Non-proteolytic ubiquitin modification of PPARγ by Smurf1 protects the liver from steatosis

Nonalcoholic fatty liver disease (NAFLD) is characterized by abnormal accumulation of triglycerides (TG) in the liver and other metabolic syndrome symptoms, but its molecular genetic causes are not completely understood. Here, we show that mice deficient for ubiquitin ligase (E3) Smad ubiquitin regulatory factor 1 (Smurf1) spontaneously develop hepatic steatosis as they age and exhibit the exacerbated phenotype under a high-fat diet (HFD). Our data indicate that loss of Smurf1 up-regulates the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes involved in lipid synthesis and fatty acid uptake. We further show that PPARγ is a direct substrate of Smurf1-mediated non-proteolytic lysine 63 (K63)-linked ubiquitin modification that suppresses its transcriptional activity, and treatment of Smurf1-deficient mice with a PPARγ antagonist, GW9662, completely reversed the lipid accumulation in the liver. Finally, we demonstrate an inverse correlation of low SMURF1 expression to high body mass index (BMI) values in human patients, thus revealing a new role of SMURF1 in NAFLD pathogenesis.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is a chronic liver condition associated with obesity, non-insulin-dependent diabetes, and hyperglyceridemia [1].Although presenting few clinical symptoms at early stages, a subset of patients with NAFLD will progress to nonalcoholic steatohepatitis (NASH) consisting of hepatic steatosis and inflammation, which can ultimately lead to cirrhosis and even liver cancer [2].Myriad social-behavioral and genetic causes of NAFLD are now known, but the roles of peroxisome proliferatoractivated receptors (PPARs) have emerged as crucial molecular underpinnings of these metabolic imbalances and targets of several investigational drugs [3][4][5].A thorough understanding of regulatory mechanisms governing PPAR activities will undoubtedly aid in the development of much-needed treatments.
PPARs are nuclear hormone receptors that heterodimerize with retinoid X receptors to modulate metabolic transcriptional programs in response to nutritional inputs [6].Of three PPARs encoded by distinct mammalian genes, PPARα, which is highly expressed in the liver, kidney, and muscle, directs expression of a network of genes that promote utilization of fat as an energy source.PPARγ, on the other hand, is normally expressed in adipose tissues, where it activates target genes involved in fatty acid uptake, transport, and lipogenesis to promote lipid storage.In the liver, PPARγ expression is normally low but becomes drastically induced as hepatic steatosis develops [7].Reports in the literature have shown that overexpression of PPARγ promotes the accumulation of lipid droplets in the liver, whereas hepatic disruption of PPARγ improves the fatty liver condition in leptin-deficient obese mice or mice that were fed on a high-fat diet (HFD) [8,9].In adipose tissues, ligand binding was reported to induce degradation of PPARγ via the ubiquitinproteasome system, whereas small ubiquitin-like modifier (SUMO)ylation of PPARγ was shown to repress its transcriptional activity [10].However, how steatogenic activities of PPARγ are regulated in the liver remains to be determined.
Smad ubiquitin regulatory factor 1 (Smurf1) and its close relative, Smurf2, are members of homologous to E6-AP carboxyl terminus (HECT) domain-containing ubiquitin ligases (E3s), which were initially identified as negative regulators of transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways [11][12][13][14].Subsequent studies broadened the repertoire of Smurf substrates and extended their function to cell differentiation, polarity, and DNA repair [15][16][17][18].During our ongoing quest for physiological functions of Smurfs, we found abnormal accumulation of lipid droplets in the livers of 9-12-month-old Smurf1 knockout (KO) mice and other signs that phenocopy NAFLD in human patients.Here, we report that Smurf1 induces non-proteolytic ubiquitination of PPARγ and inhibits PPARγ transcriptional activity in hepatocytes, thereby acting as a critical safeguard against the development of hepatic steatosis.

