The Forkhead Transcription Factor FoxO1 Regulates Proliferation and Transdifferentiation of Hepatic Stellate Cells

Background & Aims: The Forkhead box gene, group O (FoxO) family of Forkhead transcription factors is phopsphorylated and inactivated by the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and regulates a variety of cellular functions. Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. A fibrotic stimulus causes HSCs to transdifferentiate from a quiescent phenotype to a collagen-producing myofibroblast-like phenotype and to proliferate. Methods: Mutation/deletion mutants of FoxO1 were introduced into primary rat, mouse, and immortalized human HSCs and assessed for activation, proliferation, and signal transduction. The role of FoxO1 in experimental liver fibrosis was assessed in FoxO1+/− and FoxO1+/+ mice. Results: Platelet-derived growth factor (PDGF) or insulin phosphorylates FoxO1 and induces FoxO1 translocation from the nuclei to the cytosol via the PI3K/AKT pathway in HSCs. Constitutively active FoxO1 inhibits proliferation via cell cycle arrest at the G1 phase, whereas dominant-negative FoxO1 enhances proliferation of HSCs even in the presence of the PI3K inhibitor LY294002. In addition, the phosphorylation of FoxO1 is increased during transdifferentiation of HSCs. The transdifferentiation is also inhibited by constitutively active FoxO1 and is accelerated by dominant-negative FoxO1. FoxO1 directly induces the expression of p27kip1 and manganese superoxide dismutase (MnSOD). After bile duct ligation for 3 weeks, FoxO1+/− mice are more susceptible to liver fibrosis, consistent with our in vitro results. Conclusions: FoxO1 plays a crucial role in the transdifferentiation and proliferation of HSCs in liver fibrosis. Hyperinsulinemia inactivates FoxO1 in HSCs, resulting in HSC activation and may result in the fibrosis in nonalcoholic fatty liver disease. Background & Aims: The Forkhead box gene, group O (FoxO) family of Forkhead transcription factors is phopsphorylated and inactivated by the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and regulates a variety of cellular functions. Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. A fibrotic stimulus causes HSCs to transdifferentiate from a quiescent phenotype to a collagen-producing myofibroblast-like phenotype and to proliferate. Methods: Mutation/deletion mutants of FoxO1 were introduced into primary rat, mouse, and immortalized human HSCs and assessed for activation, proliferation, and signal transduction. The role of FoxO1 in experimental liver fibrosis was assessed in FoxO1+/− and FoxO1+/+ mice. Results: Platelet-derived growth factor (PDGF) or insulin phosphorylates FoxO1 and induces FoxO1 translocation from the nuclei to the cytosol via the PI3K/AKT pathway in HSCs. Constitutively active FoxO1 inhibits proliferation via cell cycle arrest at the G1 phase, whereas dominant-negative FoxO1 enhances proliferation of HSCs even in the presence of the PI3K inhibitor LY294002. In addition, the phosphorylation of FoxO1 is increased during transdifferentiation of HSCs. The transdifferentiation is also inhibited by constitutively active FoxO1 and is accelerated by dominant-negative FoxO1. FoxO1 directly induces the expression of p27kip1 and manganese superoxide dismutase (MnSOD). After bile duct ligation for 3 weeks, FoxO1+/− mice are more susceptible to liver fibrosis, consistent with our in vitro results. Conclusions: FoxO1 plays a crucial role in the transdifferentiation and proliferation of HSCs in liver fibrosis. Hyperinsulinemia inactivates FoxO1 in HSCs, resulting in HSC activation and may result in the fibrosis in nonalcoholic fatty liver disease. Hepatic fibrosis is a wound-healing response to chronic liver injury, and hepatic stellate cells (HSCs) play a crucial role in this fibrotic response.1Bataller R. Brenner D.A. Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4187) Google Scholar HSCs from normal liver show a quiescent phenotype storing vitamin A-rich fat droplets. During liver fibrosis, HSCs undergo an activation or a transdifferentiation process, which is characterized by loss of intracellular vitamin A stores and change to a myofibroblast-like cell with expression of α-smooth muscle actin (α-SMA). Transdifferentiated HSCs then remodel the extracellular matrix by secreting matrix metalloproteinases and depositing extracellular matrix, including type I collagen. In addition, HSCs migrate and proliferate in response to a variety of cytokines and growth factors elicited during liver injury. Therefore, transdifferentiation, proliferation, and collagen production of HSCs are key steps in liver fibrogenesis. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is activated by growth factors and controls a variety of cellular responses, including survival, proliferation, and metabolism.2Scheid M.P. Woodgett J.R. PKB/AKT: functional insights from genetic models.Nat Rev Mol Cell Biol. 2001; 2: 760-768Crossref PubMed Scopus (540) Google Scholar In HSC, the PI3K/AKT pathway is strongly activated by platelet-derived growth factor (PDGF), which is the most potent mitogen of HSC.3Marra F. Gentilini A. Pinzani M. Choudhury G.G. Parola M. Herbst H. Dianzani M.U. Laffi G. Abboud H.E. Gentilini P. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor’s actions on hepatic stellate cells.Gastroenterology. 1997; 112: 1297-1306Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 4Reif S. Lang A. Lindquist J.N. Yata Y. Gabele E. Scanga A. Brenner D.A. Rippe R.A. The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression.J Biol Chem. 2003; 278: 8083-8090Crossref PubMed Scopus (246) Google Scholar, 5Gabele E. Reif S. Tsukada S. Bataller R. Yata Y. Morris T. Schrum L.W. Brenner D.A. Rippe R.A. The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation.J Biol Chem. 2005; 280: 13374-13382Crossref PubMed Scopus (87) Google Scholar However, the precise mechanism by which the PI3K/AKT-signaling pathway regulates transdifferentiation and proliferation in HSCs is still unclear. The Forkhead box gene, group O (FoxO) subfamily of Forkhead transcription factors comprises functionally related proteins FoxO1, FoxO3a, and FoxO4 and plays an important role in metabolism, differentiation, survival, and proliferation.6Accili D. Arden K.C. FoxOs at the crossroads of cellular metabolism, differentiation, and transformation.Cell. 2004; 117: 421-426Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar AKT-catalyzed phosphorylation of FoxO1 results in nuclear exclusion and inhibition of FoxO-dependent gene expression.7Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar Because FoxO factors regulate proliferation and tumor growth in a variety of cells,8Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN.Mol Cell Biol. 2000; 20: 8969-8982Crossref PubMed Scopus (497) Google Scholar, 9Medema R.H. Kops G.J. Bos J.L. Burgering B.M. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1.Nature. 2000; 404: 782-787Crossref PubMed Scopus (1231) Google Scholar it is possible that FoxO1 participates in HSC proliferation downstream of the PI3K/AKT pathway. HSC transdifferentiation induces profound morphologic and molecular changes. Transdifferentiated HSCs express α-SMA and the myogenic transcription factor MyoD,10Vincent K.J. Jones E. Arthur M.J. Smart D.E. Trim J. Wright M.C. Mann D.A. Regulation of E-box DNA binding during in vivo and in vitro activation of rat and human hepatic stellate cells.Gut. 2001; 49: 713-719Crossref PubMed Scopus (28) Google Scholar whereas quiescent HSCs express adipocytic peroxisome proliferator-activated receptor-γ (PPAR-γ).11Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr, H.F. Motomura K. Anania F.A. Willson T.M. Tsukamoto H. Peroxisome proliferator-activated receptors and hepatic stellate cell activation.J Biol Chem. 2000; 275: 35715-35722Crossref PubMed Scopus (435) Google Scholar, 12Marra F. Efsen E. Romanelli R.G. Caligiuri A. Pastacaldi S. Batignani G. Bonacchi A. Caporale R. Laffi G. Pinzani M. Gentilini P. Ligands of peroxisome proliferator-activated receptor γ modulate profibrogenic and proinflammatory actions in hepatic stellate cells.Gastroenterology. 