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Cited by 159Cholesterol and Lipid MetabolismDrug Transport and Resistance MechanismsMicroRNA in disease regulation

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miR-33 controls the expression of biliary transporters, and mediates statin- and diet-induced hepatotoxicity

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Allen, Ryan M., Marquart, Tyler J., Albert, Carolyn J., Suchy, Frederick J., Wang, David Q.‐H., Ananthanarayanan, Meenakshisundaram, Ford, David A., Baldán, Ángel·2012·EMBO Molecular Medicine
DOI 10.1002/emmm.201201228PMC PMC3491822
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Abstract

Bile secretion is essential for whole body sterol homeostasis. Loss-of-function mutations in specific canalicular transporters in the hepatocyte disrupt bile flow and result in cholestasis. We show that two of these transporters, ABCB11 and ATP8B1, are functional targets of miR-33, a micro-RNA that is expressed from within an intron of SREBP-2. Consequently, manipulation of miR-33 levels in vivo with adenovirus or with antisense oligonucleotides results in changes in bile secretion and bile recovery from the gallbladder. Using radiolabelled cholesterol, we show that systemic silencing of miR-33 leads to increased sterols in bile and enhanced reverse cholesterol transport in vivo. Finally, we report that simvastatin causes, in a dose-dependent manner, profound hepatotoxicity and lethality in mice fed a lithogenic diet. These latter results are reminiscent of the recurrent cholestasis found in some patients prescribed statins. Importantly, pretreatment of mice with anti-miR-33 oligonucleotides rescues the hepatotoxic phenotype. Therefore, we conclude that miR-33 mediates some of the undesired, hepatotoxic effects of statins.

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Figure 1 Increased bile secretion following silencing of miR-33 Bile recovered from the gallbladder of mice (n = 5) on chow diet, injected with saline, and scrambled or anti-miR-33 oligonucleotides (5 mpk i.v., for 2 consecutive days). Animals were then kept for a week on chow and fasted overnight before sample collection.Levels of phosphatidylcholine (PC), cholesterol (chol) and bile acids present in gallbladder bile in the same mice.Relative expression of hepatic canalicular transporters in the same mice.A different group of mice (n = 6–8) was injected as described above. A week later, mice were anesthetized, the bile duct cannulated, and hepatic bile collected for 1 h.Levels of bile acids, phosphatidylcholine (PC), cholesterol (chol) present in bile from the last group of mice. Data shown as mean ± SEM. *p < 0.05; **p < 0.01.
Figure 1 Increased bile secretion following silencing of miR-33 Bile recovered from the gallbladder of mice (n = 5) on chow diet, injected with saline, and scrambled or anti-miR-33 oligonucleotides (5 mpk i.v., for 2 consecutive days). Animals were then kept for a week on chow and fasted overnight before sample collection.Levels of phosphatidylcholine (PC), cholesterol (chol) and bile acids present in gallbladder bile in the same mice.Relative expression of hepatic canalicular transporters in the same mice.A different group of mice (n = 6–8) was injected as described above. A week later, mice were anesthetized, the bile duct cannulated, and hepatic bile collected for 1 h.Levels of bile acids, phosphatidylcholine (PC), cholesterol (chol) present in bile from the last group of mice. Data shown as mean ± SEM. *p < 0.05; **p < 0.01.CC BY · PubMed Central
Figure 2 Functional miR-33 responsive elements in the 3′UTR of ATP8B1 and ABCB11 A,B. Conserved sequences in the 3′UTR of ATP8B1 and ABCB11 are partially complementary to miR-33. The element in human ATP8B1 is located 1877–1897 nt after the stop codon. The element in ABCB11 overlaps the stop codon in primates, while rodents show a conserved sequence 732–751 nt after the stop codon.C,D. Luciferase assays in HEK293 cells using the whole 3′UTR of human or murine ATP8B1 and ABCB11, or a single copy of the responsive elements (RE) identified above, or mutant responsive elements (RE*; where AATGCA was mutated to GGGTTG to prevent complementarity to the seed sequence of the miRNA), co-transfected with (closed bars) or without (open bars) a vector to overexpress miR-33. In grey, data from empty (negative control) and R33 (positive control containing a 100% match to miR-33) reporter vectors.E,F. Relative mRNA expression of canalicular transporters in primary murine hepatocytes (n = 4 dishes/condition) and human HuH-7 hepatoma cells (n = 3 dishes/condition) transduced 48 h with empty or miR-33 adenovirus.G. Relative protein levels in HuH7 cells transduced with empty or miR-33 adenovirus. Some cells were incubated for 16 h in the presence of FXR:RXR agonists (2 µmol/L GW4064 : 1 µmol/L 9-cis-retinoic acid) to induce ABCB11. Asterisk indicates a non-specific band. Data shown as mean ± SD; **p < 0.01.
