Autophagy and hepatic stellate cell activation – Partners in crime?

Abstract

A role for autophagy during hepatic stellate cell activationJournal of HepatologyVol. 55Issue 6PreviewAutophagy is a metabolic process that degrades and recycles intracellular organelles and proteins with many connections to human disease and physiology. We studied the role of autophagy during hepatic stellate cell (HSC) activation, a key event in liver fibrogenesis. Full-Text PDF Following liver injury, hepatic stellate cells (HSCs) lose their characteristic lipid droplets to differentiate into extracellular matrix producing myofibroblasts. Activation of HSCs is considered one of the main mechanisms contributing to the development of hepatic fibrosis in chronic liver diseases [[1]Friedman S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.Physiol Rev. 2008; 88: 125-172Crossref PubMed Scopus (2008) Google Scholar]. Although several key pathways for HSC activation, such as TGFβ and PDGF have been identified, there is still a lack of clinically applicable approaches to target HSCs for anti-fibrotic therapies. Identification of additional pathways involved in HSC activation may reveal more suitable anti-fibrotic targets than those already identified. The study by Thoen et al. in this issue of the Journal of Hepatology investigates the role of autophagy in HSC activation and the loss of HSC lipid stores, one of the most characteristic features of the HSC activation process [[2]Thoen L.F. Guimaraes E.L. Dolle L. Mannaerts I. Najimi M. Sokal E. et al.A role for autophagy during hepatic stellate cell activation.J Hepatol. 2011; 55: 1353-1360Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar]. Like most biological systems, cells need to degrade many of their components thus allowing for constant turnover and renewal of proteins and organelles, and adaptation to changing conditions. Degradation occurs through two specific machineries – the proteasome and the lysosome [[3]Mizushima N. Levine B. Cuervo A.M. Klionsky D.J. Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (5011) Google Scholar]. The proteasome degrades proteins that are specifically tagged by markers such as ubiquitin, allowing this precise proteolytic machinery to recognize proteins destined for degradation. The lysosome is able to degrade various cellular components through different processes termed macroautophagy, microautophagy, and chaperone-mediated autophagy [[4]Yang Z. Klionsky D.J. Eaten alive: a history of macroautophagy.Nat Cell Biol. 2010; 12: 814-822Crossref PubMed Scopus (1594) Google Scholar]. These three types of autophagy differ from each other in terms of the delivery method of the “cargo” to the lysosome, and their selectivity for specific types of cargo. The study by Thoen et al. focuses on macroautophagy, a catabolic process in which the cargo is first sequestered inside double-membrane vesicles called autophagosomes and then fused to lysosomes [[4]Yang Z. Klionsky D.J. Eaten alive: a history of macroautophagy.Nat Cell Biol. 2010; 12: 814-822Crossref PubMed Scopus (1594) Google Scholar]. After degradation, resulting amino acids or other small molecules are released back into the cytoplasm and can be used for various purposes including energy harvest [4Yang Z. Klionsky D.J. Eaten alive: a history of macroautophagy.Nat Cell Biol. 2010; 12: 814-822Crossref PubMed Scopus (1594) Google Scholar, 5Rabinowitz J.D. White E. Autophagy and metabolism.Science. 2010; 330: 1344-1348Crossref PubMed Scopus (1415) Google Scholar]. Accordingly, macroautophagy (referred hereafter as “autophagy”) is increased in nutrient poor states, or stress conditions [4Yang Z. Klionsky D.J. Eaten alive: a history of macroautophagy.Nat Cell Biol. 2010; 12: 814-822Crossref PubMed Scopus (1594) Google Scholar, 5Rabinowitz J.D. White E. Autophagy and metabolism.Science. 2010; 330: 1344-1348Crossref PubMed Scopus (1415) Google Scholar]. Increased autophagy has also been described in cell death and may, therefore, play a dual role in cell survival depending on conditions and stimuli. However, in many cases this is probably “cell death with autophagy rather than cell death by autophagy” [[6]Kroemer G. Levine B. Autophagic cell death: the story of a misnomer.Nat Rev Mol Cell Biol. 2008; 9: 1004-1010Crossref PubMed Scopus (1132) Google Scholar] suggesting that the autophagic response is largely a cell-protective response in mammalian cells. Accordingly, defective autophagy has been linked to common human diseases, such as neurodegenerative conditions including Alzheimer’s disease, Parkinson’s disease, metabolic disorders, such as diabetes and obesity, and aging [[3]Mizushima N. Levine B. Cuervo A.M. Klionsky D.J. Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (5011) Google Scholar]. Thoen et al. explore the relationship between autophagy and HSC activation, and introduce the idea of targeting autophagy for the prevention of HSC activation [[2]Thoen L.F. Guimaraes E.L. Dolle L. Mannaerts I. Najimi M. Sokal E. et al.A role for autophagy during hepatic stellate cell activation.J Hepatol. 2011; 55: 1353-1360Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar]. Several key findings of the study support the idea that autophagy promotes HSC activation: (i) Fibrotic livers from CCl4-treated mice displayed increased expression of LC3-II, one of the proteins involved in elongation of autophagosomes, and a useful indirect measure of autophagosomes. (ii) Autophagic flux is increased in mouse HSCs after in vitro activation as demonstrated by DsRed-GFP-LC3B transfection. With this method red punctae from DsRed (stable in an acidic lysosomal milieu) mark autophagolysosomes and yellow punctae from mixed DsRed, and GFP (whose fluorescence is quenched in the acidic lysosomal milieu) fluorescence label autophagosomes. The authors found a significant increase in red punctae during HSC activation indicating increased autophagic flux. (iii) Autophagy inhibitors bafilomycin A1, 3-methyladenine, and hydroxychloroquine efficiently suppress in vitro activation of mouse and human HSCs as evidenced by decreased expression of activation makers, such as platelet-derived growth factor receptor, Acta2, and Col1a1 mRNA expression, and α-SMA protein. Notably, disrupting bafilomycin treatment allowed HSCs to resume activation suggesting that toxic effects of bafilomycin are unlikely to be involved in the suppression of HSC activation. Despite the appealing hypothesis and the strong evidence for autophagy contributing to HSC in vitro activation, the study by Thoen et al. leaves a number of open questions. One area in which this study falls short, is the investigation of underlying mechanisms. Thoen et al. observed that bafilomycin treated cells had increased large lipid droplets, which are more characteristic of quiescent rather than activated HSCs. Additionally, treatment of HSCs with PDGF induced co-localization of lipid droplets and LC3-B fluorescence suggesting that autophagy is responsible for the metabolism lipid droplet metabolism. These findings are similar to observations in hepatocytes where autophagy negatively regulates lipid stores through a process called macrolipophagy [[7]Singh R. Kaushik S. Wang Y. Xiang Y. Novak I. Komatsu M. et al.Autophagy regulates lipid metabolism.Nature. 2009; 458: 1131-1135Crossref PubMed Scopus (2517) Google Scholar]. However, there is no solid evidence that the decrease in HSC lipid droplets promotes their activation in liver fibrosis. One study addressing this issue found that HSCs that do not contain any lipid droplets do not activate spontaneously nor do they show increased activation in response to CCl4 treatment or bile duct ligation [[8]Kluwe J. Wongsiriroj N. Troeger J.S. Gwak G.Y. Dapito D.H. Pradere J.P. et al.Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis.Gut. 2011; Crossref PubMed Scopus (95) Google Scholar]. Additionally, mTOR contributes to the activation of HSCs and promotion of liver fibrosis [9Zhu J. Wu J. Frizell E. Liu S.L. Bashey R. Rubin R. et al.Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis.Gastroenterology. 1999; 117: 1198-1204Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Gabele E. Reif S. Tsukada S. Bataller R. Yata Y. Morris T. et al.The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation.J Biol Chem. 2005; 280: 13374-13382Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Neef M. Ledermann M. Saegesser H. Schneider V. Reichen J. Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis.J Hepatol. 2006; 45: 786-796Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar] but inhibits autophagy [[12]Neufeld T.P. TOR-dependent control of autophagy: biting the hand that feeds.Curr Opin Cell Biol. 2010; 22: 157-168Crossref PubMed Scopus (192) Google Scholar]. The data from Thoen et al. seem to contradict the HSC-activating yet autophagy-inhibiting effect of mTOR. Therefore, it would be important to further delineate the relationship between mTOR, autophagy, and HSC activation. A second concern is the complete reliance of Thoen et al. on chemical inhibitors of autophagy. It cannot be excluded that these inhibitors exert non-specific effects and that the observed decrease in HSC activation is not entirely mediated by autophagy inhibition. Elegant genetic approaches such as the conditional deletion of Atg7 have been employed to study autophagy in the liver and other organs [[7]Singh R. Kaushik S. Wang Y. Xiang Y. Novak I. Komatsu M. et al.Autophagy regulates lipid metabolism.Nature. 