Nonalcoholic fatty liver disease (NAFLD), the

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1 Transforming Growth Factor Beta Signaling in Hepatocytes Participates in Steatohepatitis Through Regulation of Cell Death and Lipid Metabolism in Mice Ling Yang, 1,2 Yoon Seok Roh, 1 Jingyi Song, 1 Bi Zhang, 1 Cheng Liu, 1 Rohit Loomba, 1 and Ekihiro Seki 1 Transforming growth factor beta (TGF-b) signaling activates Smad- and TGF-b-activated kinase 1 (TAK1)-dependent signaling to regulate cell survival, proliferation, fibrosis, and tumorigenesis. The effects of TGF-b signaling on metabolic syndrome, including nonalcoholic fatty liver disease, remain elusive. Wild-type (WT) and hepatocyte-specific TGF-b receptor type II-deficient (Tgfbr2DHEP) mice were fed a choline-deficient amino acid (CDAA)-defined diet for 22 weeks to induce NASH. WT mice fed a CDAA diet displayed increased activation of Smad2/3 and had marked lipid accumulation, inflammatory cell infiltration, hepatocyte death, and fibrosis; in comparison, Tgfbr2DHEP mice fed a CDAA diet had suppressed liver steatosis, inflammation, and fibrosis. Both palmitateinduced steatotic hepatocytes and hepatocytes isolated from WT mice fed a CDAA diet had increased susceptibility to TGF-b-mediated death. TGF-b-mediated death in steatotic hepatocytes was inhibited by silencing Smad2 or blocking reactive oxygen species (ROS) production and was enhanced by inhibiting TAK1 or nuclear factor kappa B. Increased hepatic steatosis in WT mice fed a CDAA diet was associated with the increased expression of lipogenesis genes (Dgat1 and Srebp1c), whereas the decreased steatosis in Tgfbr2DHEP mice was accompanied by the increased expression of genes involved in b- oxidation (Cpt1 and Acox1). In combination with palmitate treatment, TGF-b signaling promoted lipid accumulation with induction of lipogenesis-related genes and suppression of b-oxidation-related genes in hepatocytes. Silencing Smad2 decreased TGF-b-mediated lipid accumulation and corrected altered gene expression related to lipid metabolism in hepatocytes. Finally, we confirmed that livers from patients with nonalcoholic steatohepatitis (NASH) displayed phosphorylation and nuclear translocation of Smad2/3. Conclusions: TGF-b signaling in hepatocytes contributes to hepatocyte death and lipid accumulation through Smad signaling and ROS production that promote the development of NASH. (HEPATOLOGY 2014;59: ) Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, is currently a significant health concern in the United States in both adults and children. 1-5 Obesity and type II diabetes are strongly linked to the progression of NAFLD. 6 A disease spectrum of NAFLD ranges from simple steatosis to steatosis with liver inflammation and fibrosis, referred to as nonalcoholic steatohepatitis (NASH). 5 Ten to twenty percent of NAFLD patients have NASH, and 10%-15% of Abbreviations: Ab, antibody; a-sma, alpha-smooth muscle actin; ALT, alanine aminotransferase; BHA, butylated hydroxyanisole; BSA, bovine serum albumin; CDAA, choline-deficient amino acid defined; C/EBPb, CCAAT/enhancer-binding protein beta; CSAA, choline-supplemented amino acid defined; CTGF, connective tissue growth factor; FFA, free fatty acid; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; HNE, hydroxynonenal; HSC, hepatic stellate cell; IHC, immunohistochemistry; IL, interleukin; IP, intraperitoneal; IR, insulin resistance; ITT, insulin tolerance test; JNK, c-jun N-terminal kinase; LDH, lactate dehydrogenase; mrna, messenger RNA; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; NASH, nonalcoholic steatohepatitis; NF-jB, nuclear factor kappa B; NIH, National Institutes of Health; PAI, plasminogen activator inhibitor 1; PGC-1a, PPAR-g coactivator 1 alpha; PPAR-g, peroxisome proliferator-activated receptor gamma; qpcr, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; sirna, small interfering RNA; TAK1, TGF-b-activated kinase 1; Tg, transgenic; TG, triglyceride; TGF-b, transforming growth factor-b; Tgfbr2, TGF-b receptor type II; Tgfbr2 DHep mice, hepatocyte-specific Tgfbr2 deleted mice; TIMP-1, tissue inhibitor of metalloproteinase 1; TNF-a, tumor necrosis factor alpha; TUNEL, terminal deoxynucleotidyl transferase dutp nick end labeling; UCSD, University of California San Diego; WT, wild type. From the 1 Division of Gastroenterology, Department of Medicine, School of Medicine, University of California San Diego, La Jolla, CA; and 2 Division of Gastroenterology, Department of Internal Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Received February 25, 2013; accepted August 16,

2 484 YANG ET AL. HEPATOLOGY, February 2014 NASH patients eventually progresses into liver cirrhosis that significantly increases the risk for the development of hepatocellular carcinoma. 1-4 However, it is still unclear what triggers the progression of NAFLD to NASH accompanied with inflammation and fibrosis. Though simple hepatic steatosis is considered benign, NAFLD with the presence of histologic changes consistent with NASH significantly shortens human life expectancy. NAFLD is projected to replace hepatitis C as the leading cause for liver transplantation by A deeper understanding of the molecular mechanisms underlying the development of NASH is necessary to develop effective alternative therapies for NASH that eliminate reliance on the insufficient number of donor livers. Transforming growth factor beta (TGF-b) is a pleiotropic cytokine involved in cell survival, proliferation, differentiation, angiogenesis, and wound-healing response. 7-9 TGF-b binding to type II TGF-b receptor causes type II receptor to recruit and phosphorylate type I TGF-b receptor. Type I TGF-b receptor kinase then activates a canonical Smad-dependent pathway and a noncanonical Smad-independent TGF-b-activated kinase 1 (TAK1)- or phosphoinositide 3 kinasedependent pathway. In Smad-dependent signaling, phosphorylated Smad2 and Smad3 associate with Smad4 to translocate into the nucleus to control transcriptional gene expression. In the liver, TGF-b signaling participates in fibrogenic response through hepatic stellate cell (HSC) activation. 