NAFLD-like phenotypes associated with the loss of Smurf1
We previously reported an increased bone density phenotype in aged Smurf1KO mice that were commonly observed under mixed black Swiss × 129/SvEv (BL) and C57BL/6N (B6) genetic background [18].Further analysis revealed a conspicuous accumulation of lipid droplets in the livers of aged Smurf1KO mice that was unique to the BL background (S1 Table ).The liver sections of these mice were characterized by large, clear, sharp-bordered cytoplasmic vacuoles upon hematoxylin-eosin (HE) staining (Fig 1A).The bright red staining of frozen sections by Oil Red-O confirmed the high fat and neutral lipid content therein (Fig 1A).This phenotype was observed in 12 out of 15 male and female mice examined beyond 9 months of age, implying a 75% penetrance.Microscopic quantification of HE-stained sections reaffirmed the statistically significant increase of steatosis in the livers of Smurf1KO mice compared with that of the wild-type (WT) controls (Fig 1B).Surprisingly, this steatosis phenotype was not observed in the livers of Smurf2KO mice (Fig 1A and 1B), suggesting that it is specifically associated with disruption of the Smurf1 function.To determine which lipid fractions were increased, we carried out colorimetric assays in liver lipid extracts prepared from Smurf1KO mice at 9-12 months of age, and the results showed that the level of triglycerides (TG) increased more than 3-fold compared with that of WT or Smurf2KO mice (Fig 1C).Moreover, the levels of total cholesterol (CHO) and free fatty acids (FFAs) were also increased significantly in Smurf1KO livers (Fig 1C).Compared with WT mice, Smurf1KO mice were approximately 30% heavier in body weight, bore more white adipose tissue (WAT), and had a higher liver to body weight ratio (Fig 1D).Nevertheless, despite exhibiting ostensible steatosis, the mutant livers appeared to function normally, as indicated by aspartate transaminase (AST) and alanine transaminase (ALT) activity measurements (Fig 1E).Because the manifestation of hepatic steatosis is usually accompanied by a constellation of adverse alterations in glucose metabolism, we conducted glucose tolerance and insulin resistance tests.At the fasting state, there was not much difference in plasma glucose levels between aged (9-12 months old) WT and Smurf1KO mice that had developed steatosis; however, following intraperitoneal injection of glucose, the blood glucose level of the mutant mice showed a more dramatic flash increase of the blood glucose level within 30 minutes of injection and more than 100% accumulative gain in the area under the curve (AUC) (Fig 1F).On the other hand, after an initial dip following the insulin injection, the blood glucose level in aged mutant mice recovered more rapidly and to a higher extent than that in WT controls (Fig 1G).The AUC of the insulin resistance test of aged Smurf1KO mice was 13.5% more compared with that of WT mice.Because young Smurf1KO mice (at 4-5 months of age) that had yet to develop steatosis scored no difference from their WT counterparts in both the tests (S1 Fig), the systemic change in glucose metabolism observed in aged mutant mice was most likely associated with the steatosis.Taken together, the phenotypes of hepatic steatosis, obesesity, glucose intolerance, and insulin resistance make these aged Smurf1KO mice a good mouse model of NAFLD.

Ablation of Smurf1 exacerbates HFD-induced hepatic steatosis
In rodents, difference in genetic background has a well-known influence on the susceptibility to obesity and hepatic steatosis [19][20][21].Although the spontaneous steatosis hereto described was only observed at old age, young Smurf1KO mice of the BL background were grossly normal except for a higher body fat content compared with their age-and background-matched WT counterparts and showed no sign of steatosis (Fig 2A and 2B) when fed on normal diet (ND).Mice of this strain background are notoriously known for their resistance to HFD-induced obesity, as evident by the lack of apparent gain in body weight and ratio of fat-to-lean mass in young WT mice that were put on a HFD feeding regimen beginning at 10-12 weeks of age and continuing for 8 consecutive weeks (Fig 2A   As alluded earlier, Smurf1KO mice of the B6 background did not show accumulation of lipid droplets in the liver (S1 Table ), and they were not overweight or overtly obese either (S2A Fig) .To ascertain that the steatosis pheneotype was not a mere coincidence unique to the BL background, we carried out the HFD feeding study on WT and Smurf1KO mice of the B6 background with the same regimen as for the young BL mice.In contrast to BL mice, B6 mice gained body weight and fat content on HFD as expected, regardless of the presence of Smurf1 gene (S2A Fig, S2 Table ).However, the B6-Smurf1KO mice on HFD became ostensibly more obese (S2A

Human SMURF1 expression inversely correlates with body mass index
To address if what we learned from the Smurf1KO mice is applicable to human populations, we took the advantage of the non-tumor liver tissue data sets compiled from a cohort of 247 Chinese liver cancer patients from the Liver Cancer Institute (LCI) [22].According to the SMURF1 mRNA expression levels retrieved from the gene expression profile (GEO: GSE14520), we separated non-tumor liver tissue samples into the high SMURF1 expression (top 25%) group (n = 61) and the low SMURF1 expression (bottom 25%) group (n = 59) (Fig 2D, left panel).We then graphed the body mass index (BMI) of these 120 patients against these two groups of SMURF1 expression, and found that patients with the low SMURF1 expression have a statistically significant higher BMI (Fig 2D, right panel).It is worth noting that the average BMI of the Asian population is lower than that in the United States and European countries, and an Asian with BMI > 27.5 is considered obese [23,24].This inverse correlation was further corroborated with non-tumor liver tissue data sets from the cancer genome atlas-liver hepatocellular carcinoma (TCGA-LIHC) (Fig 2E).Because there are only 37 cases of nontumor liver samples that have linked BMI values in the TCGA data set, the median SMURF1 expression level was used as the cutoff to plot BMI values (Fig 2E).Because the BMI is widely used in clinics as a surrogate prognostic indicator for fatty liver [25,26], these results suggest that low Smurf1 expression appears to be associated with high fat accumulation in humans, as well.

