GOLM1 promotes cholesterol gallstone formation via ABCG5-mediated cholesterol efflux in metabolic dysfunction-associated steatohepatitis livers
Article information
See the commentary-article "Bridging the gap: The GOLM1-OPN-ABCG5 axis in MASH and gallstone disease: Editorial on “GOLM1 promotes cholesterol gallstone formation via ABCG5-mediated cholesterol efflux in MASH livers”" on page 631.
Abstract
Background/Aims
Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation.
Methods
The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation.
Results
MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs.
Conclusions
In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.
Graphical Abstract
INTRODUCTION
Gallstones are a common digestive system disorder affecting 10–20% of adults worldwide, and more than 90% of gallstones are cholesterol gallstones (CGSs) [1]. CGS formation is caused by various factors, including imbalances in hepatic cholesterol metabolism and disruptions in gallbladder function [1]. Specifically, disturbances in hepatic cholesterol metabolism play a critical role in CGS formation [2]. Maintaining cholesterol homeostasis is highly important for preventing CGSs [3]. Metabolic dysfunction-associated steatohepatitis (MASH), the most prevalent chronic liver disease [4], is a severe stage of metabolic dysfunction-associated steatotic liver disease (MASLD) [4]. MASH is characterized by liver inflammation and metabolic disturbance and is strongly correlated with diverse metabolic disorders, including CGSs [5-8]. Furthermore, MASH is an independent risk factor for CGS formation [8,9]. However, the mechanisms linking MASH to CGSs are not fully understood.
Golgi membrane protein 1 (GOLM1) is expressed at low levels in healthy livers but is significantly upregulated in MASH livers [10]. Recent studies suggest that GOLM1 is associated with liver inflammation and fibrosis, both of which are characteristics of MASH [11,12]. Furthermore, GOLM1 expression is regulated by inflammatory cytokines, including interleukin-1β (IL-1β), which play pivotal roles in MASH development [13,14]. However, whether GOLM1 is aberrantly expressed in MASH livers and the specific mechanism of its expression remain unclear. In addition, our recent study demonstrated the involvement of GOLM1 in cholesterol metabolism [15]. GOLM1 knockdown leads to the intracellular accumulation of cholesteryl esters, indicating a potential role for GOLM1 in cholesterol efflux [16]. These findings suggest that GOLM1 may play a crucial role in gallstone formation by modulating cholesterol metabolism. Thus, we hypothesized that GOLM1 might serve as the molecular link between MASH and CGSs.
In this study, we found that GOLM1 knockout (GOLM1-/-) inhibited MASH-related CGS formation. Mechanistically, nuclear GOLM1 upregulated ATP binding cassette transporter subfamily G member 5 (ABCG5) in an osteopontin (OPN)-dependent manner, leading to biliary cholesterol supersaturation. GOLM1 upregulation required the involvement of the inflammatory factor IL-1β. Our study clarified the mechanistic relationship between MASH and CGS formation and explored the potential role of GOLM1 in MASH-related CGSs.
MATERIALS AND METHODS
Mouse models
Eight-week-old male GOLM1-/- mice (strain no. T012652) and their wild-type (WT) littermates were purchased from GemPharmatech (Nanjing, China). All the mice were maintained on a 12-h light‒12-h dark cycle at 22°C and fed either a chow diet (CD) (Dyets, AIN93G) or a high-fat diet (HFD) (Dyets, ASHF4, containing 40% kcal fat) for 15 weeks. Blood was extracted from the mice and centrifuged to collect the serum. Bile and liver samples were collected for subsequent analyses. Primary hepatocytes were isolated from the mice via a previously described method [17]. The animal experimental protocols used were approved by the Department of Laboratory Animal Science of Fudan University (No. 2020-HSYY-JS-326).
Cell lines
The human hepatoma cell line HepG2 and the human embryonic kidney cell line HEK293T were purchased from the Cell Bank of the Chinese Academy of Sciences. All the cell lines were cultured in complete media supplemented with 10% FBS at 37°C in 5% CO2.
Biochemical analysis
Bile collection and preparation were carried out according to the previous methods [18]. Cholesterol, bile acid, and phosphatidylcholine were quantified using different kits according to the manufacturer’s instructions. The cholesterol saturation index (CSI) was calculated using Carey’s critical tables [19]. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and bilirubin levels were measured with kits according to the manufacturer’s instructions.
Data source and study population
The data of the prospective cohort were collected from the UK Biobank (UKB). From 2006 to 2010, the UKB study recruited more than 500,000 participants aged 40–69 years. In this study, 297,397 participants were enrolled for further analysis after excluding participants with baseline cholelithiasis, unavailable baseline data, and record errors. MASLD was defined by having the International Classification of Diseases-10th revision codes K75.8 and K76 [20]. The NAFLD Fibrosis Score (NFS) was used to identify participants with MASH among MASLD participants [21]. All studies obtained approval from the relevant ethics committees, and all participants provided informed consent via electronic signatures at the baseline assessment. The present study was conducted using the UKB resource under application number 91660.
Human samples
Liver samples were obtained from gallstone patients or benign liver lesion patients who underwent surgical resection at the General Surgery Department of Huashan Hospital, Fudan University. Liver samples from patients diagnosed with MASH according to the nonalcoholic fatty liver disease activity score (NAS) were included in the study [22]. The NAS was determined by two proficient liver pathologists blinded to the clinical data. Gallbladder stones were categorized as CGSs on the basis of their morphological features and biochemical analysis results (cholesterol content, ≥ 50% dry weight) [23]. The participants were subsequently divided into a CGS group (n=32) and a gallstone-free (GSF) group (n=10). The study protocol was approved by the Ethics Committee of Huashan Hospital, Fudan University (No. KY2021-048) and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Each patient provided written informed consent.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). The data are shown as the mean±standard deviation or mean±standard error of the mean. Differences among groups were assessed via Student’s t-test or ANOVA test. Correlations were calculated via Pearson’s correlation coefficient. Details of the statistical comparisons are provided in the figure legends.
The detailed methodology is described in the Supplementary Methods.
RESULTS
GOLM1 participates in MASH-related CGS formation
First, we extracted data from the UKB to clarify the relationship between MASH and cholelithiasis (Fig. 1A, Supplementary Table 1). The incidence of cholelithiasis in MASH patients was greater than that in simple steatosis (SS) patients or healthy participants (Fig. 1A). Moreover, Cox analysis revealed that MASH was significantly associated with the onset of cholelithiasis (Fig. 1A). After adjustment for sex and impaired fasting glucose/diabetes mellitus (IFG/DM), the association between MASH and the onset of cholelithiasis remained significant (Supplementary Fig. 1).
GOLM1 participates in MASH-related CGS formation. (A) Left: Flow chart of the study using UK Biobank data. Middle: Cholelithiasis prevalence in healthy participants (4.3%, n=285,447), SS patients (17.5%, n=11,368), and MASH patients (19.3%, n=576). Right: Independent associations of age, sex, MASLD, MASH, SS, BMI, and IFG/DM with incident cholelithiasis. (B) Left: Schematic diagram of MASH mouse model establishment using GOLM1-/- and WT mice. Both GOLM1-/- and WT mice were randomly divided into 2 groups and fed a CD or HFD. n=12 per group at each time point. Middle left: Weight changes in the four groups. n=12 per group. Middle right: HE and Masson staining of representative livers from the four groups. Scale bar, 50 μm. Right: NASs of the four groups. n=12 per group. (C) Left and middle: Gross appearance of representative gallbladders and polarizing light microscopy examination of cholesterol crystals in the bile of the four groups. Scale bar, 50 μm. Right: Gallstone incidence in the four groups. n=12 per group. (D) IHC analysis of GOLM1 in liver tissues from the four groups. n=12 per group. (E) HE staining, Masson staining, and GOLM1 IHC staining of livers from HFD-fed mice at different time points. n=12 per group at each time point. (F) Left: GOLM1 IHC staining of livers from CD- and HFD-fed mice at different time points. Right: Gallstone incidence in normal, SS, and MASH mice. n=12 per group at each time point. ns, nonsignificant; **P<0.01, ***P<0.005. BMI, body mass index; CD, chow diet; CGS, cholesterol gallstone; GOLM1, Golgi membrane protein 1; GOLM1-/-, GOLM1 knockout; HE, hematoxylin-eosin; HFD, high-fat diet; IFG/DM, impaired fasting glucose/diabetes mellitus; IHC, immunohistochemistry; MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic dysfunction-associated steatohepatitis; NAS, nonalcoholic fatty liver disease activity score; SS, simple steatosis; WT, wild-type.
The mice were subsequently fed a HFD to establish the MASH model (Fig. 1B). Compared with CD-fed mice, HFD-fed mice presented increased body weights, liver weights, and liver-to-body weight ratios, accompanied by increased hepatic TG and TC levels (Fig. 1B, Supplementary Fig. 2A, B). The fasting blood glucose (FBG) and fasting insulin (FINS) levels were increased in HFD-fed mice (Supplementary Fig. 2C). The levels of hepatic inflammatory cytokines, including IL-1β and interleukin-6 (IL-6), were elevated in HFD-fed mice (Supplementary Fig. 2D). We then performed hematoxylin-eosin (HE) and Masson staining on mouse livers and determined the NAS, confirming the occurrence of MASH in all HFD-fed mice (Fig. 1B). Among the WT mice, 75.0% of the MASH mice developed gallstones, whereas none of the normal mice developed gallstones (Fig. 1C). Immunohistochemistry (IHC) revealed increased staining of hepatic GOLM1 in WT mice with MASH compared with that in normal WT mice (Fig. 1D).
To determine the causative role of GOLM1 in MASH-related CGS formation, we fed GOLM1-/- and WT mice a HFD. After 15 weeks of HFD feeding, body weight, liver weight, and hepatic inflammatory cytokine levels did not differ between GOLM1-/- and WT mice (Supplementary Fig. 2A, D). The histological analysis and quantification of hepatic TG and TC levels revealed that the severity of MASH was comparable between the two groups (Fig. 1B, Supplementary Fig. 2B). GOLM1-/- had no effect on FBG or FINS levels (Supplementary Fig. 2C). Notably, the incidence of CGSs in HFD-fed GOLM1-/- mice was 16.7%, whereas it was 75.0% in HFD-fed WT littermates (Fig. 1C). Next, we performed analyses to assess hepatic GOLM1 levels and the incidence of CGSs during MASH progression in WT mice (Fig. 1E, Supplementary Fig. 3). No gallstones were detected in the SS mice, and no difference in the hepatic GOLM1 level was detected between the SS and normal mice (Fig. 1E, F). However, there was a significant increase in the hepatic GOLM1 level and CGS incidence in the mice that developed MASH (Fig. 1F). These results suggest that GOLM1 participates in MASH-related CGS formation.
GOLM1 fosters cholesterol efflux to promote CGS formation
Cholesterol supersaturation is an essential factor that promotes the nucleation of cholesterol crystals [24]. To clarify how GOLM1 affects gallstone formation, we fed mice a CD or HFD for 15 weeks and collected bile samples to analyze the levels of biliary lipids (e.g., cholesterol, phospholipids, and bile acid), total lipid contents, and biliary CSI. Biliary cholesterol was lower in HFD-fed GOLM1-/- mice than in HFD-fed WT mice (Fig. 2A). However, no significant differences in biliary phospholipid, bile acid or total lipid contents were observed between the two groups (Fig. 2A). Consistently, the biliary CSI was lower in HFD-fed GOLM1-/- mice than in HFD-fed WT mice (Fig. 2A). Moreover, there was no statistically significant difference in the serum TG, TC, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, or bilirubin levels between the HFD-fed GOLM1-/- mice and the HFD-fed WT mice (Supplementary Fig. 4). These results indicate that GOLM1 increases the biliary cholesterol content and biliary CSI to promote CGS formation.
GOLM1 fosters cholesterol efflux to promote CGS formation. (A) Concentrations of biliary cholesterol, phospholipids, bile acids, and total lipid contents and CSI scores in the four groups of mice. n=12 per group. (B) qRT-PCR analysis and IHC staining of hepatic ABCG5 in the four groups of mice. n=12 per group. Scale bar, 100 μm. (C) Bile flow rates and biliary lipid outputs of the mice in the four groups. n=12 per group. (D) GOLM1 and ABCG5 protein levels and supernatant cholesterol concentrations in HepG2-shGOLM1-1/-2 and HepG2-GOLM1 cells. ns, nonsignificant; *P<0.05, **P<0.01, ***P<0.005. After 15 weeks of CD or HFD feeding, the mice were sacrificed, and samples were collected. ABCG5, ATP binding cassette transporter subfamily G member 5; CD, chow diet; CGS, cholesterol gallstone; CSI, cholesterol saturation index; GOLM1, Golgi membrane protein 1; GOLM1-/-, GOLM1 knockout; HFD, high-fat diet; HepG2-GOLM1 cells, GOLM1-overexpressing HepG2 cells; HepG2-shGOLM1 cells, GOLM1-knockdown HepG2 cells; IHC, immunohistochemistry; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild-type.