9,10 Thus, it is well established that TGF-b signaling in HSCs plays a role in progression of fibrosis in advanced NAFLD. Although previous studies demonstrated increases in hepatic TGF-b expression in NASH patients with fibrosis, 11,12 the role of TGF-b signaling in hepatocytes in the development of NASH is not well understood. In addition, the study of TGF-b signaling in metabolic disease is still limited. Previous reports demonstrated that TGF-b/Smad3 signaling is involved in insulin gene expression in pancreatic b cells. 13 TGF-b/Smad3 signaling is associated with systemic insulin resistance (IR), obesity, and hepatic steatosis through regulation of expression of PPAR-c coactivator 1 alpha (PGC-1a) and peroxisome proliferator-activated receptor gamma (PPAR-c). 14,15 Indeed, Smad3 2/2 mice were protected from IR, obesity, and hepatic steatosis induced by high-fat diet (HFD) feeding. 14,15 Because the previous study used whole-body Smad3 2/2 mice, the specific role of TGF-b signaling in hepatocytes to the development of diet-induced NASH needs to be studied. In the present study, we used choline-deficient amino acid (CDAA)-defined diet that induces hepatic steatosis, inflammation, and fibrosis, all of which are comparable pathologies to human NASH. We tested functions of hepatocyte TGF-b signaling in the progression of CDAA diet-induced NASH in mice. Materials and Methods Mouse Colonies. Albumin-Cre recombinase transgenic (Tg) mice (Albumin-Cre Tg mice) and Tgfbr2 flox/flox mice were purchased from The Jackson Laboratories (Bar Harbor, MA). The two lines were intercrossed to generate albumin-cre/ 1 Tgfbr2 flox/flox mice (Tgfbr2DHep) on C57BL6 background. Crenegativeanimalswereusedaswild-type(WT)controls. Eight-week-old male mice were divided into three groups: standard chow group (catalog no. 5053; PicoLab Rodent Diet 20 series; LabDiet, Framingham, MA); choline-supplemented L-amino-acid-defined diet group (CSAA; catalog no ; Dyets Inc., Bethlehem, PA); and CDAA-defined diet group (catalog no ; Dyets Inc.). Both CSAA and CDAA diets contain higher calories than the standard chow (Supporting Table 1) Mice were on these diets for 22 weeks, and CDAA diet intake was monitored for the first month. All mice received humane care according to National Institutes of Health (NIH; Bethesda, MD) recommendations outlined in the Guide for the Care and Use of Laboratory This study is supported by a National Institutes of Health grant (R01AA02172 [to E.S.] and R01DK [to E.S.]). R.L. is supported, in part, by the American Gastroenterological Association (AGA) Foundation-Sucampo-ASP Designated Research Award in Geriatric Gastroenterology and by a T. Franklin Williams Scholarship Award, with funding provided by: Atlantic Philanthropies, Inc., the John A. Hartford Foundation, the Association of Specialty Professors, and the AGA and grants K23-DK and P30CA L.Y. was supported, in part, by the National Natural Science Foundation of China (no and ). Address reprint requests to: Ekihiro Seki, M.D., Ph.D., Division of Gastroenterology, Department of Medicine, School of Medicine, University of California San Diego, School of Medicine, 9500 Gilman Drive, MC 0702, Leichtag Biomedical Research Building, Room 118B, La Jolla, CA ekseki@ucsd. edu; fax: Copyright VC 2013 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article.

3 HEPATOLOGY, Vol. 59, No. 2, 2014 YANG ET AL. 485 Animals. All animal experiments were approved by the University of California San Diego (UCSD) Institutional Animal Care and Use Committee (La Jolla, CA). Mouse Tissue Processing. Mouse tissues were either snap-frozen in liquid nitrogen for RNA and protein preparations or fixed in 10% or 4% neutralbuffered formalin phosphate (Fisher Scientific, Pittsburgh, PA) to be embedded in paraffin or optimal cutting temperature compound and cut into 5-lm sections for hematoxylin and eosin (H&E), Oil Red O, Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL), and Sirius Red staining TUNEL-positive cells were counted on 10 high power (2003) fields/slide. The Sirius Red-positive area was measured on 10 low power (403) fields/slide and quantified with the use of NIH imaging software. NAFLD activity score (NAS) was determined as described. Immunohistochemistry (IHC) for F4/80, alpha-smooth muscle actin (a-sma), phosphorated Smad2/3, and 4-hydroxynonenal (HNE) were performed Quantitative Real-Time Polymerase Chain Reaction. RNA extracted from livers and cells were subjected to reverse transcription and subsequently underwent quantitative real-time polymerase chain reaction (qpcr) with the use of the CFX96 real-time PCR system (Bio-Rad, Hercules, CA). PCR primer sequences are listed in Supporting Table 2. Genes were normalized to 18S RNA as an internal control. Cell Isolation and Treatment. Hepatocytes were isolated, and cells with viability above 90% were used for the experiments. 16,17 After hepatocytes attached (2 hours after plating), medium was changed to serumfree media (day 0). Then, in some experiments, cells were incubated with scramble or Smad2 small interfering RNA (sirna; sc-38375; Santa Cruz Biotechnology, Santa Cruz, CA) or adenovirus encoding control- Lac-z or IjB super-repressor for an additional 16 hours (overnight). On the following day, palmitate or oleate (200 lm) was added for 6 hours (day 1). Subsequently, cells were treated with 10 ng/ml of murine TGF-b1 (R&D Systems, Minneapolis, MN) for 24 hours (day 2) and analyzed for cell death or lipid accumulation (total, 48 hours after plating). In some experiments, 250 nm of 5z-7-oxozeaenol (TAK1 inhibitor), 45 lm of fasudil (rock inhibitor), 10 lm of SB (p38 inhibitor), 20 lm of SP (c- Jun N-terminal kinase [JNK] inhibitor), or 100 lmof butylated hydroxyanisole (BHA; Sigma-Aldrich, St Louis, MO) was added 30 minutes before TGF-b1 treatment. Albumin-conjugated palmitate used in this study was prepared by dissolving palmitate in ethanol at 50 o C and then conjugating with fatty-acid-free bovine serum albumin (BSA). To measure reactive oxygen species (ROS), cells were incubated with 10 lm of CM-H 2 DCFDA (Invitrogen, Grand Island, NY) for 30 minutes at 37 C and analyzed by a fluorescence microplate reader. 