Loss of Smurf1 activates the PPARγ pathway
To investigate the underlying causes of steatosis associated with Smurf1 loss, we compared hepatic gene expression profiles of 11-month-old Smurf1KO, Smurf2KO, and their respective matching WT mice from the BL background, and selected genes that showed either increased or decreased expression by a cutoff of 1.5-fold (false discover rate [FDR] <0.1).The results showed that 987 genes in the Smurf1KO livers were differentially expressed over their WT controls, whereas only 13 genes were differentially expressed in the Smurf2KO livers (Fig 3A,left panel,and S3 Table).This result is in line with the notion that Smurf1 plays a more prominent role in the liver than Smurf2.Many genes that are involved in the lipid metabolism were BMI, body mass index; BW, body weight; Fat/Lean, fat mass to lean mass; HE, hematoxylin-eosin; HFD, high-fat diet; KO, knockout; LCI, Liver Cancer Institute; Liver/BW, liver weight to body weight; ND, normal diet; ns, not significant; SF1KO, Smurf1 KO; Smurf, Smad ubiquitin regulatory factor; TG, triglyceride; TCGA-LIHC, the cancer genome atlas-liver hepatocellular carcinoma; WT, wild-type.https://doi.org/10.1371/journal.pbio.3000091.g002

Smurf1 regulates fatty acid uptake and lipid synthesis through PPARγ
PPARγ is a strong lipogenic factor essential for steatosis [7].Although our qRT-PCR analysis alluded that loss of Smurf1 has a direct causal effect on PPARγ1 up-regulation, further evidence is needed to confirm this finding.Toward this end, we silenced Smurf1 using short interfering RNA (siRNA)s in human hepatocarcinoma Hep3B cells and mouse normal hepatocyte AML12 cells.Relative to the effect by non-silencing control siRNA (siNS), knockdown by siSmurf1 significantly increased the level of PPARγ but not PPARα or PPARδ in both cell lines (Fig 4A).As expected, siSmurf2 had little effect in either of these two cell lines (Fig 4A).Because Pparγ is a direct transcriptional target of itself in a positive feedback loop [29], siRNAmediated silencing of Smurf1 drastically increased the expression level of Pparγ, but not other paralogous Ppars or their transcriptional regulatory partners retinoid x receptor (Rxr)α and Rxrβ (Fig 4B).In adipose tissues, transcription of Pparγ genes is under the control of CCAAT enhancer binding protein (CEBP)α/β [30,31]; however, we were unable to detect any increase of either Cebpα or Cebpβ mRNA by qRT-PCR (S4A Fig) , suggesting that the regulation of PPARγ by Smurf1 is by way of a C/EBPα/β-independent mechanism.In line with the low expression of PPARγ in AML12 cells, introducing siPPARγ showed little effect on the expression of PPARγ transcriptional target genes, Fabp1, Cd36, Acacb, and Apoc3, but siSmurf1 , thus confirming the direct causal relationship between Smurf1 and PPARγ.The fact that up-regulation caused by siSmurf1was particularly pronounced in Fabp1 and Cd36, two genes that are essential for fatty acid uptake [32,33], suggested a strong connection between Smurf1 and fatty acid uptake.Indeed, using 3 H-labelled palmitic acid as a tracer, we observed a 20% increase in fatty acid uptake by AML12 cells upon Smurf1 depletion (Fig 4D).We also measured lipid synthesis in AML12 cells by measuring the incorporation of 3 H-labelled acetate into lipids and found it was increased by siSmurf1 as well (Fig 4E).Once again, these two effects of Smurf1 loss were specifically mediated through PPARγ as they were reversed by siPPARγ (Fig 4D and 4E).
To further show if Smurf1 actually regulates lipid metablism in vivo, we injected fluorescent 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid (BODI-PY-FL-C16) into the peritoneal cavities of WT and Smurf1KO mice and found that the fatty acid uptake was greatly enhanced in the liver and WAT tissues but not the muscles of Smurf1KO mice compared with that of WT mice (Fig 4F).We also repeated the 3 H-labelled acetate incorporation experiment in primary hepatocytes isolated from WT and Smurf1KO mice and confirmed the enhancement effect of Smurf1 ablation on lipid synthesis (Fig 4G).
The increased body fat content in aged BL-Smurf1KO mice and HFD-fed young Smurf1KO mice from both background suggests that Smurf1 may also regulate adipogenesis.To determine if this was the case, we took advantage of an in vitro adipogenic differentiation system using 3T3-L1 pre-adipocytes.Following a 6-day differentiation protocol, both PPARγ1 and PPARγ2 as well as their target Cd36 were all induced, as shown by western blot analysis, and the induction was greatly enhanced by siSmurf1 but reversed by the double transfection of siS-murf1 and siPPARγ (S5A Fig) .In keeping with the western blot analysis results, Oil Red-O staining of these differentiated 3T3-L1 cells was also enhanced by siSmurf1 and reversed by siSmurf1 and siPPARγ double transfection (S5B Fig) .Finally, expression of a cohort of adipogenic target genes of PPARγ also followed the same pattern as influenced by siSmurf1 and siP-PARγ (S5C Fig) .Taken together, these data indicate that Smurf1 has an intrinsic role in controlling adipogenesis and lipid metabolism through PPARγ.

PPARγ is a direct substrate of Smurf1-mediated non-proteolytic ubiquitination
The WW domains of HECT E3 ligases recognize a PPxY (PY) motif that is present in the primary sequence of many of their targets [34].There is one such sequence motif in both human and mouse PPARγ but not in PPARα, which might potentially account for the lack of an effect on this closely related protein by the loss of Smurf1 (

Non-proteolytic modification by Smurf1 suppresses PPARγ-mediated transcription
PPARs recognize a consensus sequence of PPAR response element (PPRE) that consists of two AGGTCA-like sequences arranged in tandem with a single nucleotide spacer and is present in all PPAR target gene promoters [35,36].In AML12 cells, where PPARγ expression is very low,

PPARγ antagonist GW9662 protects Smurf1KO mice from hepatosteatosis
To directly test if the increased PPARγ activity and expression are responsible for steatosis associated with Smurf1 loss, we treated a group of WT and Smurf1KO mice from the BL background with the PPARγ antagonist GW9662 [37].The compound was administered by intraperitoneal injection starting at 7-9 months of age, and the treatment lasted for 2 months; in this time period, the steatosis was expected to fully develop in Smurf1KO mice.The GW9662 treatment decreased body weight of both WT and Smurf1KO mice (Fig 7A), but because the average beginning weight of Smurf1KO mice was higher, the reduction thereof was more dramatic than that of the WT controls (about 10% reduction versus about 5%).The body fat mass content in Smurf1KO mice was also significantly lowered, to an extent that was comparable to that of the untreated WT mice (Fig 7B).Commensurate to the systemic reduction in obesity, the lipid droplets were essentially cleared from Smurf1KO livers by GW9662 (Fig 7B and 7C).Although the GW9662 treatment caused no significant change in the serum TG and CHO levels (Fig 7D ), hepatic contents of TG, CHO, and FFA were all reduced to normal levels (Fig 7E ) and so was hepatic expression of Pparγ2, as well as several PPARγ target genes (Fig 7F).These results unequivocally demonstrated that the elevated PPARγ activity and expression account for the NAFLD phenotypes observed in Smurf1KO mice.