Figure 2 Functional miR-33 responsive elements in the 3′UTR of ATP8B1 and ABCB11 A,B. Conserved sequences in the 3′UTR of ATP8B1 and ABCB11 are partially complementary to miR-33. The element in human ATP8B1 is located 1877–1897 nt after the stop codon. The element in ABCB11 overlaps the stop codon in primates, while rodents show a conserved sequence 732–751 nt after the stop codon.C,D. Luciferase assays in HEK293 cells using the whole 3′UTR of human or murine ATP8B1 and ABCB11, or a single copy of the responsive elements (RE) identified above, or mutant responsive elements (RE*; where AATGCA was mutated to GGGTTG to prevent complementarity to the seed sequence of the miRNA), co-transfected with (closed bars) or without (open bars) a vector to overexpress miR-33. In grey, data from empty (negative control) and R33 (positive control containing a 100% match to miR-33) reporter vectors.E,F. Relative mRNA expression of canalicular transporters in primary murine hepatocytes (n = 4 dishes/condition) and human HuH-7 hepatoma cells (n = 3 dishes/condition) transduced 48 h with empty or miR-33 adenovirus.G. Relative protein levels in HuH7 cells transduced with empty or miR-33 adenovirus. Some cells were incubated for 16 h in the presence of FXR:RXR agonists (2 µmol/L GW4064 : 1 µmol/L 9-cis-retinoic acid) to induce ABCB11. Asterisk indicates a non-specific band. Data shown as mean ± SD; **p < 0.01.CC BY · PubMed Central
Figure 3 Effect of miR-33 overexpression on diet-induced cholestasis C57BL/6 mice (n = 5–7) were kept on chow diet or lithogenic diet for 7 days (lanes 1–2). A different group of animals were transduced i.v. with empty or miR-33 adenovirus (2 × 109 pfu/mouse), and then switched to the lithogenic diet (lanes 3–4). After 7 days, mice were fasted overnight and killed the following morning. Data show relative levels of hepatic miR-33.The volume of bile recovered from the gallbladder is significantly reduced in mice transduced with miR-33. Picture shows pooled bile collected from five mice each in an independent experiment.Liver to body mass ratios.Hepatic levels of bile acids; total, unesterified (UC) and esterified (EC) cholesterol; phosphatidylcholine (PC) and triglycerides (TG).The amounts of bile acids (BA), cholesterol (chol) and phosphatidylcholine (PC) were determined in bile, and expressed as mol% (mol per 100 mol). Compared to mice infused with saline or Adeno-empty, the bile from animals transduced with Adeno-miR-33 showed increased amounts of cholesterol (11.6 ± 1.1 vs. 6.6 ± 0.5 vs. 5.9 ± 0.6 mol%; miR vs. scrambled vs. saline; p = 0.009), and decreased amounts of bile acids (78.6 ± 1.7 vs. 83.7 ± 1.0 vs. 82.1 ± 1.4 mol%; miR vs. scrambled vs. saline; p = 0.03), but no change in PC contents (9.8 ± 1.3 vs. 9.7 ± 0.6 vs. 12.0 ± 0.9 mol%; miR vs. scrambled vs. saline).Relative expression of hepatic canalicular transporters in the same mice. Data shown as mean ± SD; *p < 0.05; **p < 0.01.
Figure 3 Effect of miR-33 overexpression on diet-induced cholestasis C57BL/6 mice (n = 5–7) were kept on chow diet or lithogenic diet for 7 days (lanes 1–2). A different group of animals were transduced i.v. with empty or miR-33 adenovirus (2 × 109 pfu/mouse), and then switched to the lithogenic diet (lanes 3–4). After 7 days, mice were fasted overnight and killed the following morning. Data show relative levels of hepatic miR-33.The volume of bile recovered from the gallbladder is significantly reduced in mice transduced with miR-33. Picture shows pooled bile collected from five mice each in an independent experiment.Liver to body mass ratios.Hepatic levels of bile acids; total, unesterified (UC) and esterified (EC) cholesterol; phosphatidylcholine (PC) and triglycerides (TG).The amounts of bile acids (BA), cholesterol (chol) and phosphatidylcholine (PC) were determined in bile, and expressed as mol% (mol per 100 mol). Compared to mice infused with saline or Adeno-empty, the bile from animals transduced with Adeno-miR-33 showed increased amounts of cholesterol (11.6 ± 1.1 vs. 6.6 ± 0.5 vs. 5.9 ± 0.6 mol%; miR vs. scrambled vs. saline; p = 0.009), and decreased amounts of bile acids (78.6 ± 1.7 vs. 83.7 ± 1.0 vs. 82.1 ± 1.4 mol%; miR vs. scrambled vs. saline; p = 0.03), but no change in PC contents (9.8 ± 1.3 vs. 9.7 ± 0.6 vs. 12.0 ± 0.9 mol%; miR vs. scrambled vs. saline).Relative expression of hepatic canalicular transporters in the same mice. Data shown as mean ± SD; *p < 0.05; **p < 0.01.CC BY · PubMed Central
Figure 4 Reverse cholesterol transport is enhanced after systemic miR-33 silencing C57BL/6 mice (n = 7) were infused i.v. with 5 mpk scrambled or anti-miR-33 oligonucleotides for 2 consecutive days, and 5 days later received 1 × 106 radiolabelled macrophages by i.p. injection. Mice were kept on chow for 48 h until sacrifice. A. Different parameters in mice receiving scrambled (open bars) and anti-miR-33 (closed bars) treatment at the time of sacrifice. Notice the increase in bile recovery from the gallbladder.B. Percentage of total injected dpm recovered in the plasma at different time points after injection of radiolabelled cells.C-E. Percentage of total injected dpm recovered at the time of sacrifice in liver, bile from the gallbladder and faeces of the same mice. Data shown as mean ± SD; *p < 0.05; **p < 0.01.
Figure 4 Reverse cholesterol transport is enhanced after systemic miR-33 silencing C57BL/6 mice (n = 7) were infused i.v. with 5 mpk scrambled or anti-miR-33 oligonucleotides for 2 consecutive days, and 5 days later received 1 × 106 radiolabelled macrophages by i.p. injection. Mice were kept on chow for 48 h until sacrifice. A. Different parameters in mice receiving scrambled (open bars) and anti-miR-33 (closed bars) treatment at the time of sacrifice. Notice the increase in bile recovery from the gallbladder.B. Percentage of total injected dpm recovered in the plasma at different time points after injection of radiolabelled cells.C-E. Percentage of total injected dpm recovered at the time of sacrifice in liver, bile from the gallbladder and faeces of the same mice. Data shown as mean ± SD; *p < 0.05; **p < 0.01.CC BY · PubMed Central
Figure 5 Statins induce miR-33 and reduce the mRNA expression of specific hepatic canalicular transporters A, B. C57BL/6 mice (n = 5) were gavaged daily with statins, and kept on chow diet. Samples were collected after 7 days, following an overnight fasting.C. HuH7 cells were cultured in quadruplicate for 48 h in DMEM supplemented with 2% lipoprotein-deficient serum in the presence or absence of simvastatin. Relative expression of specific genes shown as mean ± SD; *p < 0.05; **p < 0.01.