19 Lipid Isolation and Measurement. Liver extracts were prepared by homogenization in 0.25% sucrose with 1 mmol/l of ethylenediaminetetraacetic acid. Lipids were extracted using chloroform/methanol (2:1, v/v) and suspended with 5% fatty-acid-free BSA. Triglyceride (TG), total cholesterol, and free fatty acid (FFA) contents were measured with the use of the Triglyceride Reagent Set (Pointe Scientific, Canton, MI), Cholesterol E (Wako, Richmond, VA), FFA, and the half micro test (Roche, Mannheim, Germany). 16 Hepatocyte TG accumulation was quantified by extraction of hepatocyte lipids from cell homogenates using chloroform/methanol (2:1), and TG was measured. Immunofluorescence. Hepatocytes were labeled by Mito Tracker Red CMXRos (100 nm; Molecular Probes, Eugene, OR) and fixed with 4% paraformaldehyde in phosphate-buffered saline, followed by permeabilization and incubation with anti-bax antibody (Ab; Cell Signaling Technology, Danvers, MA). 20 Glucose and Insulin Tolerance Tests. For the glucose tolerance test (GTT), baseline glucose levels were measured from mice fasted for 16 hours (0 min). Then, 2 g/kg of glucose were administered by intraperitoneal (IP) injection to mice, and glucose levels were measured at 15-minute intervals over a span of 2 hours after the glucose load. For the insulin tolerance test (ITT), baseline glucose levels were measured after 4 hours of fasting. Blood-glucose concentration was monitored every minutes for 90 minutes after administration of insulin at 0.5 U/kg by IP injection. Human Liver Samples. Paraffin-embedded human liver tissues were acquired from liver biopsy samples of patients with NAFLD. All liver biopsies were read by a single hepatopathologist who was blinded to clinical data. Liver biopsies were scored using the NASH Clinical Research Network Histologic Scoring System. 21 NAS was documented that ranges from 0 to 8. NAS is a sum of three histologic scores, including steatosis (0-3), lobular inflammation (0-3), and ballooning degeneration (0-2). Patients were classified into two groups: nonalcoholic fatty liver (NAFL; NAS score between 0 and 3) without fibrosis (n 5 10) versus those with NASH with fibrosis and hepatocyte ballooning (NAS score of 5 or higher; n 5 10). Please visit the UCSD NAFLD registry for details on inclusion and exclusion criteria (fattyliver.ucsd.edu) for diagnosis of NAFLD. 22

4 486 YANG ET AL. HEPATOLOGY, February 2014 The diagnosis of NASH was defined as previously reported by the San Diego Integrated NAFLD Consortium. 23 The study was approved by the UCSD Institutional Review Board. Statistical Analysis. Differences between two groups were compared using Mann-Whitney s U test or two-tailed unpaired Student t test. Differences between multiple groups were compared using one-way analysis of variance using SPSS software (SPSS, Inc., Chicago, IL). P values <0.05 were considered significant. Results Deletion of Tgfbr2 in Hepatocytes Alleviates Steatohepatitis in Mice. Initially, we examined the activation of TGF-b signaling in mouse NASH livers induced by 22 weeks of CDAA diet feeding. NASH livers exhibited increased phosphorylation of Smad2/3 and its nuclear translocation in hepatocytes and liver nonparenchymal cells as well as increased expression of TGF-b (Fig. 1A). These results suggest an association between TGF-b signaling and NASH formation. Though TGF-b is well known to stimulate HSCs to induce fibrosis, 10,24 the present study used WT and Tgfbr2DHEP mice to investigate the specific role of TGF-b signaling in hepatocytes in the development of NASH. WT and Tgfbr2DHEP mice fed on standard chow had similar metabolic phenotypes (Supporting Table 3). Twenty-two weeks of CDAA diet feeding induced significant hepatic steatosis, hepatocyte damage, and inflammatory cell infiltration in WT livers (Fig. 1B-D) In contrast, CDAA diet Tgfbr2DHEP mice had a significant reduction in serum alanine aminotransferase (ALT) and TG levels and NAS, as determined by degree of hepatic steatosis, inflammation, and ballooning (Fig. 1B-D). Increased infiltration of hepatic macrophages and neutrophils in WT mice was reduced in Tgfbr2DHEP mice (Fig. 1B,E and Supporting Fig. 1). Consistent with histology results, hepatic expression of tumor necrosis factor alpha (TNF-a), interleukin (IL)-6, chemokine (C-C motif) ligand 2, and IL-1b was increased in WT mice fed on 22 weeks of CDAA diet, whereas these expression were decreased in Tgfbr2DHEP mice fed on the CDAA diet (Fig. 1F). These results indicate that TGF-b signaling in hepatocytes contributes to development of hepatic injury, steatosis, and inflammation in mice fed with a CDAA diet. TGF-b Signaling in Hepatocytes Promotes Liver Fibrosis in NASH. Subsequently, we analyzed liver fibrosis in WT and Tgfbr2DHEP mice fed a CDAA diet for 22 weeks. Deposition of fibrillar collagen and HSC activation were significantly increased in WT mice, but were reduced in Tgfbr2DHEP mice, as assessed by Sirius Red staining and IHC for a-sma (Fig. 2A-C). WT livers had increased messenger RNA (mrna) expression of collagena1(i), a-sma, tissue inhibitor of metalloproteinase 1 (TIMP-1), TGF-b1, connective tissue growth factor (CTGF), and plasminogen activator inhibitor 1 (PAI-1), whereas these gene expressions were suppressed in Tgfbr2DHEP mice fed with the CDAA diet (Fig. 2D). These findings demonstrate that in addition to its known profibrogenic role in HSCs, TGF-b signaling in hepatocytes also possesses profibrogenic functions, inducing liver fibrosis in NASH. TGF-b Signaling Contributes to Hepatocyte Death in NASH. Because TGF-b signaling is associated with induction of cell death, 25 we investigated whether TGF-b signaling contributes to hepatocyte death in steatotic livers. CDAA diet feeding significantly increased the number of TUNEL-positive hepatocytes and expression of cleaved caspase-3 in WT mice fed a CDAA diet; however, these increases were not observed in Tgfbr2DHEP mice (Fig. 3A,B). Proapoptotic Bax gene was increased in livers of WT mice, but not in livers of Tgfbr2DHEP mice fed a CDAA diet (Fig. 3C). To examine the direct