Since biliary cholesterol levels are affected mainly by hepatic cholesterol metabolism [25], we explored the effect of GOLM1 on this metabolic process. Hepatic mRNA expression of cholesterol metabolism-related genes was measured (Fig. 2B, Supplementary Fig. 5A). GOLM1-/- led to robust downregulation of hepatic ABCG5 but had no effect on the expression of hepatic ABCG8 (Fig. 2B, Supplementary Fig. 5B). As ABCG5 accounts for approximately 82% of hepatobiliary cholesterol efflux [26], we examined biliary lipid output in vivo. The biliary cholesterol output was markedly decreased in the GOLM1-/- mice (Fig. 2C). Consistent with these findings, ABCG5 was downregulated in GOLM1-knockdown HepG2 (HepG2-shGOLM1) cells and primary hepatocytes (H-shGOLM1) and upregulated in GOLM1-overexpressing HepG2 (HepG2-GOLM1) cells and primary hepatocytes (H-GOLM1) (Fig. 2D, Supplementary Fig. 6A). Moreover, GOLM1 knockdown reduced cholesterol levels in the cell supernatant, whereas GOLM1 overexpression had the opposite effect (Fig. 2D, Supplementary Fig. 6C). These data reveal that GOLM1 promotes cholesterol efflux through ABCG5.
GOLM1 promotes cholesterol efflux through upregulating OPN
To explore the mechanism by which GOLM1 regulates ABCG5, we conducted differential gene expression analysis on human livers in high- and low-GOLM1 groups according to the Genotype Tissue Expression (GTEx) database. OPN, which is involved in cholesterol metabolism and CGS formation [27], was differentially expressed between the two groups (Fig. 3A). Moreover, there was a significant positive relationship between GOLM1 and OPN expression (Fig. 3A). In vivo, GOLM1-/- decreased OPN expression (Fig. 3B). Additionally, OPN was downregulated in HepG2-shGOLM1 and H-shGOLM1 cells and upregulated in HepG2-GOLM1 and H-GOLM1 cells (Fig. 3C, Supplementary Fig. 6A, and Supplementary Fig. 7A, B). Furthermore, we constructed OPN-knockdown HepG2 (HepG2-shOPN) cells and primary hepatocytes (H-shOPN) to determine the effect of OPN on ABCG5. OPN knockdown decreased the expression of ABCG5 and reduced the cholesterol level in the cell supernatant (Fig. 3D, Supplementary Fig. 6B, D, and Supplementary Fig. 7C). Accordingly, upregulated ABCG5 expression and increased cholesterol levels in the cell supernatant were observed in OPN-overexpressing HepG2 (HepG2-OPN) cells and primary hepatocytes (H-OPN) (Fig. 3D, Supplementary Fig. 6B, D, and Supplementary Fig. 7D). However, the expression of GOLM1 was unaffected by OPN overexpression (Fig. 3D, Supplementary Fig. 6B, and Supplementary Fig. 7C, D). Furthermore, the decreases in ABCG5 and cholesterol in the supernatant induced by GOLM1 knockdown were reversed by OPN overexpression, and the increases in ABCG5 and cholesterol in the supernatant caused by GOLM1 overexpression were reversed by OPN knockdown (Fig. 3E, Supplementary Fig. 6E, and Supplementary Fig. 7E). These data suggest that GOLM1 promotes ABCG5-mediated cholesterol efflux by upregulating OPN.
GOLM1 promotes cholesterol efflux by upregulating OPN. (A) Left: Heatmap of the differentially expressed genes between the low- and high-GOLM1 groups. Middle: Linear regression lines showing the correlation between GOLM1 and OPN in the clinical cohort (n=110). Right: OPN mRNA expression in the low- and high-GOLM1 groups. (B) IHC staining of hepatic OPN in the four groups of mice. n=12 per group. Scale bar, 100 μm. (C) GOLM1, OPN, and ABCG5 protein expression in HepG2-shGOLM1-1/-2 and HepG2-GOLM1 cells. (D) GOLM1, OPN, and ABCG5 protein levels and supernatant cholesterol concentrations in HepG2-shOPN-1/-2 and HepG2-OPN cells. (E) GOLM1, OPN, and ABCG5 protein expression and supernatant cholesterol concentrations in shGOLM1, shGOLM1+OPN-OE, GOLM1-OE, and GOLM1-OE+shOPN HepG2 cells. ns, nonsignificant; *P<0.05, **P<0.01. ABCG5, ATP binding cassette transporter subfamily G member 5; CD, chow diet; GOLM1, Golgi membrane protein 1; GOLM1-/-, GOLM1 knockout; HepG2-GOLM1 cells, GOLM1-overexpressing HepG2 cells; HepG2-OPN cells, OPN-overexpressing HepG2 cells; HepG2-shGOLM1 cells, GOLM1-knockdown HepG2 cells; HepG2-shOPN cells, OPN-knockdown HepG2 cells; HFD, high-fat diet; IHC, immunohistochemistry; OPN, osteopontin; WT, wild-type.
GOLM1 nuclear translocation promotes OPN transcription
The physiological role of GOLM1 depends on protein modification [15,28-30], while the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that GOLM1 upregulated OPN at the transcriptional level both in vitro and in vivo (Fig. 4A, Supplementary Fig. 8A, B). To further determine the subcellular localization of GOLM1, we conducted western blotting (WB) and immunofluorescence (IF) staining analyses. WB analysis of cytosolic and nuclear proteins confirmed the presence of GOLM1 in the nucleus and the upregulation of nuclear GOLM1 in HepG2-GOLM1 and H-GOLM1 cells (Fig. 4B, Supplementary Fig. 8C, D). IF staining analysis revealed that the GOLM1 protein was present in the nuclei of HepG2 cells and primary hepatocytes (Fig. 4C, Supplementary Fig. 8E). A chromatin immunoprecipitation (ChIP) assay revealed significant enrichment of GOLM1 on the OPN promoter, which was enhanced by GOLM1 overexpression (Fig. 4D). To further corroborate these results, we performed a DNA pulldown assay, which revealed binding between the OPN promoter probe and the GOLM1 protein (Supplementary Fig. 9A). We then constructed an OPN-fused luciferase reporter plasmid to clarify the role of GOLM1 in OPN transcriptional regulation. Compared with that in control cells, luciferase activity was increased in GOLM1-overexpressing cells (Fig. 4E). Collectively, these findings imply that GOLM1 binds to the OPN promoter to activate its transcription.
GOLM1 nuclear translocation promotes OPN transcription. (A) Left: qRT-PCR analysis of OPN in the livers of the four groups of mice. n=12 per group. Middle/right: qRT-PCR analysis of OPN in HepG2-shGOLM1-1/-2 and HepG2-GOLM1 cells. (B) GOLM1 expression in the nucleus and cytoplasm of HepG2 and HepG2-GOLM1 cells. (C) IF analysis of the localization of GOLM1 in HepG2-GOLM1 and control cells. Scale bar, 20 μm. (D) ChIP assay of the binding level of the GOLM1 protein to the OPN promoter region in HepG2 and HepG2-GOLM1 cells. (E) Dual-luciferase reporter assay of luciferase activity in HepG2-GOLM1 cells. (F) Left: Schematic diagram of the serial deletion constructs of the OPN promoter. Right: Dual-luciferase reporter assay showing the luciferase activity of HepG2-GOLM1 and control cells transfected with OPN promoter deletion constructs (–500/+20, –267/+20, –127/+20, –70/+20, and –20/+20). ns, nonsignificant; *P<0.05, **P<0.01, ***P<0.005. CD, chow diet; ChIP, chromatin immunoprecipitation; GOLM1, Golgi membrane protein 1; GOLM1-/-, GOLM1 knockout; HepG2-GOLM1 cells, GOLM1-overexpressing HepG2 cells; HepG2-shGOLM1 cells, GOLM1-knockdown HepG2 cells; HFD, high-fat diet; IF, immunofluorescence; OPN, osteopontin; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild-type.
To identify the binding region of GOLM1 in the OPN promoter, we performed a dual-luciferase reporter assay with deleted fragments (–500/+20, –267/+20, –127/+20, –70/+20 and –20/+20) of the OPN promoter (Fig. 4F). Luciferase activity dramatically decreased when the –267 to –127 region of the OPN promoter was deleted (Fig. 4F), suggesting that this region may be responsible for GOLM1-induced OPN transcription.
Liver inflammation increases GOLM1 expression and nuclear translocation
GOLM1 was significantly upregulated in MASH livers but not in SS or normal livers (Fig. 1E, F). Liver inflammation is a crucial factor that can be used to differentiate between MASH and SS [31]. Therefore, we speculated that GOLM1 is regulated by MASH-related liver inflammation. We measured the levels of IL-1β and IL-6, which are major MASH-related inflammatory factors [14]. IL-1β and IL-6 levels increased as the severity of MASH increased, with IL-1β levels increasing dramatically compared with those of IL-6 (Fig. 5A). A dose-dependent increase in GOLM1 was detected in HepG2 cells and primary hepatocytes stimulated with IL-1β (Fig. 5B, Supplementary Fig. 9B, and Supplementary Fig. 10A). Moreover, IF staining and WB analysis revealed that the intensity of GOLM1 in the nucleus increased with increasing levels of IL-1β (Fig. 5C-E, Supplementary Fig. 9D, E, and Supplementary Fig. 10B, C). A dose-dependent increase in OPN was detected in HepG2 cells and primary hepatocytes stimulated with IL-1β (Fig. 5B, Supplementary Fig. 9B, 9C, and Supplementary Fig. 10A). In addition, a dual-luciferase reporter assay revealed that IL-1β promoted GOLM1-induced OPN transcription in a dose-dependent manner (Fig. 5F, Supplementary Fig. 9F). These data demonstrate that IL-1β promotes GOLM1 expression and nuclear translocation.
Liver inflammation increases GOLM1 expression and its nuclear translocation. (A) Left: Schematic diagram of the HFD feeding experiment. Middle: Relative expression of IL-1β and IL-6 in the four groups of mice. Right: Curve of the change in hepatic IL-1β with increasing HFD feeding time. n=12 per group at each time point. (B) Effect of IL-1β on the expression of GOLM1 and OPN in HepG2 cells. (C) IF analysis of the effect of IL-1β on GOLM1 localization in HepG2 cells. (D, E) GOLM1 expression in the nucleus and cytoplasm of HepG2 cells supplemented with the indicated concentrations of IL-1β. (F) Dual-luciferase reporter assay showing the luciferase activity of HepG2 cells supplemented with the indicated concentrations of IL-1β. ns, nonsignificant; *P<0.05, **P<0.01, ***P<0.005. CD, chow diet; HFD, high-fat diet; GOLM1, Golgi membrane protein 1; GOLM1-/-, GOLM1 knockout; IF, immunofluorescence; IL-1β, interleukin-1β; IL-6, interleukin-6; OPN, osteopontin; WT, wild-type.
Next, we explored the mechanisms by which IL-1β upregulates GOLM1 expression. Epithelium-specific ets transcription factor 1 (ESE-1), a member of the ESE subfamily of ETS transcription factors, is induced by proinflammatory factors such as IL-1β and regulates GOLM1 expression [13]. We speculated that IL-1β upregulates GOLM1 expression in an ESE-1-dependent manner. A dose-dependent increase in ESE-1 expression was detected in HepG2 cells and primary hepatocytes stimulated with IL-1β, demonstrating that IL-1β could upregulate ESE-1 expression (Supplementary Fig. 11A, B). The GOLM1 transcriptional level also gradually increased with increasing concentrations of IL-1β (Supplementary Fig. 11A, B). Furthermore, we constructed ESE-1-knockdown primary hepatocytes (H-shESE-1) and HepG2 (HepG2-shESE-1) cells to determine the effect of ESE-1 on GOLM1. ESE-1 knockdown decreased GOLM1 expression (Supplementary Fig. 11C, D). Accordingly, GOLM1 upregulation was observed in ESE-1-overexpressing primary hepatocytes (H-ESE-1) and HepG2 (HepG2-ESE-1) cells (Supplementary Fig. 11E, F). Finally, we assessed whether IL-1β regulated GOLM1 in an ESE-1-dependent manner. ESE-1 knockdown abolished IL-1β-induced GOLM1 upregulation in primary hepatocytes and HepG2 cells (Supplementary Fig. 11G, H). Furthermore, to demonstrate the specificity of the effects of IL-1β stimulation, we used IL-1β and IL-6 to stimulate HepG2 cells and primary hepatocytes. ESE-1, GOLM1, and OPN protein expression were slightly increased after IL-6 stimulation but significantly increased after IL-1β stimulation (Supplementary Fig. 12A, B). Nucleocytoplasmic separation experiments also revealed that IL-1β significantly increased the enrichment of the nuclear GOLM1 protein, whereas IL-6 had a weak effect (Supplementary Fig. 12C, D). In conclusion, IL-1β upregulates GOLM1 through ESE-1.
GOLM1 is upregulated in MASH patients with gallstones
Finally, to assess the impact of GOLM1 on MASH-related CGS formation in humans, we investigated the expression of GOLM1 and its target genes in liver samples. GOLM1 expression in MASH patients with gallstones was significantly greater than that in MASH patients without gallstones (Fig. 6A, B). Similarly, OPN and ABCG5 were significantly upregulated in the livers of MASH patients with gallstones compared with those without gallstones (Fig. 6C, D). Moreover, IL-1β was upregulated in the livers of MASH patients with gallstones compared with those without gallstones, suggesting that liver inflammation may contribute to the upregulation of GOLM1 expression in MASH-related gallstone patients (Fig. 6E).
GOLM1 is upregulated in MASH patients with gallstones. (A) HE staining; Masson staining; NAS; and TG, TC, AST, and ALT levels of MASH patients with gallstones (n=32) and without gallstones (n=10). Scale bar, 200 μm. (B) GOLM1 expression in the livers of MASH patients with (n=32) and without gallstones (n=10). (C) OPN expression in the livers of MASH patients with (n=32) and without gallstones (n=10). Scale bar, 100 μm. (D) ABCG5 expression in the livers of MASH patients with (n=32) and without gallstones (n=10). Scale bar, 100 μm. (E) IL-1β concentration in the livers of MASH patients with (n=32) and without gallstones (n=10). ns, nonsignificant; ***P<0.005. ABCG5, ATP binding cassette transporter subfamily G member 5; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GOLM1, Golgi membrane protein 1; HE, hematoxylin-eosin; IHC, immunohistochemistry; IL-1β, interleukin-1β; MASH, metabolic dysfunction-associated steatohepatitis; NAS, nonalcoholic fatty liver disease activity score; OPN, osteopontin; TC, total cholesterol; TG, triglyceride.
DISCUSSION
This study revealed that MASH-related liver inflammation upregulated GOLM1 expression, which contributes to CGS formation. Nuclear GOLM1 enhanced OPN transcription and upregulated ABCG5 expression, thus facilitating hepatobiliary cholesterol transport and biliary cholesterol supersaturation. The results of our study clarify the mechanistic relationship between MASH and CGS formation and indicate that GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.
The impact of MASH on gallstone occurrence has attracted increasing attention [9,32]. To address this, we collected UKB data (Supplementary Fig. 13). Analysis of large-sample prospective data from the UKB revealed a greater prevalence of cholelithiasis in the MASH group than in the SS and normal groups. Moreover, the risk of cholelithiasis in the MASH group was significantly greater than that in the other groups. Our findings are consistent with those of other studies and suggest the role of MASH in cholelithiasis [8,9]. The dataset from the UKB is a prospective cohort. Thus, the epidemiological data from the UKB are insufficient to reveal the causal relationship between MASH and cholelithiasis. Subsequent Mendelian studies may help to determine the causal relationship between MASH and CGSs. The specific mechanisms underlying MASH-related gallstone formation remain unclear. Our study revealed that hepatic GOLM1 was upregulated in MASH mice and that GOLM1-/- protected MASH mice from CGS formation. These findings indicate that GOLM1 may link MASH and CGSs. Moreover, females are more susceptible to CGSs than males are because of the effects of hormones such as estrogen [1,33]. Recent studies have shown that estrogen protects against MASH [34,35], indicating that sex effects might affect GOLM1 upregulation-induced CGS formation. Confirmation of this hypothesis requires multiple follow-up experiments.
The efflux of excessive cholesterol is the predominant cause of CGS formation [36]. Cholesterol efflux is primarily controlled by the ABCG5/8 heterodimer complex [26]. ABCG5 is responsible for approximately 82% of hepatic cholesterol excretion into the bile, and ABCG5 upregulation promotes CGS formation [26,37]. A previous study revealed aberrant accumulation of cholesteryl esters in GOLM1-knockdown cells [16], indicating that GOLM1 may promote cholesterol efflux. In the present study, GOLM1 knockdown reduced ABCG5 expression and cholesterol levels in the cell supernatant, whereas GOLM1 overexpression in vivo had the opposite effects. GOLM1-/- mice presented reduced biliary cholesterol concentrations and hepatic cholesterol output. These data indicate that GOLM1 increases cholesterol efflux through ABCG5 upregulation, thus promoting CGS formation.
Our previous study confirmed that cholesterol stabilizes GOLM1 [15]. In this study, we found that cholesterol levels in the livers of MASH patients were also increased. Thus, GOLM1 might be regulated by cholesterol in MASH livers. Despite having comparable levels of hepatic cholesterol, MASH and SS patients presented notable differences in the expression of GOLM1. These findings indicate that cholesterol enrichment is not the crucial factor contributing to increased GOLM1 expression. GOLM1 is rarely expressed in normal livers, but its expression is significantly increased during liver inflammation [38]. Consistently, our study revealed that GOLM1 was highly expressed in the livers of MASH patients, whereas its expression remained unchanged in normal controls and SS patients. Liver inflammation is one of the most distinguishing features of MASH. Therefore, GOLM1 upregulation may be associated with liver inflammation. Our study revealed that IL-1β, an essential inflammatory molecule in the progression of MASH [14], promoted GOLM1 expression and nuclear translocation. Thus, liver inflammation, which upregulates GOLM1 in MASH livers, may be a crucial factor in MASH-related CGS formation. Our study provides insight into the link between inflammatory diseases and metabolic disorders. It is worth investigating whether inflammatory liver diseases other than MASH contribute to gallstone formation. The pathophysiology of MASH involves multiple factors in addition to inflammation, such as fibrosis, impaired glucose tolerance, and metabolic syndrome [39]. GOLM1 was reported to be upregulated in the livers of patients with fibrosis and type 2 diabetes mellitus [40,41]. Our previous study confirmed that GOLM1 can be upregulated by cholesterol [15]. Thus, GOLM1 expression in MASH might be affected by these factors, and further experiments are needed to confirm this hypothesis.
GOLM1 plays a vital role in multiple digestive diseases [28,29]. Previous studies reported that GOLM1 cycles between cell membranes and organelle membranes and participates in protein sorting, modification, and transportation [11,28-30]. However, our study revealed that GOLM1 can enter the nucleus to regulate transcription. After translocating into the nucleus, GOLM1 binds to the promoter of OPN, thus activating its transcription. This discovery reveals a new mechanism by which GOLM1 regulates gene expression. Furthermore, the binding site of GOLM1 is close to that of other transcription factors, such as activating protein-1 (AP-1) [42], suggesting that GOLM1 may have DNA-binding domains similar to those of other transcription factors. Transcription factors affect various biological processes, such as immunomodulation, metabolism, and replicative senescence [43-45]. We speculate that GOLM1 may serve as a transcription factor to perform the above functions. To validate this hypothesis, protein structural analysis is likely needed. Notably, molecular expression regulation cannot fully avoid off-target effects [46]. In this study, two different shRNAs targeting distinct fragments of GOLM1 were used to avoid off-target effects, and consistent results were achieved with both shRNAs. We also performed a rescue experiment to determine whether the effect of GOLM1 in MASH-related CGSs was directly due to GOLM1 knockdown/overexpression and not to an off-target effect, and the results further confirmed our conclusions. Avoiding off-target effects in research remains challenging, and rational experimental design or improved research methods are needed to weaken off-target effects to ensure the reliability of research.
In this study, we found that GOLM1 promotes CGS formation via ABCG5-mediated cholesterol efflux in MASH livers, which indicates the role of the GOLM1-OPN-ABCG5 axis in MASH-related CGSs. Moreover, GOLM1 and OPN have been proven to participate in other metabolic disorders, such as diabetes and obesity [41,47]. As the key transporter for cholesterol efflux, ABCG5 clears cholesterol and is associated with the risk of diabetes [25,48]. Thus, the GOLM1-OPN-ABCG5 axis may play a role in a variety of metabolic diseases, and targeting this axis may provide new therapeutic options for various metabolic diseases. This axis may help clarify the connections between various metabolic diseases, aiding in the study of their interactions. In addition, studies have shown that an OPN-neutralizing antibody inhibited obesity-induced inflammation and insulin resistance and that ABCG5 could play an anti-obesity role by promoting cholesterol excretion [47,49]. Therefore, targeting the GOLM1-OPN-ABCG5 axis is expected to provide new therapeutic ideas for metabolic diseases including CGSs, obesity, and diabetes. On the other hand, GOLM1 inhibitors or neutralizing antibodies are needed to confirm the therapeutic potential of targeting GOLM1. OPN is a crucial mediator of cell adhesion and migration and plays a key role in inflammation and immune regulation; thus, interfering with OPN may have many side effects [50]. Since cholesterol metabolism is in dynamic equilibrium, the regulation of ABCG5 needs to avoid adverse effects caused by cholesterol homeostasis imbalance [25]. These are challenges to targeting the GOLM1-OPN-ABCG5 axis, and subsequent clinical and mechanistic studies are needed.
The main advantage of this study was the large, prospective cohort used to explore the association between MASH and CGSs and it revealed the role of GOLM1 in the MASH-related CGSs. We revealed a novel mechanism for the regulation of protein functions via GOLM1. Nuclear GOLM1 promoted OPN-induced ABCG5 expression to enhance cholesterol efflux, leading to CGS formation. Furthermore, GOLM1 was regulated by IL-1β, which plays a pivotal role in MASH. Thus, GOLM1 could act as a molecular hub connecting liver inflammation and cholesterol metabolism. Sex is an important demographic risk factor for CGS, with women having a higher incidence of CGS than men do [1]. The main limitation of this study was that only male mice were used in our investigation. Although this design avoids any effect of sex on the results, it prevents us from exploring the role of sex-related factors, such as estrogen, in MASH-related gallstone formation. Exploring the impact of sex on MASH-related gallstone formation is a promising direction that we will incorporate as our next research focus. Moreover, the study cohort for clinical validation was monocentric, and 42 patients were included in this study. Follow-up studies with a large sample size and multiple centers are needed to further confirm these findings.
In conclusion, our study reveals the role of GOLM1 in MASH-related CGSs. Nuclear GOLM1 induces OPN-dependent ABCG5 expression, which promotes cholesterol efflux and leads to CGS formation. Furthermore, GOLM1 is regulated by IL-1β, which plays a pivotal role in MASH. Our results indicate that GOLM1 has the potential to be a molecular hub connecting liver inflammation and cholesterol metabolism.
Notes
Authors’ contribution
Study concept and design: Yi-Tong Li, Wei-Qing Shao, Zhen-Mei Chen; acquisition of data: Yi-Tong Li, Zhen-Mei Chen, Xiao-Chen Ma; drafting of manuscript: Yi-Tong Li, Wei-Qing Shao, Zhen-Mei Chen; critical revision of manuscript: Jin-Hong Chen, Jing Lin; statistical analysis: Chen-He Yi, Bao-Rui Tao, Bo Zhang, Yue Ma, Guo Zhang, Rui Zhang, Yan Geng; study supervision: Jin-Hong Chen, Jing Lin.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant number 82070655) and the Young Scientists Fund of the National Natural Science Foundation of China (grant number 82000605). We thank the UK Biobank and the participants of the UK Biobank.
We are grateful for the drawing tools provided by Figdraw (www.figdraw.com).
Conflicts of Interest
The authors have no conflicts to disclose.
Abbreviations
ABCG5/8
ATP binding cassette transporter subfamily G member 5/8
ALT
alanine aminotransferase
AP1
activating protein-1
AST
aspartate aminotransferase
BMI
body mass index
CD
chow diet
cDNAs
complementary DNAs
CGS
cholesterol gallstone
ChIP
chromatin immunoprecipitation
CSI
cholesterol saturation index
ESE-1
epithelium-specific ets transcription factor 1
GOLM1
Golgi membrane protein 1
GOLM1-/-
GOLM1 knockout
GSF
gallstone-free
GTEx
Genotype Tissue Expression
H-ESE-1 cells
ESE-1-overexpressing primary hepatocytes
H-GOLM1 cells
GOLM1-overexpressing primary hepatocytes
H-shESE-1 cells
ESE-1-knockdown primary hepatocytes
H-shGOLM1 cells
GOLM1-knockdown primary hepatocytes
HE
hematoxylin-eosin
HepG2-ESE-1 cells
ESE-1-overexpressing HepG2 cells
HepG2-GOLM1 cells
GOLM1-overexpressing HepG2 cells
HepG2-OPN cells
OPN-overexpressing HepG2 cells
HepG2-shESE-1 cells
ESE-1-knockdown HepG2 cells
HepG2-shGOLM1 cells
GOLM1-knockdown HepG2 cells
HepG2-shOPN cells
OPN-knockdown HepG2 cells
HFD
high-fat diet
IF
immunofluorescence
IFG/DM
impaired fasting glucose/diabetes mellitus
IHC
immunohistochemistry
IL-1β
interleukin-1β
IL-6
interleukin-6
MASLD
metabolic dysfunction-associated steatotic liver disease
MASH
metabolic dysfunction-associated steatohepatitis
NAS
nonalcoholic fatty liver disease activity score
OPN
osteopontin
qRT-PCR
quantitative real-time polymerase chain reaction
TC
total cholesterol
TG
triglyceride
SS
simple steatosis
UKB
UK Biobank
WB
western blotting
WT
wild-type
SUPPLEMENTAL MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (http://www.e-cmh.org).
The independent associations of age, sex, MASLD, MASH, SS, BMI, and IFG/DM with incident cholelithiasis. (A) The independent associations of age, MASLD, MASH, SS, BMI, and IFG/DM with incident cholelithiasis after adjusting for sex. (B) The independent associations of age, sex, MASLD, MASH, SS, and BMI with incident cholelithiasis after adjusting for IFG/DM. (C) The independent associations of age, MASLD, MASH, SS, and BMI with incident cholelithiasis after adjusting for sex and IFG/DM. BMI, body mass index; IFG/DM, impaired fasting glucose/diabetes mellitus; MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic dysfunction-associated steatohepatitis; SS, simple steatosis.
Construction of MASH model and GOLM1 knockout mice. (A) Body weight, liver weight, and liver-to-body weight ratio in the four groups. n=12 mice per group. (B) Hepatic TG and TC levels in the four groups. n=12 mice per group. (C) Fasting blood glucose and insulin levels in the four groups. n=12 mice per group. (D) Hepatic IL-1β and IL-6 levels in the four groups. n=12 mice per group. (E) Verification of GOLM1 knockout in mice. (F) GOLM1 protein expressions in wild-type and GOLM1 knockout mice. ns, non-significant; ***P<0.005. CD, chow diet; GOLM1, Golgi membrane protein 1; GOLM1-/-, GOLM1 knockout; HFD, high-fat diet; IL-1β, interleukin-1β; IL-6, interleukin-6; MASH, metabolic dysfunction-associated steatohepatitis; TC, total cholesterol; TG, triglyceride; WT, wild-type.
Change of NAS, hepatic lipids, and IL-6 during MASH progression. (A) Curve of the change in NAS with increasing HFD feeding time. n=12 mice per group. (B) Curve of the change in hepatic TG with increasing HFD feeding time. n=12 mice per group. (C) Curve of the change in hepatic TC with increasing HFD feeding time. n=12 mice per group. (D) Curve of the change in hepatic IL-6 with increasing HFD feeding time. n=12 mice per group. ns, nonsignificant; **P<0.01, ***P<0.005. CD, chow diet; GOLM1-/-, GOLM1 knockout; HFD, high-fat diet; IL-6, interleukin-6; MASH, metabolic dysfunction-associated steatohepatitis; NAS, nonalcoholic fatty liver disease activity score; TC, total cholesterol; TG, triglyceride; WT, wild-type.
The serum lipid profile and bilirubin levels of mice. (A) Serum TG levels in the four groups. n=12 mice per group. (B) Serum TC levels in the four groups. n=12 mice per group. (C) Serum HDL-C levels in the four groups. n=12 mice per group. (D) Serum LDL-C in the four groups. n=12 mice per group. (E) Serum TBIL levels in the four groups. n=12 mice per group. ns, nonsignificant; ***P<0.005. CD, chow diet; GOLM1-/-, GOLM1 knockout; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TBIL, total bilirubin; TC, total cholesterol; TG, triglyceride; WT, wild-type.
Expressions of cholesterol metabolism-related genes and ABCG8 protein. (A) qRT-PCR analysis showing the hepatic metabolism-related gene expressions in the livers of CD- or HFD-fed mice. (B) IHC staining of hepatic ABCG8 in four groups of mice. n=12 mice per group. Scale bar, 100 μm. (C) GOLM1 and ABCG8 protein expression in HepG2-shGOLM1-1/-2 and HepG2-GOLM1 cells. ns, nonsignificant; ***P<0.005. ABCG8, ATP binding cassette transporter subfamily G member 8; CD, chow diet; GOLM1, Golgi membrane protein 1; GOLM1-/-, GOLM1 knockout; HepG2-GOLM1 cells, GOLM1-overexpressing HepG2 cells; HepG2-shGOLM1 cells, GOLM1-knockdown HepG2 cells; HFD, high-fat diet; IHC, immunohistochemistry; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild-type.
GOLM1 promotes cholesterol efflux through OPN in primary hepatocytes. (A) GOLM1, OPN, and ABCG5 protein expressions in H-shGOLM1-1/-2 and H-GOLM1 cells. (B) GOLM1, OPN, and ABCG5 protein expressions in H-shOPN-1/-2 and H-OPN cells. (C) Supernatant cholesterol concentration in H-shGOLM1-1/-2 and H-GOLM1 cells. (D) Supernatant cholesterol concentration in H-shOPN-1/-2 and H-OPN cells. (E) GOLM1, OPN, and ABCG5 protein expressions and supernatant cholesterol concentration in shGOLM1, shGOLM1+OPN-OE, GOLM1-OE, and GOLM1-OE+shOPN primary hepatocytes. ns, nonsignificant; ***P<0.005. ABCG5, ATP binding cassette transporter subfamily G member 5; GOLM1, Golgi membrane protein 1; H-GOLM1 cells, GOLM1-overexpressing primary hepatocytes; H-OPN cells, OPN-overexpressing primary hepatocytes; H-shGOLM1 cells, GOLM1-knockdown primary hepatocytes; H-shOPN cells, OPN-knockdown primary hepatocytes; OPN, osteopontin.