2000; 119: 466-478Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar These findings raise the possibility that HSCs undergo transdifferentiation from an adipocytic phenotype into a myofibroblastic phenotype. Because FoxO factors control cellular differentiation including adipocytes and myoblasts,13Kitamura T. Nakae J. Kitamura Y. Kido Y. Biggs III, W.H. Wright C.V. White M.F. Arden K.C. Accili D. The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic β cell growth.J Clin Invest. 2002; 110: 1839-1847Crossref PubMed Scopus (496) Google Scholar, 14Nakae J. Kitamura T. Kitamura Y. Biggs III, W.H. Arden K.C. Accili D. The forkhead transcription factor Foxo1 regulates adipocyte differentiation.Dev Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 15Hribal M.L. Nakae J. Kitamura T. Shutter J.R. Accili D. Regulation of insulin-like growth factor-dependent myoblast differentiation by Foxo forkhead transcription factors.J Cell Biol. 2003; 162: 535-541Crossref PubMed Scopus (165) Google Scholar it is possible that FoxO1 participates in the transdifferentiation process of HSCs. The present study evaluates the role of FoxO1 in proliferation and transdifferentiation of HSCs. Our functional analysis of FoxO1 revealed that both proliferation and transdifferentiation of HSCs were inhibited by transcriptionally active FoxO1 and enhanced by transcriptionally inactive FoxO1. Moreover, mice of FoxO1 haploinsufficiency (FoxO1+/−) were more sensitive to experimental liver fibrosis. Our study identifies FoxO as a key transcription factor that regulates hepatic fibrogenesis. Primary HSCs were isolated from male Sprague-Dawley rats and male Balb/c mice. Primary HSCs were isolated by a 2-step perfusion using pronase E (EMD Chemicals, Gibbstown, NJ) and collagenase D (Roche, Mannheim, Germany), followed by Nycodenz (Axis-Shield, Oslo, Norway) 2-layer discontinuous density gradient centrifugation as previously described.16Siegmund S.V. Uchinami H. Osawa Y. Brenner D.A. Schwabe R.F. Anandamide induces necrosis in primary hepatic stellate cells.Hepatology. 2005; 41: 1085-1095Crossref PubMed Scopus (157) Google Scholar Purity of rat or mouse HSC preparations was 96% and 97%, respectively, as assessed by autofluorescence at day 1. HSCs were cultured on uncoated plastic tissue culture dishes in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Growth medium was changed daily. Rat HSCs were passaged once between days 5 and 7 when cells were culture activated. The fully transdifferentiated rat HSCs were used for experiments between days 10 and 14. Mouse HSCs were not passaged and were used for experiments on days 0 to 4. In some experiments, immortalized human HSC line hTERT HSCs were cultured in DMEM supplemented with 10% FBS as previously described.17Schnabl B. Purbeck C.A. Choi Y.H. Hagedorn C.H. Brenner D. Replicative senescence of activated human hepatic stellate cells is accompanied by a pronounced inflammatory but less fibrogenic phenotype.Hepatology. 2003; 37: 653-664Crossref PubMed Scopus (165) Google Scholar Primary rat HSCs or hTERT HSCs were serum starved in serum-free DMEM for 24 hours and were treated with 20 ng/mL PDGF-BB (Roche) or 100 nmol/L insulin (Invitrogen, Auckland, New Zealand). Where indicated in results, cells were preincubated with 20 μmol/L LY294002 (Sigma-Aldrich, St. Louis, MO) for 1 hour. The adenoviral vectors encoding hemagglutinin (HA)-tagged wild-type FoxO1 (WT-FoxO1), HA-tagged constitutively active FoxO1 (ADA-FoxO1), HA-tagged dominant-negative FoxO1 (Δ256-FoxO1), HA-tagged constitutively active AKT (Myr-AKT), HA-tagged dominant-negative AKT (DN-AKT), manganese superoxide dismutase (MnSOD), green fluorescent protein (GFP), and bacterial β-galactosidase (LacZ) have been previously described.14Nakae J. Kitamura T. Kitamura Y. Biggs III, W.H. Arden K.C. Accili D. The forkhead transcription factor Foxo1 regulates adipocyte differentiation.Dev Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 16Siegmund S.V. Uchinami H. Osawa Y. Brenner D.A. Schwabe R.F. Anandamide induces necrosis in primary hepatic stellate cells.Hepatology. 