Figure 5 Statins induce miR-33 and reduce the mRNA expression of specific hepatic canalicular transporters A, B. C57BL/6 mice (n = 5) were gavaged daily with statins, and kept on chow diet. Samples were collected after 7 days, following an overnight fasting.C. HuH7 cells were cultured in quadruplicate for 48 h in DMEM supplemented with 2% lipoprotein-deficient serum in the presence or absence of simvastatin. Relative expression of specific genes shown as mean ± SD; *p < 0.05; **p < 0.01.CC BY · PubMed Central
Figure 6 Simvastatin and lithogenic diet induce liver toxicity C57BL/6 mice (n = 6) were gavaged daily with simvastatin and fed a lithogenic diet. Samples were collected after 7 days, or when mice appeared moribund. Survival is hampered by simvastatin in a dose-dependent manner.Severe hepatomegaly in mice dosed with 150 mpk simvastatin.Representative macroscopic appearance (upper and middle panels), and haematoxylin and eosin staining of paraffin-embedded sections (lower panels) from the same livers. Note the abnormally swollen cells and pale (i.e. steatotic) appearance of the livers in the 150 mpk group.Specific hepatic lipids as determined by ESI-MS, and normalized to tissue weight. FFA, free fatty acids; DAG, diacylglycerides; TAG, triacylglycerides; PC, phosphatidylcholine; UC, unesterified cholesterol; CE, cholesterol esters. **p < 0.01 versus saline.Representative samples of plasma, and levels of circulating alanine aminotransferase (ALT), aspartate aminotransferase (AST), bile acids and bilirubin.Bile was recovered from the gallbladder, pooled and the contents of phosphatidylcholine (PC), cholesterol (c) and bile acids (BA) determined with colorimetric kits.Relative expression of hepatic canalicular transporters (upper panel) and other genes involved in bile acid and sterol homeostasis (bottom panel) in samples from mice treated with 0 or 50 mpk simvastatin. Data shown as mean ± SD. **p < 0.01.
Figure 6 Simvastatin and lithogenic diet induce liver toxicity C57BL/6 mice (n = 6) were gavaged daily with simvastatin and fed a lithogenic diet. Samples were collected after 7 days, or when mice appeared moribund. Survival is hampered by simvastatin in a dose-dependent manner.Severe hepatomegaly in mice dosed with 150 mpk simvastatin.Representative macroscopic appearance (upper and middle panels), and haematoxylin and eosin staining of paraffin-embedded sections (lower panels) from the same livers. Note the abnormally swollen cells and pale (i.e. steatotic) appearance of the livers in the 150 mpk group.Specific hepatic lipids as determined by ESI-MS, and normalized to tissue weight. FFA, free fatty acids; DAG, diacylglycerides; TAG, triacylglycerides; PC, phosphatidylcholine; UC, unesterified cholesterol; CE, cholesterol esters. **p < 0.01 versus saline.Representative samples of plasma, and levels of circulating alanine aminotransferase (ALT), aspartate aminotransferase (AST), bile acids and bilirubin.Bile was recovered from the gallbladder, pooled and the contents of phosphatidylcholine (PC), cholesterol (c) and bile acids (BA) determined with colorimetric kits.Relative expression of hepatic canalicular transporters (upper panel) and other genes involved in bile acid and sterol homeostasis (bottom panel) in samples from mice treated with 0 or 50 mpk simvastatin. Data shown as mean ± SD. **p < 0.01.CC BY · PubMed Central
Figure 7 Silencing miR-33 rescues the liver damage induced by simvastatin and lithogenic diet C57BL/6 mice (n = 12) were injected i.v. with 5 mpk scrambled or anti-miR-33 oligonucleotides for two consecutive days, and then treated as in Fig 5. Survival curves.Body weight, expressed as % compared to mass on the day of first injection.Representative macroscopic appearance of the livers, and haematoxylin and eosin staining of paraffin-embedded sections.Liver to total body mass ratios. **p < 0.01 versus moribund mice.Representative samples of plasma. Note the normalization in colour in the anti-miR-33 group.Amounts of specific hepatic lipids as determined by ESI-MS, normalized to tissue weight (n = 4). Acronyms for lipid classes as defined in Fig 5. **p < 0.01 versus scrambled.Relative expression of hepatic canalicular transporters (upper panel) and other genes involved in bile acid and sterol homeostasis (bottom panel) (n = 5 for scrambled treatment; n = 10 for anti-miR-33 treatment). Data shown as mean ± SEM. **p < 0.01 versus moribund scrambled mice; *p < 0.05 versus surviving scrambled mice.
