1
Supporting Materials and Methods
Reagents
Reagents were obtained as follows: an irreversible PPAR antagonist, GW9662, was
from Sigma (St. Louis, MO, USA). PPARoverexpression vector (pCI-PPARwas
kindly provided by Drs. M. Seo and I. Inoue (Saitama Medical School, Saitama, Japan).
Its control vector (pCI-neo mammalian expression vector) was from Promega (Madison,
WI, USA).
Animal Studies
For the in vivo study using antagomirs against miR33a (Anti-miR33 oligonucleotide;
Operon Biotechnologies, Huntsville, AL, USA), 9-wk-old male C57BL/6J mice were
given CCl4 at a dose of 5 l (10% CCl4 in corn oil)/g body weight, by intraperitoneal
injection twice a week for 4 weeks, during which time they the mice also received 2
subcutaneous injections of 10 mg/kg anti-miR33 or an equivalent volume of PBS the first
week, spaced 2 days apart, and weekly injections of 10 mg/kg anti-miR33 (or PBS),
thereafter, for 3 weeks.2
2
Biochemical and Histological Analysis
Serum concentrations of ALT, TG, glucose, and cholesterol were determined as
previously described.3 A commercially available enzyme-linked immunosorbent assay
kit (R&D Systems) was used to determine serum insulin. Hepatic TG content was
measured as previously described.3 Liver cholesterol levels or the cholesterol content of
HSCs were measured using the Cholesterol/Cholesteryl Ester Quantitation Kit
(BioVision, Mountain View, CA), following the manufacturer’s instructions. We
determined the cholesterol content of HSCs immediately after isolation. Liver tissues
were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with H&E and a
Masson's trichrome solution. For protein or RNA analysis, tissues were frozen in liquid
nitrogen and stored at 80°C until needed.
Kupffer cell depletion
Male 9-week-old male C57BL/6J mice were injected dichloromethylene diphosphonic
acid (DMDP, Clodronate)-loaded or PBS-loaded liposomes (Encapsula NanoSciences)
intravenously (200 l per mouse). Thereafter, animals were fed a CE-2 diet, HC diet,
MCD diet, or MCD + HC diet for 8 weeks. At 2, 4, and 6 weeks after start of feeding, they
were injected with liposomal clodronate or vehicle (100 l per mouse).
3
Immunohistochemistry
Paraffinized sections were deparaffinized, rehydrated, blocked with normal horse serum,
and incubated with anti-SMA monoclonal antibody (mAb) 1A4 (Dako Japan, Kyoto,
Japan) or anti-F4/80 mAb (Serotec, Oxford, UK), overnight at 4°C. The mouse F4/80
antigen is a 160-kDa glycoprotein expressed by mouse macrophages; anti-mouse F4/80
antibody binds mouse monocytes/macrophages and Kupffer cells. The antigen is not
expressed by either lymphocytes or polymorphonuclear cells. Antibody binding was
detected by incubation with biotinylated antimouse IgG antibody and visualized with a
Vectastain Elite ABC Kit (Vector Laboratories, Inc. Burlingame, California, USA) by
reaction with Vectastatin DAB Substrate (Vector).
Fresh frozen liver sections were cut 6-mm thick on a cryostat, collected on slides, and
immediately dried. The sections were fixed with acetone. The slides were incubated
overnight with anti-CD68 Ab (Serotec), followed by incubation with Histofine Simple
Stain Mouse MAX-PO (Nichirei, Tokyo, Japan) for 1 hour.
Detection of apoptosis
4
TUNEL staining (Chemicon International, Temecula, CA) was performed on specimens
to assess apoptosis. Apoptosis was quantified by counting positively stained cells in 10
random fields at 200x magnification. Apoptosis was measured for each specimen as a
percentage of total cells per field. Antibody binding was detected by incubation with
biotinylated anti-mouse IgG antibody and visualized with a Vectastain Elite ABC Kit
(Vector) by reaction with Vectastatin DAB Substrate (Vector).