2005; 41: 1085-1095Crossref PubMed Scopus (157) Google Scholar, 18Osawa Y. Hannun Y.A. Proia R.L. Brenner D.A. Roles of AKT and sphingosine kinase in the antiapoptotic effects of bile duct ligation in mouse liver.Hepatology. 2005; 42: 1320-1328Crossref PubMed Scopus (40) Google Scholar, 19Nakae J. Kitamura T. Silver D.L. Accili D. The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression.J Clin Invest. 2001; 108: 1359-1367Crossref PubMed Scopus (510) Google Scholar, 20Lehmann T.G. Wheeler M.D. Froh M. Schwabe R.F. Bunzendahl H. Samulski R.J. Lemasters J.J. Brenner D.A. Thurman R.G. Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat.Transplantation. 2003; 76: 28-37Crossref PubMed Scopus (56) Google Scholar ADA-FoxO1 contains mutation in all of the 3 AKT phosphorylation sites, resulting in constitutively active FoxO1-dependent gene transcription.19Nakae J. Kitamura T. Silver D.L. Accili D. The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression.J Clin Invest. 2001; 108: 1359-1367Crossref PubMed Scopus (510) Google Scholar Δ256-FoxO1 contains a DNA-binding domain but lacks a transactivation domain, resulting in inhibition of FoxO1-dependent gene transcription.19Nakae J. Kitamura T. Silver D.L. Accili D. The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression.J Clin Invest. 2001; 108: 1359-1367Crossref PubMed Scopus (510) Google Scholar The adenovirus vector encoding p27kip1 short interfering RNA (siRNA) was created by using the pRNAT-H1.1/Adeno shuttle vector (GenScript, Piscataway, NJ) and AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) according to the manufacturers’ protocol. Oligonucleotide sequences were modified from the sequences by Tamamori-Adachi M et al21Tamamori-Adachi M. Hayashida K. Nobori K. Omizu C. Yamada K. Sakamoto N. Kamura T. Fukuda K. Ogawa S. Nakayama K.I. Kitajima S. Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4 Evidence for impaired Skp2-dependent degradation of p27 in terminal differentiation.J Biol Chem. 2004; 279: 50429-50436Crossref PubMed Scopus (33) Google Scholar: 5′-CGC GTG TGG GAG TGT TTA ATG GGA ACG TGT GCT GTC CGT TCC CGT TAG ACA CTC TCA CTT TTT A-3′ and 5′-AGC TTA AAA AGT GAG AGT GTC TAA CGG GAA CGG ACA GCA CAC GTT CCC ATT AAA CAC TCC CAC A-3′. Cells were transduced with each adenovirus at a multiplicity of infection (MOI) of 100 (primary mouse HSCs), 200 (primary rat HSCs or hTERT HSCs), or otherwise instructed to achieve transduction rates of greater than 80%. DNA synthesis was estimated as the amount of methyl-3H-thymidine as previously described.22Schnabl B. Kweon Y.O. Frederick J.P. Wang X.F. Rippe R.A. Brenner D.A. The role of Smad3 in mediating mouse hepatic stellate cell activation.Hepatology. 2001; 34: 89-100Crossref PubMed Scopus (247) Google Scholar HSCs were incubated with 1 μCi/mL 3H-thymidine (Amersham, Piscataway, NJ) for 18 or 24 hours followed by trichloroacetic precipitation, lysis, and measurement in a scintillation counter. Twenty-four hours after treatment with PDGF or insulin, rat HSCs were harvested by scraping and fixed with cold ethanol (50%) in phosphate-buffered saline (PBS) for 1 hour. Cells were then washed with PBS and treated with 0.5 mg/mL RNase A (Qiagen, Valencia, CA) for 1 hour at 37°C. Cells were incubated with 20 μg/mL propidium iodide (Sigma-Aldrich) at 4°C in the dark, and cell cycle state was assessed by flow cytometry using a fluorescent-activated cell sorting (FACS) instrument (FACSCalibur, BD Biosciences, San Jose, CA). Electrophoresis of protein extracts and subsequent blotting were performed as previously described.18Osawa Y. Hannun Y.A. Proia R.L. Brenner D.A. Roles of AKT and sphingosine kinase in the antiapoptotic effects of bile duct ligation in mouse liver.Hepatology. 