Discussion
PPARγ is a nuclear hormone receptor with principle functions of increasing insulin sensitivity and promoting lipid storage in adipose tissues [6].In the liver, the physiological function of PPARγ is less clear, although its expression is associated with injury-induced activation of hepatostellate cells and provides an anti-fibrogenic protection [3,5].PPARγ up-regulation is also a known property of steatotic livers, and liver-specific disruption of PPARγ was reported to protect leptin-deficient mice or HFD-fed mice from developing fatty liver [7][8][9].Here, we show that mice deficient for HECT-domain E3 ligase Smurf1 in the mixed BL genetic background develop hepatosteatosis spontaneously as they age or are more susceptible to HFDinduced hepatosteatosis.These mutant mice are overweight and obese, as well as glucose intolerant and insulin resistant.These NAFLD phenotypes can be attributed to the heightened transcriptional activity of PPARγ, which in turn increases the expression of itself and genes involved in lipogenesis and fatty acid transport via a positive feedback loop.We further demonstrate that Smurf1 catalyzes the K63-linked non-proteolytic ubiquitination that normally attenuates PPARγ transcriptional activity, and show an inverse correlation between low  Different HECT E3 ligases are known to catalyze ubiquitination with different ubiquitin chains that mark modified protein substrates for different fates [38].Members of the neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) family E3 ligases preferentially support monoubiquitin modification or K63-linked chains associated with nonproteolytic functions, but can also assemble lysine 48 (K48)-linked chains that target proteins for proteasome-mediated degradation [39].As members of this E3 ligase family, Smurf1 and Smurf2 have been shown to target many proteins for K48-linked ubiquitination and degradation [16].Smurf2 was also shown to induce multi-monoubiquitin modification of Smad3, thereby inhibiting Smad3 activity [40], but the K63-linked ubiquitination by Smurfs has not been reported in mammalian species.Recently, NEDD4 itself was shown to induce both K48and K63-linked ubiquitination of PPARγ in adipocytes, with different functional outcomes [41].In our study, Smurf1 inhibits PPARγ activity, and deletion of Smurf1 enhances PPARγ activity and up-regulates PPARγ levels through a positive feedback mechanism.In contrast, NEDD4 was shown to stabilize PPARγ, and knockdown of NEDD4 reduced PPARγ expression [41].Moreover, the PY motif in PPARγ played a role in mediating interaction with Smurf1, but it was not demonstrated for NEDD4 as such.Perhaps these apparent discrepancies reflect the differences in experimental conditions conducted in different cell types, or the mixed linkages in ubiquitin chains formed by NEDD4 could have compounded the functions of modified PPARγ.In any event, the steatosis observed in Smurf1KO mice is consistent with the heightened PPARγ activity in the liver.Despite the conspicuous steatosis, overweight, and obesity that were present in 75% aged BL-Smurf1KO mice, their liver functions were nevertheless normal.Because these animals were well shielded from inflammatory insults by their accommodative housing facility, it is likely that the elevated PPARγ activity unleashed by the loss of Smurf1 was only sufficient to manifest a restricted impact in bringing about the earlystage NAFLD phenotypes.Future studies are necessary to ascertain the tissue origin of the steatogenic effect of Smurf1 ablation using conditional knockout approaches and to determine if and how BL-Smurf1 mice could be enticed to progress through NASH or even liver cancer to model the entire NAFLD disease spectrum.Regulation of PPARγ by the Smurf1-mediated K63-linked ubiquitin modification centers on its transcriptional activity.Because PPARγ is also a transcriptional target of its own, a disturbance of Smurf1 would create an "all or none" effect: a rise or fall of Smurf1 across a threshold level would either maximize or minimize PPARγ activity.This scenario may normally operate to keep the lipogenic activity of PPARγ to a minimum in the liver but maximized in the adipose tissues.
Epidemiology studies indicate that an estimated 27%-34% of the general population within North America have NAFLD [42], for which there is no approved treatment available at present.Current NAFLD drug developmental effort centers on repurposing fibric acid derivatives, which are lipid-lowering PPARα agonists and insulin sensitivity-improving PPARγ agonists, thiazolidinediones, but the clinical trials yielded mixed results [3,4].Because of the opposite actions of PPARα and PPARγ on hepatic steatosis, the "spillover" effects of these PPAR agonists might prevent a net gain in their ability to reduce TG accumulation in the liver.As to metabolism genes in the livers of GW9662-treated mice.Data are presented as mean ± SD; statistical significance of difference is indicated by � p < 0.05, �� p < 0.01, ��� p < 0.001.Original raw data can be found in S1 Data.BL, mixed black Swiss × 129/SvEv background; BW, body weight; CHO, total cholesterol; Ctrl, control; Fat/Lean, fat mass to lean mass; FFA, free fatty acid; GW, GW9662; HE, hematoxylin-eosin; KO, knockout; Liver/BW, liver weight to BW; ns, not significant; PPAR, peroxisome proliferator-activated receptor; qRT-PCR, quantitative real-time PCR; SF1KO, Smurf1 KO; Smurf, Smad ubiquitin regulatory factor; TG, triglyceride; WT, wild-type.https://doi.org/10.1371/journal.pbio.3000091.g007 PPARγ agonists, although clinical trials for rosiglitazone in patients with type 2 diabetes reported improvement of steatosis by a median of 20% during the first year, no further improvement was found after 2 additional years of treatment, and the trials exposed severe cardiovascular risks and weight gain [43].Intuitively, it is possible that the benefit is derived from the systemic lipid clearance by increased fat storage in adipose tissue, because PPARγ is normally expressed in adipose tissues, and its activation in the liver was clearly linked to fatty liver formation.Given our current finding of Smurf1 in protecting the liver from steatosis, a viable strategy to treat NAFLD may be to curtail the transcriptional activity of PPARγ by turning on Smurf1-mediated non-proteolytic ubiquitin modification.

Ethics statement
All mice were maintained and handled under protocols (LCMB-014, ASP 10-214, 13-214, 16-214) approved by the Animal Care and Use Committee of the National Cancer Institute, National Institutes of Health (NIH), according to NIH guidelines.