Plasmid Construction
Mouse Insig-1 and Insig-2 cDNAs were obtained by RT-PCR by using mouse liver RNA
and the following primers: Insig-1, 5'-CTG GAC GCC GAT GCC CAG GC and 5'-GTC
ACT GTG AGG CTT TTC CG; Insig-2, 5'-CTG GAC GCC GAT GCC CAG GC and
5'-GTC ACT GTG AGG CTT TTC CG. Each of the PCR products was cloned into
pCAGGS vector and the sequence was verified by DNA sequencing (pCAGGS-Insig1
and pCAGGS-Insig2). Empty vector was used as a control (pCAGGS-control).
HSC Isolation and Cell Culture
For FC accmulation in HSCs, HSCs were incubated with LDL (200g/ml) or 10% FBS
for 16 hours. 25-HC (1 g/ml) and MCD/cholesterol complex (15 g/ml) were added to
5
HSC cultures, as previously described.4 LPS (100 ng/ml) and/or TGF(1 ng/ml) were
added to HSC cultures, as previously described.3 Chloroquine (10 M) and/or MG-132
(40 M) were added to HSC cultures, as previouly described.5 6 Transfection with
si-RNAs (100 nM), anti-miR33a, pre-miR33a, control-miR33a, pCAGGS vectors, or pCI
vectors was done using Lipofectamine RNAiMAX (Invitrogen), according to the
manufacturer's instructions. PPAR antagonist, GW9662, or vehicle was added to HSC
cultures, as previously described.7
Hepatocyte Isolation and Cell Culture
Primary cultured hepatocytes were prepared from the livers of mice and plated on
six-well collagen-coated culture plates, as previously described.8
Trypsin Cleavage Assay of Scap
This assay was performed on the 20,000 × g membrane suspension prepared from HSCs
or hepatocytes isolated from the livers of hamsters (Sankyo labo Service Corporation,
Tokyo, Japan), as previously described.9 The following primary antibodies were used for
immunoblot analyses: 5 g/ml IgG-9D5, a mouse monoclonal antibody against hamster
Scap (amino acids 540-707) (Santa Cruz Biotechnology), as previously described.9
6
Real time quantitative and reverse transcription polymerase chain reaction analysis
for quantification of mRNA expression
Total RNA was extracted from total liver homogenates or HSCs using RNeasy Mini Kit
(Qiagen, Dusseldorf, Germany). Reverse transcription was performed as previously
described.8 For quantitation of mRNA expression, the following real-time PCR
amplifications were performed in duplicate, using the SYBR Premix Ex Taq (Perfect Real
Time) kit (TaKaRa Bio, Ohtsu, Japan) in a Thermal Cycler Dice Real Time system
(TaKaRa Bio).
Quantitative RT-PCR Measurement of miRNA
Total RNA was isolated from total liver homogenates or HSCs using RNeasy Mini Kit
(Qiagen). cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit
(Applied Biosystems, Foster City, CA), according to manufacturer's instructions.
Real-time PCR amplification was performed in duplicate, using TaqMan MicroRNA
Assay (Applied Biosystems). Relative quantitation (RQ) was calculated by the ddCT
method normalizing miRNA expression to the endogenous control SnoRNA202 (Applied
Biosystems).
7
Nuclear Extraction
Nuclear extracts from liver tissues, HSCs, or hepatocytes were prepared using NE-PER
Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce Biotechnology, Rockford, IL).
Isolation of Mitochondria and Late Endosomes/Lysosomes from HSCs
The mitochondrial fractions were enriched from HSCs and liver specimens with a
Mitochondria Isolation Kit (Sigma) by two consecutive centrifugation steps at 600 × g
and 11,000 × g, following the manufacturer’s instructions. Late endosomal/lysosomal
fractions were also prepared from HSCs using the lysosome isolation kit (Sigma),
following the manufacturer’s instructions.
Western Blot Analysis
Western blotting was performed as described,3 using the following antibodies: TLR4,
PPAR, SREBP2, and Insig-1 (Abcam, Cambridge, UK), Scap (Santa Cruz
Biotechnology, Santa Cruz, CA), Insig-2 (Santa Cruz Biotechnology), and -actin
(Sigma).