2005; 42: 1320-1328Crossref PubMed Scopus (40) Google Scholar Blots were incubated with antibodies against phospho-FoxO1 (Ser-256), phospho-AKT (Ser-473), hemagglutinin (both Cell Signaling, Beverly, MA), FoxO1, AKT (both Santa Cruz, Santa Cruz, CA), p27kip1 (BD Biosciences), MnSOD (Stressgen, Victoria BC, Canada), α-SMA, β-actin (both Sigma-Aldrich), and desmin (DAKO, Glostrup, Denmark) overnight at 4°C. For detection of insulin receptor substrate-2 (IRS-2) tyrosine phosphorylation, cell lysates from insulin-treated rat HSCs were immunoprecipitated with anti-IRS-2 antibody (Upstate, Lake Placid, NY) and protein A-agarose (Santa Cruz). Immunoprecipitates were then subjected to SDS-PAGE, and Western blot analysis was performed using antiphosphotyrosine antibody (Upstate) or anti-IRS-2 antibody. One hour after the treatment with PDGF or insulin, primary rat HSCs were fixed with −10°C methanol for 5 minutes and blocked with PBS containing 4% FBS. Cells were incubated with anti-HA antibody for 1 hour and then incubated with rhodamine-conjugated anti-mouse IgG antibody (Pierce, Rockford, IL). For nuclear counterstaining, 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes, Eugene, OR) was used according to the manufacturer’s instruction. Cells were observed under a fluorescence microscope with appropriate filters. Reactive oxygen species (ROS) production was tested in activated HSCs using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA)-based fluorescence. DCFDA-associated ROS production was measured in a time course of 30 minutes using multiwell platereader (BMG Optima, Durham, NC).23Bataller R. Schwabe R.F. Choi Y.H. Yang L. Paik Y.H. Lindquist J. Qian T. Schoonhoven R. Hagedorn C.H. Lemasters J.J. Brenner D.A. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (496) Google Scholar Extracted RNA from the liver and the cells was reverse transcribed (First-Strand cDNA Synthesis Kit; Amersham), and quantitative real-time polymerase chain reaction (PCR) with the probe-primers sets of human p27kip1 (Hs00153277_m1), mouse p27kip1 (Mm00438167_g1), human MnSOD (SOD2, Hs00167309_m1), mouse MnSOD (Mm00449726_m1), mouse collagen α1(I) (Mm00801666_g1), mouse PPAR-γ (Mm00440945_m1), mouse TIMP-1 (Mm00801666_g1), and 18S ribosomal RNA (Hs99999901_s1) (Applied Biosystems, Foster City, CA) was performed using Taqman analysis (ABI Prism 7000 Sequence Detection System, Applied Biosystems). The changes were normalized to 18S. Chromatin immunoprecipitation (ChIP) assays were performed by using the ChIP assay kit (Upstate) with some modification as previously described.24Daitoku H. Yamagata K. Matsuzaki H. Hatta M. Fukamizu A. Regulation of PGC-1 promoter activity by protein kinase B and the forkhead transcription factor FKHR.Diabetes. 2003; 52: 642-649Crossref PubMed Scopus (229) Google Scholar hTERT HSCs were transduced with WT-FoxO1 or ADA-FoxO1 adenoviruses. ChIP was performed with anti-HA antibody or normal mouse IgG as a negative control. Precipitated DNA was analyzed by PCR using specific primers for promoter regions containing the Forkhead binding element (FBE) of p27kip1: 5′-TGCGCGCTCCTAGAGCTC-3′ and 5′-TTTCTCCCGGGTCTGCAC-3′, MnSOD: 5′-GTCCCAGCCTGAATTTCC-3′ and 5′-CTAGGCTTCCGGTAAGTG-3′, and β-actin coding region: 5′-CAAGAGATGGCCACGGCTGC-3′ and 5′-CTAGAAGCATTTGCGGTGGACG-3′.25Daitoku H. Hatta M. Matsuzaki H. Aratani S. Ohshima T. Miyagishi M. Nakajima T. Fukamizu A. Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity.Proc Natl Acad Sci U S A. 2004; 101: 10042-10047Crossref PubMed Scopus (511) Google Scholar FoxO1+/− mice and wild-type littermates (FoxO1+/+ mice) (mixed background) were bred for studies as previously described.26Nakae J. Biggs III, W.H. Kitamura T. Cavenee W.K. Wright C.V. Arden K.C. Accili D. Regulation of insulin action and pancreatic β-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1.Nat Genet. 