are also smaller than the wild type. Green fluorescent protein-labeled tissue-specific markers indicate that unc-51 and bec-1 mutant nematodes have the same number of somatic cells as the wild-type ones. Together, our results imply that at least two autophagy-related genes, unc-51/Atg1 and bec-1/Atg6, are required for cell growth in C. elegans and perhaps in other eukaryotic species too. We suggest that identifying autophagy genes as mediators of cell growth in C. elegans will be essential to understand how genetic lesions and adverse environmental conditions affecting growth factor signaling lead to human malignancies. Autophagy genes also contribute to diverse cellular functions, e.g., endocytosis, protein trafficking and membrane remodeling. Thus, it remains an important question whether autophagy itself is implicated in cell growth. Autophagosome formation is compromised

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Cell growth—the primary determinant of cell size—has an intimate relationship with proliferation; cells divide only after they reach a critical size. Despite its developmental and medical significance, little is known about cellular pathways that mediate the growth of cells. Accumulating evidence demonstrates a role for autophagy—a mechanism of eukaryotic cells to digest their own constituents during development or starvation—in cell size control. Increasing autophagic activity by prolonged starvation, rapamycin treatment inhibiting TOR (target of rapamycin) signaling, or genetic intervention, causes cellular atrophy in worms, flies and mammalian cell cultures. In contrast, we have shown that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes, unc-51/Atg1 and bec-1/Atg6, confers reduced cell size. We argue that physiological levels of autophagy are required for normal cell size, whereas both insufficient and excessive levels of autophagy lead to retarded cell growth. Furthermore, we discuss data suggesting that the insulin/IGF-1 (insulin-like growth factor receptor-1) and TGF-β (transforming growth factor-beta) signaling systems acting as major growth regulatory pathways converge on autophagy genes to control cell size. Thus, autophagy may act as a central regulatory mechanism of cell growth.

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Key words: autophagy, cell growth, cell size, Ceanorhabditis elegans, insulin/IGF-1 signaling, TGFβ signaling

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Autophagy Genes are Required for Normal Cell Size

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In multicellular organisms, cell growth is intimately linked to nutrient availability and growth factor signaling including insulin/ IGF-1, TGF-β and TOR, as well as requires a well-controlled balance in macromolecule and organelle turnover (i.e., between synthesis and degradation).1-3 Cellular mechanisms for degrading aberrant, damaged constituents of cytosol involve the non-lysosomal ubiquitinproteasome system and autophagy, which is a lysosome-mediated process.4 During autophagy, subcellular membrane structures are formed to sequester cargo into lysosomes for breakdown by acid hydrolases.5,6 Autophagy is activated upon nutrient deprivation and

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*Correspondence to: Tibor Vellai; Department of Genetics; Eötvös Loránd University; Pázmány Péter sétány 1/C; Budapest H-1117 Hungary; Tel.: +36.1.209.0555/8684; Fax: +36.1.209.0555/1841; Email: [email protected] Submitted: 01/22/08; Revised: 01/30/08; Accepted: 02/04/08 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/5670

Addendum to: Aladzsity I, Tóth ML, Sigmond T, Szabó E, Bicsák B, Barna J, Regós A, Orosz L, Kovács AL, Vellai T. Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans. Genetics 2007; 177:655–60, DOI: 10.1534/genetics.107.075762. www.landesbioscience.com

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Autophagy regulates cell growth

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Figure 1. Autophagy genes function downstream of daf-16/Foxo in the insulin/IGF-1 and TGFβ signaling pathways to regulate development and cell growth in C. elegans. DAF-2, insulin/IGF-1 receptor; DAF-1, type I TGFβ receptor; DAF-4, type II TGFβ receptor; DAF-7, TGFβ ligand; DBL-1, TGFβ ligand; DAF-16, FoxO-like forkhead transcription factor; TOR, target of rapamycin kinase. Arrows indicate activations, bars indicate negative regulatory interactions. It is unknown (question mark) whether LON-1/PK acts upstream or downstream of TOR in cell growth control.

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in unc-51 and bec-1 mutant worms.14,15 This however does not exclude the possibility that in unc-51 and bec-1 mutant nematodes cell growth becomes retarded independently of abnormal autophagy. Thus, monitoring cell size in organisms in which autophagosome formation is specifically blocked with chemicals is a challenging area for future investigations.

Table 1 DAF-16 is required for constitutive dauer development in mutants with aberrant TGFβ signaling Genotype

% dauers

n

Wild type

0

many

daf-16(m26)

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daf-16(mu86)

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daf-1(m40)

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Cell growth is controlled by the insulin/IGF-1-TOR growth factor signaling axis and the TGFβ growth factor pathway.1,2 For example, mutations reducing the activity of daf-2, which encodes an IGF-1 receptor, and genetic manipulation increasing the dosage of dbl-1, which encodes a TGFβ growth factor, each confer a Lon (long body size) phenotype in C. elegans via enhancing cell growth. We have examined whether unc-51 and bec-1 interact with these hormonal systems to influence cell size.14 According to our epistasis (double mutant) analysis, mutational inactivation of unc-51 and bec1 suppresses the Lon phenotype of daf-2(-) mutant and dbl-1(+++) animals; double mutants defective for any of the tested autophagy genes and daf-2, and animals defective for unc-51 or bec-1, but hyperactive for dbl-1, are almost as small as the corresponding single autophagy mutants. Thus, the insulin/IGF-1 and TGFβ pathways converge on autophagy genes to regulate cell growth: unc-51 and bec-1 function downstream of, and are inhibited by, these signal transduction systems in growth control (Fig. 1). An important question is, which parts of the insulin/IGF-1 and TGF-β cascades interact with autophagy genes? In C. elegans, both pathways promote reproductive growth by inhibiting the developing animals from entering into a developmental diapause called dauer

daf-7(e1372)

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daf-1(m40); daf-16(m26)

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daf-1(m40); daf-16(mu86)

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daf-7(e1372); daf-16(m26)

55.6

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daf-7(e1372); daf-16(mu86)

2.5

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The Insulin/IGF-1 and TGFβ Signaling Pathways Converge on Autophagy Genes to Control Cell Growth

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Animals were maintained at 25°C, and tested for % dauers 2 weeks after hatching.

larval stage, which is triggered by starvation and crowding in the wild type. Strong loss-of-function mutations in DAF-2/IGF-1 and TGFβ signaling cause constitutive dauer development even under favorable conditions and this process, similar to cell growth, is known to require the activity of autophagy genes.8 We wondered whether the FoxO-like forkhead transcription factor DAF-16 that is required for dauer development in daf-2 mutant animals also mediates dauer development in mutants with aberrant TGF-β signaling. We found that daf-1 and daf-7 mutant animals defective for a type I TGFβ receptor and a TGFβ ligand, respectively, develop as abnormal dauers in the daf-16 mutant background (Table 1). Although double mutants were able to enter into the dauer stage, after a few days they recovered and continued normal development. These data show that

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Autophagy regulates cell growth

References

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Figure 2. Both downregulation and hyperactivation of autophagy genes cause defects in various cellular functions. Changing a balance in the autophagic pathway in either direction perturbs cell growth, cell survival and the rate at which cells age.

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1. Saucedo LJ, BA Edgar. Why size matters: altering cell size. Curr Opin Genet Dev 2002; 12:565-71. 2. Oldham S, Hafen E. Insulin/IGF and target of rapamycin signaling: a TOR de force in growth control. Trends Cell Biol 2003; 13:79-85. 3. Neufeld TP. Body building: regulation of shape and size by PI3K/TOR signaling during development. Mech Dev 2003; 120:1283-96. 4. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000; 290:1717-21. 5. Kovács AL, Pálfia Z, Réz G, Vellai T, Kovács J. Sequestration revisited: integrating traditional electron microscopy, de novo assembly and new results. Autophagy 2007; 3:655-62. 6. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008; 4: 1-25. 7. Cutler NS, Pan X, Heitman J, Cardenas ME. The TOR signal transduction cascade controls cellular differentiation in response to nutrients. Mol Cell Biol 2001; 12:4103-13. 8. Meléndez A, Tallóczy Z, Seaman M, Eskelinen E-L, Hall DH, Levine B. Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 2003; 301:1387-91. 9. Hosokawa N, Hara Y, Mizushima N. Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett 2006; 580:2623-9. 10. Mörck C, Pilon M. C. elegans feeding defective mutants have shorter body lengths and increased autophagy. BMC Dev Biol 2006; 6:39. 11. Mörck C, Pilon M. Caloric restriction and autophagy in Caenorhabditis elegans. Autophagy 2007; 3:51-3. 12. Scott RC, Juhász G, Neufeld TP. Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr Biol 2007; 17:1-11. 13. Neufeld TP. Contribution of Atg1-dependent autophagy to TOR-mediated cell growth and survival. Autophagy 2007; 3:477-9. 14. Aladzsity I, Tóth ML, Sigmond T, Szabó E, Bicsák B, Barna J, Regós Á, Orosz L, Kovács AL, Vellai T. Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans. Genetics 2007; 177:655-60. 15. Tóth ML, Sigmond T, Borsos É, Barna J, Erdélyi P, Takács-Vellai K, Orosz L, Kovács AL, Csikós G, Sass M, Vellai T. Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans. Autophagy 2008; in press. 16. Jia K, Chen D, Riddle DL. The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span. Development 2004; 131:3897-906. 17. Takács-Vellai K, Vellai T, Puoti A, Passannante M, Wicky C, Streit A, Kovács AL, Müller F. Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Curr Biol 2005; 15:1513-7. 18. Takács-Vellai K, Bayci A, Vellai T. Autophagy in neuronal cell loss: a road to death. BioEssays 2006; 28:1126-31. 19. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006; 441:885-9. 20. Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E, Tanaka K. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 2006; 441:880-4. 21. Tóth ML, Simon P, Kovács AL, Vellai T. Influence of autophagy genes on ion-channeldependent neuronal degeneration in Caenorhabditis elegans. J Cell Sci 2007; 120:1134-41. 22. Vellai T, Tóth ML, Kovács AL. Janus-faced autophagy. A dual role for cellular self-eating in neurodegeneration? Autophagy 2007; 3:461-3. 23. Kang C, You Y-J, Avery L. Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation. Genes Dev 2007; 21:2161-71. 24. Simonsen A, Cumming RC, Brech A, Isakson P, Schubert DR, Finley KD. Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila. Autophagy 2008; 4: in press. 25. Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 2006; 443:780-6.

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DAF-16/FoxO already links the insulin/IGF-1 and TGFβ pathways. Since DAF-16 transcriptionally regulates daf-1516 whose protein product (Raptor) binds to and functions with TOR, a negative regulator of autophagy, UNC-51 and BEC-1 might operate downstream of DAF-16 in insulin/IGF-1 and TGFβ signaling to control development and cell growth (Fig. 1).

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Both Insufficient and Excessive Levels of Autophagy Distrupt Several Cellular Functions

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Cell growth requires a balance in the autophagic pathway; altering this balance in either direction perturbs the growth process. Indeed, both mutational inactivation of autophagy genes and their excessive activity due to prolonged starvation, rapamycin treatment or genetic manipulation result in retarded cell growth.10,12,14 Interestingly, other cellular processes influenced by autophagy genes, such as aging, apoptosis and necrosis, also rely on the physiological levels of the autophagic pathway (Fig. 2). Both insufficient and excessive levels of autophagy proteins appear to inhibit survival, and trigger apoptotic or necrotic cell death in organisms from worms to mammals.12,15,17-24 Thus, suggestions for therapeutic interventions that involve rapamycin treatment, a potent inducer of autophagy,25 should be approached with caution. Acknowledgements

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This work was supported by grants from the Ministry of Health (167/2006) and Hungarian Scientific Research Funds (OTKA K68372 and T04741). T.V. is a grantee of the János Bolyai scholarship.

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