Animals and treatment
Generation of Smurf1KO and Smurf2KO mice in the mixed BL and pure C57BL/6N (B6) background was described previously [18,40].For spontaneous hepatosteosis development, animals were maintained on a ND, monitored weekly, and euthanized and necropsied at 9-12 months of age.For the HFD treatment, male mice were maintained on a ND until 10-12 weeks of age before they were given HFD (Research Diets, Cat# D12266B) containing 16.8% kcal protein, 31.8%kcal fat, and 51.4% kcal carbohydrate for 8 weeks.For the GW9662 treatment, a dose of 1 mg/kg of GW9662 dissolved in DMSO was injected intraperitoneally (i.p.) into 7-9-month-old BL-WT and BL-Smurf1KO mice for 5 days per week for 2 months.Age and sex of mice used in these studies are listed in S1 Table.

Measurement of body composition and histology
Body composition was determined using an EchoMRI mouse scanner (EchoMRI, Houston, TX).Mouse liver and epididymal fat pad were dissected, weighed, then either snap-frozen in liquid N 2 or fixed in 10% neutral buffered formalin prior to paraffin embedding.Frozen liver tissues were used for Oil Red-O staining.Liver and fat tissue histology were read by board-certified veterinary pathologists in the Pathology and Histotechnology Laboratory of the Frederick National Laboratory for Cancer Research.

Blood and liver biochemical analysis
Serum TG, CHO, and albumin concentrations, as well as ALT and AST activities were measured by standard methods with a Vitro 250 dry slide analyzer (Ortho Clinical Diagnostics) in the Pathology and Histotechnology Laboratory of the Frederick National Laboratory for Cancer Research.Liver TG, CHO, and FFA concentrations were determined using the EnzyChrom TG, CHO, and FFA assay kits (Bioassay Systems) after extracting total lipids from 50-mg liver tissues as described [44].

Glucose tolerance test and insulin tolerance test
To perform the gluclose tolerance test (GTT) or insulin tolerance test (ITT), mice were fasted overnight before receiving an i.p. injection of 20% glucose (2 g/kg body weight) or recombinant insulin (Humulin R, 0.75 U/kg; Lily), respectively.Blood samples were collected from the

RNA extraction and qRT-PCR
Total RNA from AML12 cells or liver tissues was extracted by RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.High Capacity Reverse Transcription Kit (ABI, Life Tech) was used to generate cDNA from RNA (500-2,000 ng).qRT-PCR was performed with Power SYBR Green PCR Master Mix (Life Technologies) using specific oligonucleotide primers as specified (S4 Table ).

ChIP and luciferase reporter assays
ChIP assays were carried out with an EZ-ChIP Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions.Immunoprecipitations were carried out using anti-PPARγ antibody (Abcam, A3409A) and an isotype-matched IgG as the control.Reporter assays were performed in 12-well plates using PPRE-Luc (0.5 μg) and pRL-TK (0.2 μg) reporter plasmids, and the luciferase activities were determined using Dual Luciferase Reporter Assay System (Promega).

Microarray, KEGG pathway, and metabolomic profile analysis
Microarray experiments for mouse liver tissues were performed on Affymetrix GeneChip Mouse Gene 1.0 ST arrays according to the standard Affymetrix GeneChip protocol at the Affymetrix service core in the Frederick National Laboratory for Cancer Research.The raw array data were then analyzed with packages oligo and lima under R platform, as described before [50,51], to identify differentially expressed genes among groups (fold > 1.5, FDR cutoff = 0.1), and results were visualized using VennDiagram (https://cran.r-project.org/web/packages/VennDiagram) and gplots (https://cran.r-project.org/web/packages/gplots)under R platform.Data were submitted to GEO (accession number GSE113995).KEGG pathway analysis was performed by gage package, as described [52], to identify significantly enriched pathway (FDR q-value cutoff = 0.1) between Smurf1KO and WT liver samples.
The microarray analysis for human liver tissues from the LCI cohort of 247 Chinese patients was previously published [22] and data are accessible through GEO (accession number GSE14520).TCGA non-tumor liver tissue gene expression data were downloaded from TCGA-LIHC (https://portal.gdc.cancer.gov).

Statistical analysis
Unless indicated in the figure legends, two-tailed Student t test was used for statistical analysis.

Fig 2 .
Fig 2. Inverse correlation of low Smurf1 expression to high fat accumulation.(A-C) Loss of Smurf1 exacerbates HFD-induced steatosis in mice.(A) BW, Fat/Lean ratio, Liver/BW ratio, and histological scores of steatosis in male WT and SF1KO mice from BL background reared on either a ND (n = 7 per group) or HFD (n = 8 per group), beginning at 10-12 weeks of age for 8 weeks.Liver sections were scored as in Fig 1B.(B) HE staining of representative liver sections of the above BL mice at the end of diet treatment.Bar = 100 μm.(C) Liver TG levels of BL mice from (A).Data are presented as mean ± SD; statistical significance of differences is indicated as � p < 0.05, �� p < 0.01, ��� p < 0.001.Original raw data can be found in S1 Data.(D-E) Low SMURF1 expression in human livers is inversely correlated to high BMI values.(D) Human liver tissues from the LCI cohort with low levels of SMURF1 expression show a significantly higher BMI than those with high SMURF1 expression.Non-tumor liver samples from the LCI cohort were grouped into SMURF1 High (top 25%, n = 61) and SMURF1 Low (bottom 25%, n = 59) groups.(E) Inverse correlation between SMURF1 low and BMI high was also observed in non-tumor liver samples from the TCGA-LIHC cohort.Non-tumor liver samples (n = 37) from the TCGA-LIHC cohort were grouped into SMURF1 High (above the median, n = 18) and SMURF1 Low (below the median, n = 19) groups.Data are presented using box and whisker plot; centerline represents the median, whiskers represent 10th-90th percentile.Nonparametic t tests between the two groups were performed; p-values are indicated in the graph.BL, mixed black Swiss × 129/SvEv background; Fig) and showed more severe lipid droplet accumulation in the liver compared to WT mice of the same background (S2A and S2B Fig).In addition, the increase in the liver TG content was also more pronounced (S2C Fig).Thus, the steatosis associated with Smurf1 loss is likely the result of an overall gain in body fat content in both strain backgrounds, suggesting that Smurf1 may have a systemic role in regulating lipid metabolism.

Fig 3 .
Fig 3. Up-regulation of PPARγ and other relevant lipid metabolic pathways associated with Smurf1 loss.(A) Venn diagram of genes that were differentially expressed (fold > 1.5, FDR < 0.1) in Smurf1KO and Smurf2KO livers from the BL background relative to their WT counterparts at 11 months of age.For a detailed list, see S3 Table.(B)Heat map of a group of lipid metabolism-related genes that were differentially expressed in Smurf1KO livers and the KEGG pathway analysis of differentially expressed genes from BL-Smurf1KO livers.Enrichment score (p-value (−log10)) is indicated on the x-axis.(C) qRT-PCR analyses of Pparγ1, Pparγ2, and Pparα in livers from BL-WT, Smurf1KO, and Smurf2KO mice (n = 7 per group).(D) Western blot showing the increase of the PPARγ protein in livers of BL-Smurf1KO mice.Quantitation of PPARγ expression against loading control Hsc70 is shown at the bottom.(E) qRT-PCR analyses showing the increase of total Pparγ mRNA in livers and epididymal WAT but not skeletal muscle of BL-Smurf1KO mice, n = 7 per group.(F) qRT-PCR analyses showing up-regulation of a group of PPARγ target genes in livers of BL-Smurf1KO mice (n = 7 per group).Data from qRT-PCR are presented using box and whisker plot showing all points; centerline represents the median.All mice were analyzed at 9-11 months of age, and statistical significance of difference between WT and Smurf1KO is indicated as � p < 0.05, �� p < 0.01, and ��� p < 0.001.Original raw data can be found in S1 Data.BL, mixed black Swiss × 129/SvEv background; FDR, false discover rate; Hsc70, heat shock 70 kDa protein 8; KEGG, Kyoto Fig 3. Up-regulation of PPARγ and other relevant lipid metabolic pathways associated with Smurf1 loss.(A) Venn diagram of genes that were differentially expressed (fold > 1.5, FDR < 0.1) in Smurf1KO and Smurf2KO livers from the BL background relative to their WT counterparts at 11 months of age.For a detailed list, see S3 Table.(B)Heat map of a group of lipid metabolism-related genes that were differentially expressed in Smurf1KO livers and the KEGG pathway analysis of differentially expressed genes from BL-Smurf1KO livers.Enrichment score (p-value (−log10)) is indicated on the x-axis.(C) qRT-PCR analyses of Pparγ1, Pparγ2, and Pparα in livers from BL-WT, Smurf1KO, and Smurf2KO mice (n = 7 per group).(D) Western blot showing the increase of the PPARγ protein in livers of BL-Smurf1KO mice.Quantitation of PPARγ expression against loading control Hsc70 is shown at the bottom.(E) qRT-PCR analyses showing the increase of total Pparγ mRNA in livers and epididymal WAT but not skeletal muscle of BL-Smurf1KO mice, n = 7 per group.(F) qRT-PCR analyses showing up-regulation of a group of PPARγ target genes in livers of BL-Smurf1KO mice (n = 7 per group).Data from qRT-PCR are presented using box and whisker plot showing all points; centerline represents the median.All mice were analyzed at 9-11 months of age, and statistical significance of difference between WT and Smurf1KO is indicated as � p < 0.05, �� p < 0.01, and ��� p < 0.001.Original raw data can be found in S1 Data.BL, mixed black Swiss × 129/SvEv background; FDR, false discover rate; Hsc70, heat shock 70 kDa protein 8; KEGG, Kyoto

Fig 4 .
Fig 4. Smurf1 regulates fatty acid uptake and lipid synthesis through PPARγ.(A) Western blots showing that knockdown of Smurf1 but not Smurf2 in Hep3B and AML12 cells increased PPARγ protein level.(B) qRT-PCR analyses showing that knockdown of Smurf1 but not Smurf2 increased Pparγ mRNA level in AML12 cells.(C) qRT-PCR analyses showing that knockdown of Smurf1 in AML12 cells increased expression of Fabp1, Cd36, Acacb, and Apoc3 in a PPARγ-dependent manner.(D) Fatty acid uptake in AML12 cells as measured by 3 H-palmitate incorporation (n = 3).(E) Lipid synthesis in AML12 cells as measured by incorporation of 3 Hacetate into lipid (n = 3).(F) In vivo fatty acid uptake after intraperitoneal injection of BODIPY-FL-C16.The BODIPY-FL-C16 accumulation in the liver, epididymal WAT, and skeletal muscle was normalized to tissue weight (n = 8 per group).(G) Lipogenesis in primary hepatocytes as measured by the incorporation of 3 H-acetate into lipid (n = 6 per group).Data are presented as mean ± SD; statistical significance of difference is indicated as � p < 0.05, �� p < 0.01, ��� p < 0.001.Original raw data can be found in S1 Data.BODIPY-FL-C16, 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid; eWAT, epididymal WAT; HSC70, heat shock cognate 71 kDa protein; NS, non-silencing control; PPAR, peroxisome proliferator-activated receptor; qRT-PCR, quantitative real-time PCR; RFU, relative fluorescence units; Rxr, retinoid x receptor; SF1KO, Smurf1 KO; siNS, non-silencing control siRNA; Smurf, Smad ubiquitin regulatory factor; WAT, white adipose tissue; WT, wild-type.https://doi.org/10.1371/journal.pbio.3000091.g004 Fig 3C).By co-immunoprecipitation experiments, we found that endogenous Smurf1 interacted specifically with PPARγ in the AML12 cells (Fig 5A), and the PY motif of PPARγ contributed to the interaction, because removing it considerably weakened the interaction between Myc-tagged Smurf1 and FLAGtagged PPARγ, as assayed in transiently transfected AML12 cells (Fig 5B).Also in AML12 cells, Smurf1 but not Smurf2 showed the propensity to ubiquitinate both PPARγ1 and PPARγ2 isoforms (Fig 5C).The substrate and enzyme relationship was further demonstrated in Smurf1KO mouse embryonic fibroblasts (MEFs), in which exogenous Smurf1 but not the catalytically inactive Smurf1 C699A (CA) mutant ubiquitinated PPARγ (Fig 5D), as well as in a reconstituted in vitro reaction with recombinant Smurf1 and PPARγ (Fig 5E).Finally, the ubiquitin chain of the modified PPARγ is likely of the K63 linkage, as only the ubiquitin mutant with a single lysine residue at the amino acid residue position 63 supported the polyubiquitination of PPARγ in the reconstituted in vitro reaction, whereas other single-lysine ubiquitin mutants with lysine at other positions did not (Fig 5F).In light of this result and the fact that co-expressing Smurf1 with PPARγ did not alter the stability of the latter (Fig 5B and 5C), we concluded that Smurf1 mediates a non-proteolytic ubiquitin modification of PPARγ.

Fig 7 .
Fig 7. Treatment with PPAR antagonist GW9662 protects BL-Smurf1KO mice from steatosis.(A) BW of male mice from the BL background before (age 7-9 months) and after 60 days (age 9-11 months) of GW9662 treatment (n = 8 per group).(B) Fat/Lean and Liver/BW ratios and histological score of steatosis of the above mice after GW9662 treatment, compared with those of untreated control mice at the same age (n = 8 per group).(C) Representative HE-stained sections of the above mice.Bar = 100 μm.(D) Serum TG and CHO contents of the above mice, n = 8 per group.(E) Liver TG, CHO, and FFA contents of the above GW9662-treated mice.(F) qRT-PCR analysis of expression of Pparγ2 and relevant lipid , S2 Table).In contrast, HFD feeding significantly increased fat content in the Smurf1KO mice (Fig 2A, S2 Table).Despite a lack of significant weight gain, HFD feeding did cause mild steatosis (Fig 2A and 2B), as well as an