2002; 32: 245-253Crossref PubMed Scopus (535) Google Scholar Liver fibrosis was induced by bile duct ligation (BDL).23Bataller R. Schwabe R.F. Choi Y.H. Yang L. Paik Y.H. Lindquist J. Qian T. Schoonhoven R. Hagedorn C.H. Lemasters J.J. Brenner D.A. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (496) Google Scholar The mice were killed 3 weeks after BDL, and collagen α1(I) mRNA levels and protein levels for α-SMA and desmin were determined as previously described.23Bataller R. Schwabe R.F. Choi Y.H. Yang L. Paik Y.H. Lindquist J. Qian T. Schoonhoven R. Hagedorn C.H. Lemasters J.J. Brenner D.A. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (496) Google Scholar Collagen deposition was stained with Sirius red (saturated picric acid containing 0.1% Direct Red 80 and 0.1% FastGreen FCF). For measurement of hydroxyproline content, the extracted protein from the liver was hydrolyzed for 24 hours at 110°C in 6 mol/L HCl. The samples were oxidized with Chloramine-T (Sigma-Aldrich) for 25 minutes and then incubated with Ehrich’s percholic acid solution containing 4-(Dimethylamino)benzaldehyde (Sigma-Aldrich) at 65°C for 20 minutes. The sample absorbance was measured at 560 nm. The acute liver injury produced by anti-Fas antibody (Jo2; BD Pharmingen, San Diego, CA) was also assessed in FoxO1+/− and FoxO1+/+ mice. Jo2 (100 μg/mouse) was administrated intravenously. Treated animals were anesthetized and killed 2.5 hours after Jo2 administration. All procedures for HSC isolation and BDL were approved by the Investigation and Ethics Committee and Institutional Animal Care and Use Committee of the Columbia University. Results are expressed as mean ± SD. The results were analyzed using the unpaired Student t test. A P value of less than .05 was considered statistically significant. Because the PI3K/AKT pathway induces proliferation and migration of HSCs,4Reif S. Lang A. Lindquist J.N. Yata Y. Gabele E. Scanga A. Brenner D.A. Rippe R.A. The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression.J Biol Chem. 2003; 278: 8083-8090Crossref PubMed Scopus (246) Google Scholar, 5Gabele E. Reif S. Tsukada S. Bataller R. Yata Y. Morris T. Schrum L.W. Brenner D.A. Rippe R.A. The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation.J Biol Chem. 2005; 280: 13374-13382Crossref PubMed Scopus (87) Google Scholar we first sought to elucidate the role of FoxO1 in the proliferation of transdifferentiated HSCs. PDGF (20 ng/mL) or insulin (100 nmol/L) induced phosphorylation of FoxO1 in parallel with AKT phosphorylation in serum-starved primary rat HSCs. The phosphorylation of FoxO1 or AKT was completely inhibited by the pretreatment with PI3K inhibitor LY294002 (Figure 1A). Insulin also phosphorylated IRS-2 (Figure 1B). Moreover, phosphorylation of FoxO1 by PDGF or insulin was completely inhibited by overexpression of a dominant-negative form of AKT (DN-AKT) (Figure 1C, left). In contrast, constitutively active AKT-transduced HSCs induce phosphorylation of FoxO1 during serum starvation (Figure 1C, right). These results indicate that PDGF or insulin induces phosphorylation of endogenous FoxO1 via the PI3K/AKT pathway in primary rat HSCs. AKT-dependent phosphorylation of FoxO transcription factors induces nuclear exclusion, resulting in inhibition of their transcriptional activity.27Datta S.R. Brunet A. Greenberg M.E. Cellular survival: a play in three Akts.Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar, 28Kops G.J. Medema R.H. Glassford J. Essers M.A. Dijkers P.F. Coffer P.J. Lam E.W. Burgering B.M. Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors.Mol Cell Biol. 2002; 22: 2025-2036Crossref PubMed Scopus (374) Google Scholar To investigate the subcellular distribution of FoxO1 after PDGF or insulin, we transduced rat HSC with WT-FoxO1 or ADA-FoxO1 and immunostained with anti-HA antibody. In serum-starved HSCs, FoxO1 protein had a nuclear distribution (Figure 2A). Addition of PDGF or insulin resulted in a cytosolic redistribution of FoxO1, which was blocked by LY294002 (Figure 2A). Phosphorylation-resistant ADA-FoxO1 showed nuclear localization even after PDGF or insulin (Figure 2B and C). These results indicate that PDGF- or insulin-induced FoxO1 phosphorylation results in nuclear exclusion of FoxO1 via the PI3K/AKT pathway. To investigate the role of FoxO1 in the proliferation of HSCs, 3H-thymidine incorporation was assessed. PDGF or insulin increased 3H-thymidine incorporation, which was significantly inhibited in ADA-FoxO1-transduced HSCs (Figure 3A). In contrast, HSCs transduced with Δ256-FoxO1 showed an increase in 3H-thymidine incorporation (Figure 3B), and PDGF or insulin did not further increase 3H-thymidine in Δ256-FoxO1-transduced cells (data not shown). LY294002 inhibited PDGF- or insulin-induced 3Marra F. Gentilini A. Pinzani M. Choudhury G.G. Parola M. Herbst H. Dianzani M.U. Laffi G. Abboud H.E. Gentilini P. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor’s actions on hepatic stellate cells.Gastroenterology. 1997; 112: 1297-1306Abstract Full Text PDF PubMed Scopus (187) Google ScholarH-thymidine incorporation, whereas Δ256-FoxO1 reversed this inhibition (Figure 3B), indicating that FoxO1 is a crucial downstream target of the PI3K/AKT pathway to control HSC proliferation. To investigate further the mechanisms by which FoxO1 controls proliferation, FACS analyses were performed to examine the cell cycle. PDGF, insulin or Δ256-FoxO1 induced cells to accumulate in the S/G2/M phase of the cell cycle. In contrast, ADA-FoxO1 resulted in the accumulation of cells in the G0/G1 phase, even after treatment with PDGF or insulin (Figure 3C and D). These results indicate that active (nuclear) FoxO1 inhibits proliferation by inducing cell cycle arrest in the G1 phase. FoxO factors control a variety of target genes, including antioxidant genes29Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress.Nature. 2002; 419: 316-321Crossref PubMed Scopus (1282) Google Scholar and regulators of metabolism,19Nakae J. Kitamura T. Silver D.L. Accili D. The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression.J Clin Invest. 2001; 108: 1359-1367Crossref PubMed Scopus (510) Google Scholar cell cycle,9Medema R.H. Kops G.J. Bos J.L. Burgering B.M. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1.Nature. 2000; 404: 782-787Crossref PubMed Scopus (1231) Google Scholar, 30Schmidt M. Fernandez de Mattos S. van der Horst A. Klompmaker R. Kops G.J. Lam E.W. Burgering B.M. Medema R.H. Cell cycle inhibition by FoxO forkhead transcription factors involves down-regulation of cyclin D.Mol Cell Biol. 2002; 22: 7842-7852Crossref PubMed Scopus (472) Google Scholar and cell death.7Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 31Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1.Curr Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar To investigate the mechanism by which FoxO1 inhibits proliferation via cell cycle arrest at G1, target genes of FoxO1 were assessed with hTERT HSCs.17Schnabl B. Purbeck C.A. Choi Y.H. Hagedorn C.H. Brenner D. Replicative senescence of activated human hepatic stellate cells is accompanied by a pronounced inflammatory but less fibrogenic phenotype.Hepatology. 2003; 37: 653-664Crossref PubMed Scopus (165) Google Scholar hTERT HSCs showed the same results as shown in primary rat HSCs, including phosphorylation of FoxO1 by PDGF or insulin and inhibition of proliferation by ADA-FoxO1 (data not shown). Among the FoxO1 target genes tested, p27kip1 and MnSOD were significantly decreased by PDGF, insulin, or Δ256-FoxO1. In contrast, ADA-FoxO1 markedly increased mRNA levels of p27kip1 and MnSOD, which were not inhibited by PDGF or insulin (Figure 4A and see