effect of TGF-b signaling in hepatocyte death, we purified primary hepatocytes from WT mice fed either a control CSAA diet or CDAA diet and challenged them with recombinant TGF-b. Similar viabilities of these hepatocytes were confirmed before TGF-b treatment. Expression of TGF-b receptor type I, but not type II, was increased in hepatocytes of CDAA-fed mice (Supporting Fig. 2). TGF-b treatment increased cell death only in lipid-accumulated hepatocytes purified from mice fed a CDAA diet, but not in hepatocytes with less lipid accumulation, purified from mice fed a CSAA diet (Fig. 3D). In response to TGF-b, lactate dehydrogenase (LDH) concentrations in the supernatant of hepatocytes from CDAA-diet-fed mice were elevated, when compared to supernatant from hepatocytes from CSAA-diet-fed mice (Fig. 3E). TGF-b treatment induced up-regulation and translocation of Bax into mitochondria in steatotic hepatocytes (Fig. 3F). These results indicate that fat accumulation increases susceptibility of hepatocytes to TGF-b-mediated cell death. TGF-b Induces Cell Death in Lipid-Accumulated Hepatocytes Through Smad Signaling, but Independent of TAK1/Nuclear Factor Kappa B Signaling. Then, we tested whether TGF-b signaling induces cell death in steatotic hepatocytes developed by in vitro treatment with palmitate. As expected,

5 HEPATOLOGY, Vol. 59, No. 2, 2014 YANG ET AL. 487 Fig. 1. TGF-b signaling in hepatocytes promotes NASH. (A-F) WT and Tgfbr2DHEP mice were fed a CSAA (CS; n 5 10) or CDAA (CD; n 5 8) diet for 22 weeks. (A, left) IHC staining of phosphorylated Smad2/3 in liver tissues from WT and Tgfbr2DHEP mice fed with 22 weeks of a CDAA diet. Original magnification: (A, middle) Immunoblots for phosphorylated Smad2 and Smad3, total Smad2, and Smad3 in liver tissues from WT and Tgfbr2DHEP mice are shown. (A, right) Hepatic Tgfb1 expression in WT mice fed CSAA and CDAA diets was measured by qpcr. (B) Liver sections were stained with H&E, Oil Red O, and IHC staining for F4/80. Original magnification: 2003 for H&E and F4/80 staining and 1003 for Oil Red O. (C, left) ALT level. (C, right) NAS. (D) Hepatic TG levels were measured. (E) F4/80-positive area was calculated. (F) Hepatic mrna levels of inflammatory genes (Il1b, Il6, Tnf, Ccl2) were determined by qpcr. Data represent mean 6 standard error of the mean. Open bar: WT mice; closed bar: Tgfbr2DHEP mice. *P < 0.05; **P < TGF-b stimulation dramatically increased hepatocyte death with LDH released in palmitate-induced steatotic hepatocytes, but not in normal hepatocytes and oleate-treated hepatocytes (Fig. 4A and Supporting Fig. 3A,B). We found that TGF-b treatment induced Smad2 phosphorylation in normal hepatocytes, which was further potentiated in palmitate-treated steatotic hepatocytes (Fig. 4B). This led us to examine whether Smad activation is involved in TGF-b-induced cell death in fat-rich hepatocytes. We knocked down Smad2 by incubation of hepatocytes with sirna- Smad2. When Smad2 was inactivated, increased TGFb-induced cell death in lipid-accumulated hepatocytes was significantly suppressed with the decrease of LDH

6 488 YANG ET AL. HEPATOLOGY, February 2014 Fig. 2. Tgfbr2 deficiency in hepatocytes attenuates liver fibrosis. (A-D) WT and Tgfbr2DHEP mice were fed a CSAA (CS; n 5 10) or CDAA (CD; n 5 8) diet for 22 weeks. (A) Liver sections were stained with Sirius Red and IHC staining for a-sma. Original magnification: (B and C) Quantifications of Sirius Red staining (B) and staining for a-sma (C). (D) Hepatic mrna levels of fibrogenic markers (Col1a1, Acta2, Timp1, Tgfb1, Ctgf, and Pai1) were determined by qpcr. Data represent mean 6 standard error of the mean. Open bar: WT mice; closed bar: Tgfbr2DHEP mice. *P < 0.05; **P < levels in culture supernatant (Fig. 4A). Thus, Smad signaling is an important pathway to contribute to cell death in lipid-accumulated hepatocytes in response to TGF-b. Then, we tested whether a Smad-independent TAK1-dependent pathway in TGF-b-mediated hepatocyte death is important. TAK1 activity was blocked in hepatocytes by treatment with 5z-7-oxozeaenol, a specific TAK1 inhibitor. 26 TAK1 inhibition increased cell death in TGF-b-treated hepatocytes and further increased cell death in hepatocytes treated with both palmitate and TGF-b (Fig. 4C). This result suggests that the TAK1-dependent pathway acts as a negative regulator for TGF-b-induced cell death in steatotic hepatocytes. Because TAK1 regulates activation of nuclear factor kappa B (NF-jB), JNK, and p38 in TGF-b signaling, 27,28 we examined which downstream pathways contribute to the enhancement of TGF-bmediated hepatocyte death. We blocked activation of NF-jB, JNK, and p38 by treatment with adenoviral IjB super-repressor, SP600125, and SB203580, respectively. Whereas inhibition of NF-jB increased TGF-b-induced death in steatotic hepatocytes, inhibition of JNK or p38 did not affect hepatocyte death (Fig. 4D and Supporting Fig. 4). Because TGF-b also activates the Rho/ROCK pathway, we examined its importance in TGF-b-mediated hepatocyte death. 29 Fasudil, a ROCK inhibitor, suppressed TGF-bmediated death in steatotic hepatocytes (Fig. 4E). These results suggest that TGF-b-induced cell death in steatotic hepatocytes is dependent on Smad and Rho/ ROCK signaling, but is negatively regulated by TAK1/ NF-jB pathway. TGF-b and Palmitate Synergistically Produce ROS That Contributes to Hepatocyte Death. To investigate whether TGF-b-mediated ROS production in hepatocytes is involved in development of NASH, we examined lipid peroxidation in WT and Tgfbr2DHEP mice fed a CDAA diet by IHC for 4- HNE. Hepatic expression of 4-HNE increased in WT mice, but not in Tgfbr2DHEP mice fed a CDAA diet (Fig. 5A,B). Then, we investigated the effects of TGFb-induced ROS in hepatocytes. Whereas TGF-b or palmitate alone induced ROS production, cotreatment with TGF-b and palmitate synergistically increased ROS production in hepatocytes (Fig. 5C). Antioxidant BHA inhibited ROS production and cell death induced by cotreatment with TGF-b and palmitate (Fig. 5D,E), suggesting that ROS production induced by TGF-b and palmitate cotreatment contributes to hepatocyte death.