8
Immunoprecipitation
200 g lysates were immunoprecipitated with anti-Scap antibody using the Catch &
Release Immunoprecipitation kit (Millipore, Billerica, MA) as recommended by the
manufacturer, then subjected to SDS/PAGE electrophoresis and transferred to a PVDF
membrane. Membranes were probed with anti-Insig-1 antibody or anti-Insig-2 antibody.
9
References
1.
Inoue I, Hayashi K, Yagasaki F, Nakamura K, Matsunaga T, Xu H,
Inukai K, et al. Apoptosis of endothelial cells may be mediated by genes of
peroxisome proliferator-activated receptor gamma 1 (PPARgamma 1) and
PPARalpha genes. J Atheroscler Thromb 2003;10:99-108.
2.
Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S,
van Gils JM, et al. Antagonism of miR-33 in mice promotes reverse cholesterol
transport and regression of atherosclerosis. J Clin Invest 2011;121:2921-2931.
3.
Teratani T, Tomita K, Suzuki T, Oshikawa T, Yokoyama H, Shimamura
K, Tominaga S, et al. A high-cholesterol diet exacerbates liver fibrosis in mice
via accumulation of free cholesterol in hepatic stellate cells. Gastroenterology
2012;142:152-164 e110.
4.
Abi-Mosleh L, Infante RE, Radhakrishnan A, Goldstein JL, Brown MS.
Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport
of cholesterol in Niemann-Pick type C cells. Proc Natl Acad Sci U S A
2009;106:19316-19321.
5.
Chuang TH, Ulevitch RJ. Triad3A, an E3 ubiquitin-protein ligase
regulating Toll-like receptors. Nat Immunol 2004;5:495-502.
10
6.
Husebye H, Halaas O, Stenmark H, Tunheim G, Sandanger O, Bogen B,
Brech A, et al. Endocytic pathways regulate Toll-like receptor 4 signaling and
link innate and adaptive immunity. EMBO J 2006;25:683-692.
7.
Paik YH, Kim JK, Lee JI, Kang SH, Kim DY, An SH, Lee SJ, et al.
Celecoxib induces hepatic stellate cell apoptosis through inhibition of Akt
activation and suppresses hepatic fibrosis in rats. Gut 2009;58:1517-1527.
8.
Tomita K, Oike Y, Teratani T, Taguchi T, Noguchi M, Suzuki T,
Mizutani A, et al. Hepatic AdipoR2 signaling plays a protective role against
progression of nonalcoholic steatohepatitis in mice. Hepatology
2008;48:458-473.
9.
Brown AJ, Sun L, Feramisco JD, Brown MS, Goldstein JL. Cholesterol
addition to ER membranes alters conformation of SCAP, the SREBP escort
protein that regulates cholesterol metabolism. Mol Cell 2002;10:237-245.
11
Supplemental Figure legends
Supplementary Figure 1. Accelerated liver fibrosis after increased cholesterol
intake in the two mouse models of NASH.
To clarify the precise role of cholesterol in the pathophysiology of NASH, we used the
methionine–choline deficient (MCD) diet-induced mouse model of NASH1 and the high
fat (HF) diet-induced mouse model of NASH, which show the typical features of the
disease, including macrovesicular steatosis, cellular inflammatory infiltrate, and
pericellular fibrosis. C57BL/6 mice (9 weeks old, male) were fed (A–C) the control (n=6),
HC (n=7), MCD (n=9), or MCD+HC (n=8) diet for 12 weeks or (D–F) the control (n=6),
HC (n=7), HF (n=8), or HF+HC (n=8) diet for 24 weeks. (A and D) H&E-stained,
Masson's trichrome-stained, and SMA-immunostained sections of representative liver
samples. (B and E) Quantification of Masson's trichrome staining (upper panel) and
SMA immunostaining (lower panel). (C and F) Quantification of hepatic collagen 11,
collagen 12, SMA, and TGF mRNA. The livers of the MCD+HC diet-fed mice,
compared with the MCD diet-fed mice, significantly enhanced mRNA expressions of
collagen 1α1, collagen 1α2, andαSMA. TGFβ mRNA levels were not significantly
different between these groups. As observed in the MCD diet-induced NASH model,
12
mRNA expressions of collagen 1α1, collagen 1α2, and αSMA were significantly
enhanced in the livers of the HF+HC diet-fed mice, compared with the HF diet-fed mice.