Figure 7 Silencing miR-33 rescues the liver damage induced by simvastatin and lithogenic diet C57BL/6 mice (n = 12) were injected i.v. with 5 mpk scrambled or anti-miR-33 oligonucleotides for two consecutive days, and then treated as in Fig 5. Survival curves.Body weight, expressed as % compared to mass on the day of first injection.Representative macroscopic appearance of the livers, and haematoxylin and eosin staining of paraffin-embedded sections.Liver to total body mass ratios. **p < 0.01 versus moribund mice.Representative samples of plasma. Note the normalization in colour in the anti-miR-33 group.Amounts of specific hepatic lipids as determined by ESI-MS, normalized to tissue weight (n = 4). Acronyms for lipid classes as defined in Fig 5. **p < 0.01 versus scrambled.Relative expression of hepatic canalicular transporters (upper panel) and other genes involved in bile acid and sterol homeostasis (bottom panel) (n = 5 for scrambled treatment; n = 10 for anti-miR-33 treatment). Data shown as mean ± SEM. **p < 0.01 versus moribund scrambled mice; *p < 0.05 versus surviving scrambled mice.CC BY · PubMed Central
Figure 8 miR-33 limits the mobilization of sterols in hepatocytes through both the sinusoidal and the canalicular membrane miR-33 mediates the cross-talk between the SREBP-2, LXR and FXR pathways by directly modulating the expression of ATP8B1, ABCB11, ABCA1 and ABCG1 (* in mice, but not in humans). Data suggest that miR-33 might also indirectly affect the expression of ABCG5/8 (see main text for details). §Decreased ATP8B1 activity in human, but not mouse, hepatocytes results in PKC-dependent inactivating phosphorylation of FXR.During episodes of low intracellular cholesterol, or following treatment with statin drugs, miR-33 is transcriptionally induced and reduces the expression of sterol transporters.
Figure 8 miR-33 limits the mobilization of sterols in hepatocytes through both the sinusoidal and the canalicular membrane miR-33 mediates the cross-talk between the SREBP-2, LXR and FXR pathways by directly modulating the expression of ATP8B1, ABCB11, ABCA1 and ABCG1 (* in mice, but not in humans). Data suggest that miR-33 might also indirectly affect the expression of ABCG5/8 (see main text for details). §Decreased ATP8B1 activity in human, but not mouse, hepatocytes results in PKC-dependent inactivating phosphorylation of FXR.During episodes of low intracellular cholesterol, or following treatment with statin drugs, miR-33 is transcriptionally induced and reduces the expression of sterol transporters.CC BY · PubMed Central

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Authors
Allen, R. M., Marquart, T. J., Albert, C. J., Suchy, F. J., Wang, D. Q. ‐., Ananthanarayanan, M., Ford, D. A., & Baldán, Á.
Affiliations
  • Saint Louis University (US)
  • University of Colorado Anschutz (US)
  • Yale University (US)
Corresponding author
Baldán, Ángel
ORCID
Venue
EMBO Molecular Medicine
Publisher
Springer Science and Business Media LLC
Published
2012-07-05
Volume
4
Issue
9
Pages
882-895
ISSN
  • 1757-4676 (ISSN-L)
  • 1757-4684
DOI
10.1002/emmm.201201228
PMC
PMC3491822
OpenAlex
W2159167853
Cited by
159
References
70
Citations by year
2026: 1 · 2025: 6 · 2024: 5 · 2023: 5 · 2022: 3 · 2021: 13 · 2020: 9 · 2019: 10 · 2018: 15 · 2017: 12 · 2016: 16 · 2015: 19 · 2014: 24 · 2013: 18 · 2012: 3
FWCI
20.90
Type
journal article
Language
en
Open access
Licence
Locations
  • EMBO Molecular Medicine (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • AHA (11SDG5030012)
  • NIH (HL107794 (to Á.B.), HL074214, HL098907, RR019232)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
Keywords
miR-33, ABCB11, ATP8B1, simvastatin, lithogenic diet, cholestasis, bile secretion, sterol homeostasis, reverse cholesterol transport, SREBP2, antisense oligonucleotides, gallbladder
MeSH terms
ATP Binding Cassette Transporter, Subfamily B, Member 11, Adenosine Triphosphatases, Animals, Bile, Cells, Cultured, Diet, Gene Expression Regulation, Liver, Male, Mice, Inbred C57BL, Models, Biological, ATP-Binding Cassette Transporters, Simvastatin, Hepatocytes, MicroRNAs, Phospholipid Transfer Proteins, Mice

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