7 HEPATOLOGY, Vol. 59, No. 2, 2014 YANG ET AL. 489 Fig. 3. TGF-b signaling promotes hepatocyte death in NAFLD. (A and B) WT and Tgfbr2DHEP mice were fed a CSAA (CS; n 5 10) or CDAA (CD; n 5 8) for 22 weeks. (A) TUNEL staining of liver tissues from WT and Tgfbr2DHEP mice fed with a CDAA diet. Original magnification: Representative pictures (left panel) and their quantification (right panel). (B) Immunoblots for cleaved and full-length caspase-3 of liver tissues of WT and Tgfbr2DHEP mice. (C) Hepatic expression of Bax mrna was determined by qpcr. Open bar: WT mice; closed bar: Tgfbr2DHEP mice. (D-F) Primary hepatocytes were isolated from WT mice fed with a CDAA or CSAA diet and then treated with 10 ng/ml of TGF-b for 24 hours. (D) TUNEL staining of hepatocytes (left) and its quantification are shown (right). Pre, cell viability before TGF-b stimulation. (E) Concentrations of LDH released to supernatants were measured. (F, left) mrna expression of Bax mrna was determined by qpcr. (F, right) Hepatocytes were stained with Mito Tracker and anti-bax Ab followed by assessment with confocal microscopy (original magnification: 6003). Data represent mean 6 standard error of the mean; n 5 3 for each group. *P < 0.05; **P < TGF-b Signaling Is Involved in Lipid Metabolism in Hepatocytes. Because hepatic lipid accumulation was suppressed in Tgfbr2DHEP mice, compared to WT mice, we analyzed the gene expression involved in lipid metabolism in the liver. Upon CDAA diet feeding, WT livers increased the gene expression associated with lipogenesis, including Dgat1, Pparg, and Srebp1c, whereas livers of Tgfbr2DHEP did not (Fig. 6A). In contrast, gene expression related to b-oxidation, such as Acox1, Cpt1, and Hmgcs2, increased in Tgfbr2DHEP mice, but not in WT mice (Fig. 6B). These findings prompted us to examine whether TGF-b signaling

8 490 YANG ET AL. HEPATOLOGY, February 2014 Fig. 4. TGF-b-Smad signaling induces cell death in lipid-accumulated hepatocytes. (A-E) WT primary hepatocytes were treated with 10 ng/ml of TGF-b for 24 hours after pretretment with palmitate. (A) sirna Smad2 was used for silencing Smad2 expression in hepatocytes. Quantification of TUNEL staining (left). Concentrations of LDH released to supernatants were measured (right). (B) Immunoblots for phosphorylated and total Smad2 and b-actin are shown. (C) TAK1 activity was inhibited by 250 nm of 5z-7-oxozeaenol. Quantifications of TUNEL staining (left) and concentrations of LDH in supernatants are shown (right). (D) Adenovirus IjB superrepressor (IjB-sr) was used for blocking NF-jB activation in hepatocytes. Quantifications of TUNEL staining (left) and concentrations of LDH in supernatants are shown (right). (E) Rho/ROCK activity was inhibited by 45 lm of fasudil. Quantifications of TUNEL staining (left) and concentrations of LDH in supernatants are shown (right). Data represent mean 6 standard error of the mean; n 5 3 for each group. *P < 0.05; **P < 0.01.