2009; 458: 1131-1135Crossref PubMed Scopus (2517) Google Scholar], and should be used to confirm pharmacological approaches. Finally, the study on HSC activation is entirely based on in vitro models of HSC activation. The only in vivo part of this study is on whole liver extracts from CCl4-treated mice and lacks an investigation of autophagy regulation in HSCs and other cell types in the fibrotic liver. In vitro activation of mouse and human HSCs differs considerably from in vivo activation due to the absence of various cell–cell interactions and soluble mediators that are typically present in the injured liver [13Sancho-Bru P. Bataller R. Gasull X. Colmenero J. Khurdayan V. Gual A. et al.Genomic and functional characterization of stellate cells isolated from human cirrhotic livers.J Hepatol. 2005; 43: 272-282Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 14De Minicis S. Seki E. Uchinami H. Kluwe J. Zhang Y. Brenner D.A. et al.Gene expression profiles during hepatic stellate cell activation in culture and in vivo.Gastroenterology. 2007; 132: 1937-1946Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar]. To exclude that the observed induction of autophagic flux is merely a result of cell culture conditions, confirmatory in vivo studies are needed that compare autophagic flux in quiescent and activated HSCs directly in the liver, or between HSCs isolated from normal or fibrotic livers. Along this line, it should be pointed out that the comparison of autophagic flux in quiescent and culture-activated HSCs by DsRed and GFP fluorescence is challenging due to different time intervals between transfection and analysis under these conditions. When considering the potential therapeutic implications of the study by Thoen et al., one needs not only to consider the often protective role of autophagy in human physiology and pathophysiology [[3]Mizushima N. Levine B. Cuervo A.M. Klionsky D.J. Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (5011) Google Scholar], but also its functions in hepatic cell populations besides HSCs [[15]Rautou P.E. Mansouri A. Lebrec D. Durand F. Valla D. Moreau R. Autophagy in liver diseases.J Hepatol. 2010; 53: 1123-1134Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar]. In the liver, autophagy appears to predominantly exert protective functions including the promotion of hepatic function during aging, protection from hepatocellular carcinoma, protection from liver disease due to α1-antitrypsin deficiency, and protection from Mallory-Denk body formation and liver injury in alcoholic liver disease [16Zhang C. Cuervo A.M. Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function.Nat Med. 2008; 14: 959-965Crossref PubMed Scopus (377) Google Scholar, 17Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. et al.Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.J Clin Invest. 2003; 112: 1809-1820Crossref PubMed Scopus (1826) Google Scholar, 18Harada M. Hanada S. Toivola D.M. Ghori N. Omary M.B. Autophagy activation by rapamycin eliminates mouse Mallory-Denk bodies and blocks their proteasome inhibitor-mediated formation.Hepatology. 2008; 47: 2026-2035Crossref PubMed Scopus (108) Google Scholar, 19Hidvegi T. Ewing M. Hale P. Dippold C. Beckett C. Kemp C. et al.An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis.Science. 2010; 329: 229-232Crossref PubMed Scopus (467) Google Scholar]. However, under some conditions the autophagic machinery may also promote liver disease – e.g. in patients with viral hepatitis where HBV and HCV hijack this machinery for their own benefit [[15]Rautou P.E. Mansouri A. Lebrec D. Durand F. Valla D. Moreau R. Autophagy in liver diseases.J Hepatol. 2010; 53: 1123-1134Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar]. In conclusion, targeting autophagy for anti-fibrotic therapies is likely to have broad and many unwanted effects in the liver and other organs, and currently does not appear to be an attractive target for anti-fibrotic therapies. Further in vitro and in vivo studies are required to confirm and understand the role and targets of autophagy in HSCs in the fibrotic liver, and may reveal novel players in the HSC activation process. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.