Protein quantitation for western blotting. (A) Quantification of GOLM1, OPN, and ABCG5 protein levels in HepG2-shGOLM1-1/-2 cells. (B) Quantification of GOLM1, OPN, and ABCG5 protein levels in HepG2-GOLM1 cells. (C) Quantification of GOLM1, OPN, and ABCG5 protein levels in HepG2-shOPN-1/-2 cells. (D) Quantification of GOLM1, OPN, and ABCG5 protein levels in HepG2-OPN cells. (E) Quantification of GOLM1, OPN, and ABCG5 protein levels in shGOLM1, shGOLM1+OPN-OE, GOLM1-OE, and GOLM1-OE+shOPN HepG2 cells. ns, nonsignificant; ***P<0.005. ABCG5, ATP binding cassette transporter subfamily G member 5; GOLM1, Golgi membrane protein 1; HepG2-GOLM1 cells, GOLM1-overexpressing HepG2 cells; HepG2-OPN cells, OPN-overexpressing HepG2 cells; HepG2-shGOLM1 cells, GOLM1-knockdown HepG2 cells; HepG2-shOPN cells, OPN-knockdown HepG2 cells; OPN, osteopontin.
Nuclear GOLM1 promotes OPN transcription in primary hepatocytes. (A) qRT-PCR analysis of OPN in H-shGOLM1-1/-2 cells. (B) qRT-PCR analysis of OPN in H-GOLM1 cells. (C) GOLM1 expression in nucleus and cytoplasm of primary hepatocytes. (D) GOLM1 expression in nucleus and cytoplasm of H-GOLM1 cells. (E) IF analysis of the localization of GOLM1 protein in H-GOLM1 and control cells. Scale bar, 20 μm. **P<0.01, ***P<0.005. ESE-1, epithelium-specific ets transcription factor 1; GOLM1, Golgi membrane protein 1; H-GOLM1 cells, GOLM1-overexpressing primary hepatocytes; H-shGOLM1 cells, GOLM1-knockdown primary hepatocytes; IF, immunofluorescence; OPN, osteopontin; qRT-PCR, quantitative real-time polymerase chain reaction.
IL-1β promotes GOLM1 expression and nuclear translocation. (A) DNA pull-down assay detecting the enrichment of GOLM1 protein in OPN promoter. (B) The effect of IL-1β on the protein expressions of GOLM1 and OPN in HepG2-GOLM1 cells. (C) The effect of IL-1β on OPN mRNA expression in HepG2-GOLM1 cells. (D) IF analysis showed the effect of IL-1β on GOLM1 localization in HepG2-GOLM1 cells. Scale bar, 20 μm. (E) GOLM1 expression in nucleus and cytoplasm of HepG2-GOLM1 cells supplemented with indicated concentrations of IL-1β. (F) Dual luciferase reporter assay of the luciferase activity of HepG2-GOLM1 cells supplemented with indicated concentrations of IL-1β. ns, nonsignificant; *P<0.05, **P<0.01, ***P<0.005. GOLM1, Golgi membrane protein 1; HepG2-GOLM1 cells, GOLM1-overexpressing HepG2 cells; IL-1β, interleukin-1β; OPN, osteopontin.
IL-1β boosts GOLM1 expression and its nuclear translocation in primary hepatocytes. (A) The effect of IL-1β on the expressions of GOLM1 and OPN in primary hepatocytes. (B) GOLM1 expression in nucleus and cytoplasm of primary hepatocytes supplemented with indicated concentrations of IL-1β. (C) IF analysis of the effect of IL-1β on GOLM1 localization in primary hepatocytes. Scale bar, 20 μm. ns, nonsignificant; *P<0.05, **P<0.01, ***P<0.005. GOLM1, Golgi membrane protein 1; IF, immunofluorescence; IL-1β, interleukin-1β; OPN, osteopontin.
IL-1β promotes GOLM1 expression through ESE-1. (A) The effect of IL-1β on the expressions of ESE-1 and GOLM1 in HepG2 cells. (B) The effect of IL-1β on the expressions of ESE-1 and GOLM1 in primary hepatocytes. (C) ESE-1 and GOLM1 protein expressions in HepG2-shESE-1-1/-2 cells. (D) ESE-1 and GOLM1 protein expressions in H-shESE-1-1/-2 cells. (E) ESE-1 and GOLM1 protein expressions in HepG2-ESE-1 cells. (F) ESE-1 and GOLM1 protein expressions in H-ESE-1 cells. (G) ESE-1 and GOLM1 protein expressions in IL-1β (-)+shESE-1 (-), IL-1β (-)+shESE-1 (+), IL-1β (+)+shESE-1 (-), and IL-1β (+)+shESE-1 (+) HepG2 cells. (H) ESE-1 and GOLM1 protein expressions in IL-1β (-)+shESE-1 (-), IL-1β (-)+shESE-1 (+), IL-1β (+)+shESE-1 (-), and IL-1β (+)+shESE-1 (+) primary hepatocytes. ns, nonsignificant; *P<0.05, **P<0.01, ***P<0.005. ESE-1, epithelium-specific ets transcription factor 1; GOLM1, Golgi membrane protein 1; H-ESE-1 cells, ESE-1-overexpressing primary hepatocytes; H-shESE-1, ESE-1-knockdown primary hepatocytes; HepG2-ESE-1 cells, ESE-1-overexpressing HepG2 cells; HepG2-shESE-1 cells, ESE-1-knockdown HepG2 cells; IL-1β, interleukin-1β.
Effects of IL-1β and IL-6 on ESE-1, GOLM1, and OPN expression and GOLM1 nuclear translocation. (A) Effect of IL-1β and IL-6 on the expression of ESE-1, GOLM1, and OPN in HepG2 cells. (B) Effect of IL-1β and IL-6 on the expression of ESE-1, GOLM1, and OPN in primary hepatocytes. (C) GOLM1 expression in the nucleus and cytoplasm of HepG2 cells supplemented with of IL-1β and IL-6. (D) GOLM1 expression in the nucleus and cytoplasm of primary hepatocytes supplemented with of IL-1β and IL-6. ***P<0.005. ESE-1, epithelium-specific ets transcription factor 1; GOLM1, Golgi membrane protein 1; IL-1β, interleukin-1β; IL-6, interleukin-6; OPN, osteopontin.
Distribution of baseline characteristics. Data distribution of age, BMI, fasting glucose, AST, ALT, platelet, and albumin. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index.
Baseline characteristics
Reagents used in the present study
Oligonucleotide sequences used in quantitative real-time polymerase chain reaction
References
Article information Continued
Notes
Study Highlights
• GOLM1 knockout attenuates MASH-related gallstone formation by inhibiting ABCG5 in an OPN-dependent manner.
• IL-1β promotes GOLM1 expression and nuclear translocation.
• GOLM1 acts as a molecular hub connecting liver inflammation and cholesterol metabolism.