9 HEPATOLOGY, Vol. 59, No. 2, 2014 YANG ET AL. 491 Fig. 5. TGF-b-induced ROS production mediates hepatocyte death in NASH. (A and B) WT and Tgfbr2DHEP mice were fed a CSAA (CS; n 5 10) or CDAA (CD; n 5 8) for 22 weeks. (A) IHC for 4-HNE of liver tissues from WT and Tgfbr2DHEP mice fed with a CDAA diet. Original magnification: (B) Quantification of 4-HNE staining. (C) Primary WT hepatocytes were incubated with DCFDA (10 lm) and then stimulated with 10 ng/ml of TGF-b. Hepatocytes were preincubated with or without antioxidant BHA (100 lm) for 30 minutes before TGF-b stimulation. Fluorescent signals were quantified for 30 minutes using a fluorometer at excitation and emission wavelengths of 488 and 520 nm, respectively. (D and E) WT primary hepatocytes were treated with 10 ng/ml of TGF-b for 24 hours after pretretment with palmitate. (D) ROS production was inhibited by BHA (100 lm). Quantifications of TUNEL staining and (E) concentrations of LDH in supernatants are shown. DCFDA, dichlorofluorescin diacetate. affects lipid accumulation in primary cultured hepatocytes. TGF-b stimulation alone had little effect on lipid accumulation. Interestingly, TGF-b stimulation potentiated hepatocyte lipid accumulation induced by palmitate (Fig. 6C). Consistent with lipid accumulation, hepatocyte TG levels were also potentiated by cotreatment with TGF-b and palmitate, but not oleate (Fig. 6D and Supporting Fig. 3C). These results indicate that TGF-b signaling has an effect on modulating lipid metabolism in hepatocytes. Smad Signaling Is Crucial for TGF-b-Mediated Modulation of Lipid Metabolism in Hepatocytes. Next, we determined whether the TGF-b/Smad pathway is required for regulation of lipid metabolism in hepatocytes. Increased lipid accumulation and TG levels in hepatocytes treated with TGF-b and palmitate were decreased when Smad2 was knocked down by sirna (Fig. 7A,B). In the context of decreased lipid accumulation and TG levels in Smad2-silenced cells, gene expression involved in lipogenesis, including Dgat1, Fas, and Srebp1c, were suppressed, whereas gene expression related to b-oxidation (Ppara, Acox1, and Cpt1) were increased in hepatocytes treated with TGF-b and palmitate when Smad2 was inactivated (Fig. 7C,D). These results suggest that TGF-b/Smad signaling regulates lipogenesis and b-oxidation-related gene expression that affects palmitate-induced lipid accumulation in hepatocytes. Hepatic-Specific Deletion of Tgfbr2 Influences Systemic Insulin Sensitivity and Metabolic Phenotypes in CDAA-Fed Mice. To test whether hepatocyte TGF-b signaling affects systemic IR in mice fed on 22 weeks of a CDAA diet, GTTs and ITTs were examined. GTT demonstrated that increased blood-sugar levels of CDAA-diet fed WT mice started to decrease after 60 minutes of glucose administration and did not decrease to basal levels within the 120-minute time point. In contrast, Tgfbr2DHEP mice displayed a rapid reduction in blood-sugar levels, compared to WT mice, after glucose challenge (Supporting Fig. 5A). Moreover, WT mice fed a CDAA diet exhibited IR, but Tgfbr2DHEP mice fed a CDAA diet were more sensitive to insulin than WT mice (Supporting Fig. 5B). Whereas Tgfbr2DHEP mice exhibited lower body weight and liver/body-weight ratio than WT mice fed a CDAA diet (Supporting Table 3), food intake was

10 492 YANG ET AL. HEPATOLOGY, February 2014 Fig. 6. TGF-b signaling is associated with gene expression involved in lipogenesis and b-oxidation in fatty liver disease. (A-D) WT and Tgfbr2DHEP mice were fed a CSAA (CS; n 5 10) or CDAA (CD; n 5 8) for 22 weeks. (A and B) Hepatic mrna expression of Dgat1, Pparg, Srebp1c (A), Acox1, Cpt1, and Hmgcs2 (B) were determined by qpcr. Open bar: WT mice; closed bar: Tgfbr2DHEP mice. (C and D) WT primary hepatocytes were treated with 10 ng/ml of TGF-b for 24 hours after pretretment with palmitate. (C) Representative pictures of Oil Red O staining are shown. Original magnifications: (D) TG concentrations in hepatocytes were measured. Data represent mean 6 standard error of the mean. *P < 0.05; **P < similar between WT and Tgfbr2DHEP mice (Supporting Fig. 6). These results suggest that TGF-b signaling in hepatocytes may contribute to systemic IR and increased body weight in mice fed a CDAA diet during development of NASH. TGF-b Signaling in Hepatocytes Is Activated in Human NASH Livers. Last, we examined activation of TGF-b signaling in liver biopsy specimens of patients with NAFL (NAS score between 0 and 3) without fibrosis and patients with NASH (NAS score of 5 or higher) with severe fibrosis and hepatocyte ballooning by IHC for phospho-smad2/3. In liver tissue from mild NAFL patients, we observed weak and no phosphorylation of Smad2/3 in hepatocytes (Fig. 8). In contrast, we observed increased phosphorylation and nuclear translocation of Smad2/3 in liver parenchymal cells in liver tissue of NASH patients (Fig. 8), confirming the clinical relevance of activation of TGF-b signaling in hepatocytes in human NASH. Discussion Activation of TGF-b signaling is associated with formation of fibrotic scar tissue in the liver. 30 Activated HSCs are key players in responding to TGF-b, producing scar tissues consisting of collagen fiber. Alternatively, phosphorylation and nuclear translocation of Smad2/3 was increased in hepatocytes treated with TGF-b as well as in mouse and human NASH livers (Figs. 1, 4 and 8). 25 However, it was unclear that whether TGF-b signaling specifically in hepatocytes contributes to progression of NASH and whether TGF-b-mediated hepatic phenotypes affect systemic metabolic manifestations. The present study demonstrated that TGF-b signaling in hepatocytes promoted hepatic steatosis, hepatocyte damage, inflammatory cell infiltration, inflammatory cytokine production, HSC activation, and fibrosis. TGF-b signaling participated in ROS production and hepatocyte death in lipid-accumulated hepatocytes (Figs. 4 and 5). TGF-b signaling was also involved in lipid metabolism by

11 HEPATOLOGY, Vol. 59, No. 2, 2014 YANG ET AL. 493 Fig. 7. TGF-b-Smad signaling regulates lipid metabolism in lipid-accumulated hepatocytes. (A-D) WT primary hepatocytes were treated with 10 ng/ml of TGF-b for 24 hours after pretretment with palmitate. sirna Smad2 was used for silencing Smad2 expression in hepatocytes. (A) Representative pictures of Oil Red O staining. Original magnifications: (B) TG concentrations in hepatocytes. (C and D) mrna expression of Dgat1, Fas, Srebp1c (C), Ppara, Acox1, and Cpt1 (D) were measured by qpcr. n 5 3 for each group. *P < 0.05; **P < regulation of gene expression involved in lipogenesis and fatty acid b-oxidation. Intriguingly, hepatocyte s TGF-b signaling influenced systemic IR and body-weight gain. Previous studies demonstrated that Smad3 deficiency results in decreased obesity and systemic IR on an HFD. 14,15 Systemic Smad3 deficiency does not Fig. 8. Human NASH livers displayed activation of TGF-b signaling. IHC for phospho-smad2/3 in liver biopsy samples from patients with mild NAFLD without fibrosis (n 5 10) and samples from patients with NASH with severe fibrosis (n 5 10) was performed. (A) Representative pictures are shown. Original magnification: (B) Quantification of phospho-smad2/ 3-positive hepatocytes.