Again, TGFβ mRNA levels showed no significant differences between these groups. **P
< 0.01, compared with the control diet group. All data are expressed as means (SEM).
Supplementary Figure 2. Hepatocellular damage in the two mouse models of NASH.
C57BL/6 mice (9 weeks old, male) were fed (A–C) the control (n=6), HC (n=7), MCD
(n=9), or MCD+HC (n=8) diet for 12 weeks or (D–F) the control (n=6), HC (n=7), HF
(n=8), or HF+HC (n=8) diet for 24 weeks. (A and D) Serum alanine aminotransferase
(ALT) activity. The levels of serum ALT, a biological marker of hepatocellular damage,
were increased significantly in the mice fed the MCD and HF diets, compared with those
fed the corresponding control diets; these increases were not affected by the increased
intake of cholesterol. **P < 0.01, compared with the control diet group. (B and E)
Percentage of terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end
labeling (TUNEL)-positive hepatocytes and the representative sections. Increased
cholesterol intake did not significantly impact the numbers of TUNEL-positive
hepatocytes in the livers of the mice fed the MCD and HF diets. **P < 0.01, compared
with the control diet group. (C and F) Quantification of hepatic FC and CE (left panel)
13
and TG (right panel). Although increased intake of cholesterol significantly raised the
hepatic content of FC, it did not significantly impact the hepatic triglyceride
concentration. **P < 0.01, compared with FC in the control diet group, #P < 0.05,
compared with CE in the control diet group. All data are expressed as means (SEM).
Supplementary Figure 3. Hepatic Cyp27a1 expression and mitochondrial FC
content in the two mouse models of NASH.
C57BL/6 mice (9 weeks old, male; n = 6–9/group) were fed (A and C) the control, HC,
MCD, or MCD+HC diet for 12 weeks or (B and D) the control, HC, HF, or HF+HC diet
for 24 weeks. (A and B) Quantification of hepatic Cyp27a1 mRNA. (C and D)
Quantification of mitochondrial FC and CE in livers.
Supplementary Figure 4. Kupffer cell activation in the two mouse models of NASH.
C57BL/6 mice (9 weeks old, male; n = 6–9/group) were fed (A–C) the control, HC, MCD,
or MCD+HC diet for 12 weeks or (D–F) the control, HC, HF, or HF+HC diet for 24
weeks. (A and D) Immunohistochemical detection of F4/80-positive cells in the mouse
livers. (B and E) Quantification of F4/80 immunostaining and mRNA. Macrophage
infiltration into the liver, evaluated by immunohistochemical staining using the F4/80
14
antibody, was significantly enhanced in the mice fed the MCD and HF diets, compared
with those fed the corresponding control diets, and these increases were not influenced by
the increased intake of cholesterol. (C and F) Quantification of hepatic TNF, VCAM-1,
ICAM-1, CD68, and CD11b mRNA. Although MCD and HF diet feeding significantly
promoted the mRNA expression of tumor necrosis factor α (TNFα), mainly produced by
Kupffer cells in the liver,2 the increased intake of cholesterol did not enhance this
expression. In addition, MCD and HF diet feeding significantly increased the mRNA
expressions of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion
molecule-1 (ICAM-1), CD68, and CD11b, but these increases were not impacted by the
increased intake of cholesterol. **P < 0.01 and *P < 0.05, compared with the control diet
group. All data are expressed as means (SEM).
Supplementary Figure 5. Foam cell formation, Cyp27a1 expression, and
mitochondrial or late endosomal/lysosomal cholesterol contents in Kupffer cells in
the two mouse models of NASH.
Immunohistochemical detection of CD68-positive cells in representative liver samples
from C57BL/6 mice (9 weeks old, male) fed (A upper panel) the control, HC, MCD, or
MCD+HC diet for 12 weeks or (A lower panel) the control, HC, HF, or HF+HC diet for
15
24 weeks. (B) Quantification of Cyp27a1 mRNA in Kupffer cells isolated from the mice
in each group. (C) Quantification of cellular FC and CE in mitochondria in Kupffer cells
from the mice in each group. (D) Quantification of cellular FC and CE in late
endosomes/lysosomes in Kupffer cells from the mice in each group.
Supplementary Figure 6. Effects of increased intake of cholesterol on liver fibrosis
in Kupffer cell-depleted mice with NASH.
To determine whether the aggravation of liver fibrosis resulting from the increased intake
of cholesterol required the presence of Kupffer cells, C57BL/6 mice (9 weeks old, male; n
= 5–7/group) depleted of Kupffer cells with liposomal clodronate were fed the control,
HC, MCD, or MCD+HC diet for 8 weeks. (A) Immunohistochemical detection of
F4/80-positive cells and quantification of F4/80 immunostaining in the mouse livers. (B)
Quantification of hepatic F4/80, CD68, and TNF mRNA. (C) Serum ALT activity.
Liposomal clodronate achieved almost complete depletion of Kupffer cells, along with
the suppression of TNFα. However, it did not impact hepatocellular injury in these
mice.**P < 0.01 and *P < 0.05, compared with the control-diet vehicle-treated group. (D)
(Left panel) H&E-stained, Masson's trichrome-stained, and SMA-immunostained
sections of representative liver samples. Quantification of Masson's trichrome staining
16
(right upper panel) and SMA immunostaining (right lower panel). **P < 0.01,
compared with the control-diet clodronate-treated group. (E) Quantification of hepatic
collagen 11, collagen 12, and SMA mRNA. (F) Serum ALT activity. The mRNA
expressions of collagen 1α1, collagen 1α2, and αSMA were significantly enhanced in the
livers of the MCD+HC diet-fed mice, compared with the MCD diet. Serum ALT levels,
however, were not affected by the increased intake of cholesterol. **P < 0.01, compared
with the control-diet clodronate-treated group. All data are expressed as means (SEM).
Supplementary Figure 7. Enhanced FC accumulation in HSCs and the consequent
upregulation of TLR4 expression and downregulation of Bambi expression in the
two mouse models of NASH.
C57BL/6 mice (9 weeks old, male; n = 6–9/group) were fed (A–C) the control, HC, MCD,
or MCD+HC diet for 12 weeks or (D–F) the control, HC, HF, or HF+HC diet for 24
weeks. To examine the effects of increased cholesterol intake on HSCs, these cells were
isolated from mice in each experimental group. (A and D) Quantification of cellular FC
and cholesterol ester (CE) in HSCs immediately after isolation from the mice in each
group. **P < 0.01 versus FC in the control diet group. #P < 0.05 versus CE in the control
diet group. (B and E) Quantification of Bambi, TGFβ receptor-1 (TGFβR1), and TGFR2
17
mRNA in HSCs immediately after isolation from the mice in each group. **P < 0.01 and
*P < 0.05, compared with the control diet group. (C and F) Quantification of TLR4
mRNA (left panel) and expression of TLR4 protein (right panel) in HSCs isolated from
the mice in each group. The relative levels of TLR4 to -actin are indicated below the
corresponding bands. All data are expressed as means (SEM).
Supplementary Figure 8. Treatment with antagomirs against miR33a significantly
alleviates the activation of HSCs in a mouse model of liver fibrosis induced by CCl4.
C57BL/6 (9 weeks old, male; n = 5/group) were given CCl4 twice a week for 4 weeks,
along with and in the absence of anti-miR33 treatment.
(A) Quantification of cellular FC and CE in HSCs isolated from the mice in each group.
(B) Expression and quantification of TLR4 protein in HSCs isolated from the mice in
each group. **P < 0.01, compared with the control group. (C) Quantification of collagen
1α1, collagen 1α2, αSMA, and Bambi mRNA and miR-33a in HSCs isolated from the
mice in each group. *P < 0.05, compared with the control group. All data are expressed as
means (SEM).
Supplementary Figure 9. SREBP2-mediated feedback regulation of cholesterol
18
homeostasis in the two mouse models of NASH.
C57BL/6 mice (9 weeks old, male; n = 6–9/group) were fed (left panel of A, and B) the
control, HC, MCD, or MCD+HC diet for 12 weeks or (right panel of A, and C) the control,
HC, HF, or HF+HC diet for 24 weeks. (A) Expression and quantification of total and
nuclear SREBP2 protein in whole livers. **P < 0.01, compared with the control diet
group. (B and C) Quantification of hepatic LDLR and HMGCR mRNA. **P < 0.01, *P <
0.05, and #P = 0.10, compared with the control diet group. All data are expressed as
means (SEM).
Supplementary Figure 10. The cholesterol-induced Scap conformational change in
hepatocytes and HSCs.
(A) Altered trypsin digestion of Scap after incubation of hamster hepatocytes, qHSCs, or
aHSCs with MCD/cholesterol complex or not. The relative intensities of the upper and
lower bands are indicated below the corresponding bands. (B) Expression and
quantification of the relative intensities of the upper and lower bands following the
trypsin cleavage assay in hamster hepatocytes or aHSCs, incubated with
MCD/cholesterol complex. All data are expressed as means (SEM).
19
Supplementary Figure 11. The feedback regulation system of cholesterol
homeostasis impacts the sensitization of HSCs to TGFβ-induced activation.
(A) Quantification of Bambi mRNA in HSCs, treated with Scap-siRNA, Insig-1 siRNA,
or control-siRNA in the presence of LPS and/or LDL. **P < 0.01, compared with the
control culture. (B) Quantification of collagen 11 and collagen 12 mRNA in HSCs,
treated with Scap-siRNA, Insig-1 siRNA, or control-siRNA in the presence of LPS,
TGF, and/or LDL. **P < 0.01, compared with the control culture. (C) Quantification of
Bambi mRNA in HSCs, treated with Insig-2-O/E vector or control vector in the presence
of LPS and/or LDL. **P < 0.01 and *P < 0.05, compared with the control culture. (D)
Quantification of collagen 11 and collagen 12 mRNA in HSCs, treated with
Insig-2-O/E vector or control vector in the presence of LPS, TGF, and/or LDL. **P <
0.01 and *P < 0.05, compared with the control culture. (E) Quantification of Bambi
mRNA in HSCs, treated with Insig-1-O/E vector, Insig-2-O/E vector, or control vector in
the presence of LPS and/or LDL. **P < 0.01 and *P < 0.05, compared with the control
culture. (F) Quantification of collagen 11 and collagen 12 mRNA in HSCs, treated
with Insig-1-O/E vector, Insig-2-O/E vector, or control vector in the presence of LPS,
TGF, and/or LDL. **P < 0.01, compared with the control culture. All data are expressed
as means (SEM).
20
Supplementary Figure 12. Downregulation of Insig-1 expression by HSC activation.
C57BL/6 mice (9 weeks old, male; n = 6–9/group) were fed (A) the control, HC, MCD, or
MCD+HC diet for 12 weeks or (B) the control, HC, HF, or HF+HC diet for 24 weeks. (A
and B) Quantification of Scap and Insig-1 mRNA in HSCs isolated from the mice in each
group. **P < 0.01 and *P < 0.05, compared with the control diet group. (C) Expression of
Insig-1 and Scap protein in HSCs cultured for 1, 3, 5, or 7 days after isolation from
C57BL/6 mice. The relative protein levels are indicated below the corresponding bands
(upper column). Quantification of Insig-1 and Scap mRNA in HSCs cultured for 1, 3, 5,
or 7 days after isolation from C57BL/6 mice (lower column). **P < 0.01, compared with
the day 1 cultures. All data are expressed as means (SEM).
21
References
1.
Tomita K, Tamiya G, Ando S, Ohsumi K, Chiyo T, Mizutani A,
Kitamura N, et al. Tumour necrosis factor alpha signalling through activation of
Kupffer cells plays an essential role in liver fibrosis of non-alcoholic
steatohepatitis in mice. Gut 2006;55:415-424.
2.
Su GL. Lipopolysaccharides in liver injury: molecular mechanisms of
Kupffer cell activation. Am J Physiol Gastrointest Liver Physiol
2002;283:G256-265.