12 494 YANG ET AL. HEPATOLOGY, February 2014 modulate the gene expression associated with b- oxidation and lipolysis in skeletal muscles and the liver. The responsible site of Smad3 signaling for systemic metabolic manifestations is peripheral adipose tissue in HFD-fed mice. 15 In white adipose tissues of Smad3-deficient mice, expression of lipogenic PPAR-c, sterol regulatory element-binding protein 1c, and fatty acid synthase was suppressed, whereas expression of molecules related to b-oxidation and lipolysis, such as Acyl-coenzyme A oxidase 1, carnitine palmitoyl transferase 1, and PPAR-d, was increased. In WT adipocytes, CCAAT/enhancer-binding protein beta (C/ EBPb) interacts with histone deacetylase 1, a transcriptional repressor, which binds to PPAR-d promoter to inhibit expression of b-oxidation-related genes. 15 C/ EBPb also binds to PPAR-c to increase lipogenesis gene expression that results in adipogenesis in WT adipocytes. 15 In contrast, Smad3 deficiency increases C/ EBP homologous protein-10 expression, which interferes with interaction of C/EBPb to the promoter of PPAR-d or PPAR-c, leading to inhibition of adipogenesis. 15 Moreover, Smad3 can directly bind to the PGC-1a promoter to repress its promoter activity, contributing to mitochondrial biogenesis, including b- oxidation. 14 Alternatively, Smad3 deficiency leads to changes in phenotype of macrophages in adipose tissues from M1 to M2, suggesting that Smad3 signaling in macrophages is also important for adipogenesis. 14 In addition to previous studies, our results clearly showed that TGF-b signaling regulates lipid metabolism through induction of lipogenesis genes and suppression of b-oxidation-related genes. TGF-b signaling itself did not possess a significant effect on hepatocyte lipid metabolism. In combination with FFA challenges, TGF-b signaling modulated the gene expression involved in lipogenesis and fatty acid b-oxidation, which resulted in increased TG synthesis and lipid accumulation in hepatocytes. TGF-b signaling activates the Smad2/3/4-signaling pathway, and inhibition of these pathways abolished changes in gene expression associated with lipid metabolism. In alcoholic liver disease, TGF-b signaling increases hepatic lipid accumulation through down-regulation of alcohol dehydrogenase In conclusion, these evidences indicate that hepatocytic TGF-b signaling enhances fat deposition in hepatocytes induced by excessive fat loading. TGF-b signaling is also involved in hepatocyte death. Treatment with TGF-b alone does not cause hepatocyte death, but induces significant death in fat-accumulated hepatocytes. Inhibition of Smad2 inhibited TGF-b-induced death in fat-accumulated hepatocytes. As previously reported, inhibition of NF-jB increases susceptibility of hepatocytes to death induced by TNF-a or IL-1b. 31 Similarly, TGF-b is able to induce hepatocyte death by inhibition of NFjB or TAK1, which is further enhanced in lipidaccumulated hepatocytes. 32 Importantly, expression of TGF-b receptor type I was increased in lipidaccumulated hepatocytes (Supporting Fig. 2). Moreover, TGF-b-induced ROS production was dramatically increased in palmitate-treated hepatocytes, and inhibition of ROS suppressed TGF-b-mediated hepatocyte death (Fig. 5). These findings suggest that lipid-accumulated hepatocytes enhance TGF-bmediated ROS production, resulting in the enhancement of Smad activation and hepatocyte death. 33 In addition to the conventional intracellular signaling pathways activated by TGF-b, such as Smad- and TAK1-dependent pathways, a previous study demonstrated that p53-mediated signaling is also required for TGF-b-mediated hepatocyte death. 34 We additionally determined that Rho/ROCK signaling is an important component for TGF-b-mediated hepatocyte death in fatty liver cells. The present study suggests that TGF-b-mediated liver pathology could influence body-weight gain and systemic IR, as analyzed by GTTs and ITTs. It has been shown that systemic inflammation influences metabolic manifestations, including weight gain and IR. 35,36 Thus, liver inflammation caused by hepatocyte TGF-b-Smad signaling may influence systemic or adipose tissue inflammation that affect body weight and systemic IR. This may further contribute to increased liver steatosis and inflammation. Interestingly, whereas Tgfbr2DHEP mice are resistance to GTTs and ITTs, there are no significant differences in fasting bloodsugar levels between WT and Tgfbr2DHEP mice. This suggests that TGF-b signaling in adipose tissues is essential to regulate fasting blood-sugar levels, but hepatic IR determined by hepatic TGF-b signaling may contribute to systemic IR. In summary, the results from our study revealed that TGF-b signaling in hepatocytes contributes to lipid accumulation by deregulation of lipid metabolism and enhancement of cell death in lipid-accumulated hepatocytes, resulting in the development of hepatic steatosis, hepatocyte death, inflammation, and fibrosis. Currently, fresolimumab, a human monoclonal Ab against TGF-b, is being tested for clinical trials for idiopathic pulmonary fibrosis, renal disease, and cancer. 37,38 Our study provides evidence that targeting TGF-b signaling could be an effective therapy of NASH.

13 HEPATOLOGY, Vol. 59, No. 2, 2014 YANG ET AL. 495 References 1. Day CP. Non-alcoholic fatty liver disease: a massive problem. Clin Med 2011;11: Kneeman JM, Misdraji J, Corey KE. Secondary causes of nonalcoholic fatty liver disease. Therap Adv Gastroenterol 2012;5: Ratziu V, Bellentani S, Cortez-Pinto H, Day C, Marchesini G. A position statement on NAFLD/NASH based on the EASL 2009 special conference. J Hepatol 2010;53: Sanyal AJ, Brunt EM, Kleiner DE, Kowdley KV, Chalasani N, Lavine JE, et al. Endpoints and clinical trial design for nonalcoholic steatohepatitis. HEPATOLOGY 2011;54: Tiniakos DG, Vos MB, Brunt EM. Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol 2010;5: Loomba R, Abraham M, Unalp A, Wilson L, Lavine J, Doo E, Bass NM. Association between diabetes, family history of diabetes, and risk of nonalcoholic steatohepatitis and fibrosis. HEPATOLOGY 2012;56: Achyut BR, Yang L. Transforming growth factor-beta in the gastrointestinal and hepatic tumor microenvironment. Gastroenterology 2011; 141: Dooley S, Weng H, Mertens PR. Hypotheses on the role of transforming growth factor-beta in the onset and progression of hepatocellular carcinoma. Dig Dis 2009;27: Dooley S, ten Dijke P. TGF-beta in progression of liver disease. Cell Tissue Res 2012;347: Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115: Klein S, Mittendorfer B, Eagon JC, Patterson B, Grant L, Feirt N, et al. Gastric bypass surgery improves metabolic and hepatic abnormalities associated with nonalcoholic fatty liver disease. Gastroenterology 2006;130: Cayon A, Crespo J, Mayorga M, Guerra A, Pons-Romero F. Increased expression of Ob-Rb and its relationship with the overexpression of TGF-beta1 and the stage of fibrosis in patients with nonalcoholic steatohepatitis. Liver Int 2006;26: Lin HM, Lee JH, Yadav H, Kamaraju AK, Liu E, Zhigang D, et al. Transforming growth factor-beta/smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function. J Biol Chem 2009; 284: Yadav H, Quijano C, Kamaraju AK, Gavrilova O, Malek R, Chen W, et al. Protection from obesity and diabetes by blockade of TGF-beta/ Smad3 signaling. Cell Metab 2011;14: Tan CK, Leuenberger N, Tan MJ, Yan YW, Chen Y, Kambadur R, et al. Smad3 deficiency in mice protects against insulin resistance and obesity induced by a high-fat diet. Diabetes 2011;60: Miura K, Kodama Y, Inokuchi S, Schnabl B, Aoyama T, Ohnishi H, et al. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 2010;139: e Miura K, Yang L, van Rooijen N, Brenner DA, Ohnishi H, Seki E. TLR2 and palmitic acid cooperatively contribute to the development of nonalcoholic steatohepatitis through inflammasome activation. HEPATO- LOGY 2013;57: Miura K, Yang L, van Rooijen N, Ohnishi H, Seki E. Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2. Am J Physiol Gastrointest Liver Physiol 2012;302:G De Minicis S, Seki E, Oesterreicher C, Schnabl B, Schwabe RF, Brenner DA. Reduced nicotinamide adenine dinucleotide phosphate oxidase mediates fibrotic and inflammatory effects of leptin on hepatic stellate cells. HEPATOLOGY 2008;48: Cazanave SC, Mott JL, Elmi NA, Bronk SF, Werneburg NW, Akazawa Y, et al. JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis. J Biol Chem 2009;284: Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. HEPATOLOGY 2005;41: Permutt Z, Le TA, Peterson MR, Seki E, Brenner DA, Sirlin C, Loomba R. Correlation between liver histology and novel magnetic resonance imaging in adult patients with non-alcoholic fatty liver disease MRI accurately quantifies hepatic steatosis in NAFLD. Aliment Pharmacol Ther 2012;36: Le TA, Chen J, Changchien C, Peterson MR, Kono Y, Patton H, et al. Effect of colesevelam on liver fat quantified by magnetic resonance in nonalcoholic steatohepatitis: a randomized controlled trial. HEPATOLOGY 2012;56: Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, Schwabe RF. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med 2007;13: Ciuclan L, Ehnert S, Ilkavets I, Weng HL, Gaitantzi H, Tsukamoto H, et al. TGF-beta enhances alcohol dependent hepatocyte damage via down-regulation of alcohol dehydrogenase I. J Hepatol 2010;52: Ninomiya-Tsuji J, Kajino T, Ono K, Ohtomo T, Matsumoto M, Shiina M, et al. A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase. J Biol Chem 2003;278: Shim JH, Xiao C, Paschal AE, Bailey ST, Rao P, Hayden MS, et al. TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo. Genes Dev 2005;19: Sato S, Sanjo H, Takeda K, Ninomiya-Tsuji J, Yamamoto M, Kawai T, et al. Essential function for the kinase TAK1 in innate and adaptive immune responses. Nat Immunol 2005;6: Kita T, Hata Y, Arita R, Kawahara S, Miura M, Nakao S, et al. Role of TGF-beta in proliferative vitreoretinal diseases and ROCK as a therapeutic target. Proc Natl Acad Sci U S A 2008;105: Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGFbeta in hepatic fibrosis. Front Biosci 2002;7:d793-d Kodama Y, Taura K, Miura K, Schnabl B, Osawa Y, Brenner DA. Antiapoptotic effect of c-jun N-terminal kinase-1 through Mcl-1 stabilization in TNF-induced hepatocyte apoptosis. Gastroenterology 2009; 136: Yang L, Inokuchi S, Roh YS, Song J, Loomba R, Park EJ, Seki E. Transforming growth factor-beta signaling in hepatocytes promotes hepatic fibrosis and carcinogenesis in mice with hepatocyte-specific deletion of TAK1. Gastroenterology 2013;144: e Carnesecchi S, Deffert C, Donati Y, Basset O, Hinz B, Preynat-Seauve O, et al. A key role for NOX4 in epithelial cell death during development of lung fibrosis. Antioxid Redox Signal 2011;15: Tomita K, Teratani T, Suzuki T, Oshikawa T, Yokoyama H, Shimamura K, et al. p53/p66shc-mediated signaling contributes to the progression of non-alcoholic steatohepatitis in humans and mice. J Hepatol 2012;57: Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 2007;56: Lumeng CN, Saltiel AR. Inflammatory links between obesity and metabolic disease. J Clin Invest 2011;121: Ling H, Li X, Jha S, Wang W, Karetskaya L, Pratt B, Ledbetter S. Therapeutic role of TGF-beta-neutralizing antibody in mouse cyclosporin A nephropathy: morphologic improvement associated with functional preservation. J Am Soc Nephrol 2003;14: Nam JS, Terabe M, Mamura M, Kang MJ, Chae H, Stuelten C, et al. An anti-transforming growth factor beta antibody suppresses metastasis via cooperative effects on multiple cell compartments. Cancer Res 2008;68: