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The role of estrogen-related receptor alpha (NR3B1) in nonalcoholic fatty liver diseases
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The role of estrogen-related receptor alpha (NR3B1) in nonalcoholic fatty liver diseases
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Content
The Role of Estrogen-Related Receptor Alpha (NR3B1)
in Nonalcoholic Fatty Liver Diseases
By CHIEN-YU CHEN
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2019
Copyright 2019
Dedication
to my mother, Julie Oun
i
Acknowledgements
My sincere thanks to my advisor, Dr. Bangyan Stiles, for her guidance throughout
my Ph.D. study at USC. You have always encouraged us to think critically like a scientist
and supported us to pursue our goals in research. The passion and wisdom that I have
learned from you not only further my education but also benefit my future.
I also would like to thank my dissertation committee, Dr. Andrew Stolz, Dr. Jean
Shih, and Dr. Enrique Cadenas. Thank you for staying with me from the beginning to the
last moment in the thesis defense. The academic advice and expertise you offered are
invaluable to me and have laid a foundation for this project.
Gratefully thank Dr. Peter Dervan and his group in Caltech for the resources and
insights to this study. It is your help that greatly advances our project to the next level.
Special thanks to my co-workers in Stiles’ Lab at USC. Dr. Yang Li, Jingyu
Chen, Taojian Tu, Lina He and many others who have helped me technically and
emotionally for making my everyday laboratory life delightful.
My most heartfelt acknowledgment to Peiyee Lim, my family, and mother. Your
unconditional love and companionship have been the greatest backing to my whole life.
Joshua Chien-yu Chen
University of Southern California
July 2019
ii
Table of Contents
Acknowledgements ............................................................................................................ i
Table of Contents .............................................................................................................. ii
List of Figures ................................................................................................................... iv
List of Tables ..................................................................................................................... vi
Abstract ............................................................................................................................ vii
Chapter I. Introduction .................................................................................................... 1
I-1: Prevalence of NAFLD .......................................................................................................... 1
I-2: Histopathogenesis................................................................................................................. 1
I-3: Risk factors and metabolic basis .......................................................................................... 3
I-4: Potential therapeutic targets ................................................................................................. 4
Chapter II: NR3B1 - a potential therapeutic target for NAFLD/NASH ..................... 6
II-1: NR3B as a regulator for lipid homeostasis ......................................................................... 6
II-2: Design of a polyamide molecule to target NR3B DNA-binding activity ......................... 13
II-3: NR3B-PA as an effective NR3B1-targeting inhibitor ....................................................... 21
II-4: NR3B-PA’s specificity toward NR3B1 downstream gene targets .................................... 26
II-5: Conclusion ........................................................................................................................ 28
iii
Chapter III: Inhibition of NR3B1 blocks hepatic steatosis caused by dietary and
genetic induction ............................................................................................................. 29
III-1: PTEN-null model for NAFLD/NASH ............................................................................. 31
III-2: Inhibiting NR3B1 with NR3B-PA prevents in vivo NAFLD development ..................... 35
III-3: NR3B1 inhibition leads to suppressed de novo lipogenesis and lipid oxidation ............. 37
III-4: Inhibiting NR3B1 with NR3B-PA reverses the hepatic steatosis induced by dietary
treatment or PTEN deletion ...................................................................................................... 40
III-5: NR3B-PA treatment ameliorates NASH phenotype ........................................................ 42
III-6: Conclusion ....................................................................................................................... 45
Chapter IV: Mechanistic study of NR3B1 in triacylglycerol biosynthesis ................. 46
IV-1: Downregulation of glycerolipid biosynthesis genes by NR3B1-KD .............................. 48
IV-2: The effect of NR3B1 inhibition is partially mediated via CEBPb .................................. 53
IV-3: GPAT4 is a novel target of NR3B1.................................................................................. 59
IV-4: Conclusion ....................................................................................................................... 66
Chapter V: Materials and Methods ............................................................................... 69
Bibliography .................................................................................................................... 77
Supplement ...................................................................................................................... 84
iv
List of Figures
Figure II-1. NR3B is the master regulator for mitochondrial functions and biogenesis..... 8
Figure II-2. The DNA binding motif sequence for the nuclear receptor NR3B. .............. 10
Figure II-3. The general binding model for a hairpin polyamide to Watson-Crick base-
paring DNA. ...................................................................................................................... 14
Figure II-4. Structure of Polyamide 1 (NR3B-PA) to target DNA sequence 5’-
AAGGTCA-3’. ................................................................................................................. 16
Figure II-5. The Polyamide 1 (NR3B-PA) reduces NR3B1 binding to the ERRE motif. 18
Figure II-6. NR3B-PA suppresses the NR3B1-dependent luciferase expression. ............ 20
Figure II-7. NR3B-PA suppresses NR3B1 function and reduces mitochondria respiration
similar to siNR3B1-KD and NR3B1 inverse agonist XCT. ............................................. 23
Figure II-8. NR3B-PA suppresses NR3B1-depdent CytC expression .............................. 25
Figure II-9. NR3B-PA and siNR3B1 share common downstream gene targets in Huh7
cells ................................................................................................................................... 27
Figure III-10. The insulin/PI3K/AKT signaling pathway ................................................. 30
Figure III-11. The liver specific PTEN deletion mouse model ......................................... 33
Figure III-12. Down-regulation of PTEN correlated with the up-regulation of NR3B1 is
associated with human liver diseases. ............................................................................... 33
Figure III-13. NR3B1 is robustly expressed in Pm hepatocyte from the NAFLD/NASH
mouse model. .................................................................................................................... 34
Figure III-14. NR3B1 inhibition prevents the PTEN loss-induced NAFLD development.
........................................................................................................................................... 36
v
Figure III-15. NR3B1 inhibition prevents fatty liver development by reducing the de novo
lipogenesis......................................................................................................................... 39
Figure III-16. NR3B-PA blocks hepatic lipogenesis induced by high carbohydrate diet
(HCD). .............................................................................................................................. 41
Figure III-17. NR3B-PA reverss the hepatic steatosis and reducs macrophage infiltration
in the NASH mice induced by PTEN loss ........................................................................ 44
Figure IV-18. Hepatic lipid droplet accumulation results from an imbalance in lipid
synthesis, storage, secretion and fatty acid oxidation. ...................................................... 47
Figure IV-19. NR3B1 inhibition downregulates the glycerolipid biosynthesis ............... 52
Figure IV-20. Candidate transcription factors, CEBPb and PGC-1β, are highly expressed
in liver. .............................................................................................................................. 55
Figure IV-21. The glycerolipid biosynthetic genes are independent of PGC-1β in Huh7
cells ................................................................................................................................. 57
Figure IV-22.The glycerolipid genes under the regulation of NR3B1 are dependent on
CEBPb expression ............................................................................................................ 58
Figure IV-23. NR3B1 binds to the Dgat1 and Gpat4 promoter regions and regulates their
expression. ........................................................................................................................ 63
Figure IV-24. Master regulator of NR3B1 in hepatic triacylglycerol biosynthesis .......... 68
vi
List of Tables
Table II-1. NR3B1 directly targets the lipogenic genes and lipogenesis-related
transcriptional factors ....................................................................................................... 12
Table II-2. NR3B1 active expression in human liver diseases ......................................... 12
Table IV-3. TG biosynthesis-related transcription factors that are modulated by siNR3B1
........................................................................................................................................... 54
Table IV-4. Transcription factors that are under NR3B1 transcriptional control ............. 54
Table S-5. Genes regulated by 1 μM NR3B-PA (>2 fold) ................................................ 84
Table S-6. Genes regulated by 0.1 μM NR3B-PA (>2 fold) ............................................. 86
Table S-7. Genes regulated by siNR3B1 (>2 fold) ........................................................... 87
vii
Abstract
The nuclear receptor NR3B1 plays an important role in energy homeostasis
especially the lipid metabolism. Using a liver-conditional PTEN deletion model where
the activation of its downstream PI3K/AKT signaling pathway led to fatty liver and
steatohepatitis, we reported previously that the NAFLD/NASH development was
accompanied by elevated mitochondrial bioenergetics and dramatic induction of NR3B1.
In this project, a polyamide (NR3B-PA) that recognized the promoters in NR3B target
genes was designed. It was shown that NR3B-PA blocked the NR3B1 binding to the
estrogen-related orphan receptor response element (ERRE) and inhibited NR3B1’s
transcriptional activity. In the mouse models, we discovered that inhibiting NR3B1 not
only prevented the early fatty liver development but also reversed the steatosis in NASH.
We further demonstrated that NR3B-PA inhibited NAFLD/NASH by suppressing the key
metabolic pathways for de novo lipogenesis and glycerolipid biosynthesis.
Mechanistically, NR3B1 directly regulated the transcription of fatty acid synthase (Fas),
acetyl-CoA carboxylase (Acc), and diacylglycerol acyltransferase (Dgat) genes; through
the downstream transcription factor CEBPb, NR3B1 also modulated other glycerolipid
genes such as Gpam, Dgat1 and Dgat2. Our result also for the first time identified
glycerol-3-phosphate acyltransferase (Gpat4) as a transcriptional target of NR3B1.
Collectively, NR3B1 is the master regulator for lipid biosynthesis and plays an important
role in the progression of hepatic steatosis. Our study underscores the NR3B1’s essential
role in hepatic triacylglycerol production and its therapeutic potential in fatty liver
diseases.
1
Chapter I. Introduction
I-1: Prevalence of NAFLD
Non-alcoholic fatty live disease (NAFLD) is the most common type of liver
disease in the developed world. It has various manifestations described in both sexes in
all ethnicities (Younossi et al., 2016). Over the last 20 years, the prevalence of NAFLD
has generally increased in all areas. The worldwide prevalence of NAFLD
is estimated at up to 1 billion, affecting around 80 to 100 million people in the United
States, with nearly 25% of patients with NAFLD progressing towards a more severe form
of nonalcoholic steatohepatitis (NASH) (Loomba & Sanyal, 2013). In Europe, the
median prevalence is approximately 26% with wide variations in different populations
(Bedogni et al., 2007). Asia is facing with the highest epidemic of obesity, and therefore
the prevalence of NAFLD was not surprisingly increased rapidly (Farrell, Wong, &
Chitturi, 2013). Since westernized diet has been introduced, adolescents in China have a
prevalence of NAFLD in excess of 25%. In Korea, Japan and Taiwan, they have an
increasing prevalence of NAFLD from 11 to 26 % (Farrell et al., 2013). As a result of the
high prevalence, it is expected that NAFLD will become the leading cause of
hepatocellular carcinoma (HCC) and liver transplant in the next decade (Calzadilla Bertot
& Adams, 2016).
I-2: Histopathogenesis
NAFLD is characterized by abnormal accumulation of fat in the liver (hepatic
steatosis) and is a continuum of liver abnormalities, from simple lipid accumulation to
2
steatohepatitis, a state in which steatosis is combined with inflammation. One of the
reasons behind NAFLD's high prevalence is its strong association with obesity, a global
epidemic mainly associated with hypercaloric diet and low physical activity and its
direct consequences, insulin resistance, and dyslipidemia. NAFLD usually presents in
patients with an unhealthy dietary pattern that has the characteristics of high
consumption of esterified fat with cholesterol and fructose. However, fatty liver can
also occur in lean insulin resistance patients, or in patients with genetic liver metabolic
disorders (Kim & Kim, 2017). The liver may remain fatty without disturbing liver
functions. However, via various mechanisms and possible insults to the liver, it may also
progress into NASH, hepatocellular ballooning and sometimes fibrosis (Calzadilla Bertot
& Adams, 2016). As NASH evolves, over time it can result in excessive scarring in the
liver known as hepatic fibrosis, a natural response to injury, which can lead to liver
cirrhosis or liver cancer. NAFLD's pathophysiology has yet to be clarified. Nonetheless,
primary NAFLD has a strong relationship with metabolic syndrome and insulin
resistance (Utzschneider & Kahn, 2006). The insulin resistance may be defined as a
condition in which a normal metabolic response requires more concentrations of insulin
or in which normal concentration of insulin does not achieve a normal metabolic
response. When insulin binds to the insulin receptor in cell membrane, it induces the
phosphorylation of the insulin receptor, leading to the activation of insulin receptor
substrate proteins (IRS). There are two primary insulin signal cascades: the MAPK/ERK
pathway, which controls gene expression and works with the PI3K to manage cell growth
and differentiation, and the PI3K/AKT pathway, which is responsible for most of insulin's
metabolic actions (Boucher, Kleinridders, & Kahn, 2014). AKT is a serine / threonine
3
kinase that induces the glucose transporter to translocate to the plasma membrane, thus
enabling cellular uptake of glucose (Wang et al., 1999). Glycogen synthase kinase-3
(GSK3) is also phosphorylated by AKT, resulting in activation of glycogenesis (Beurel,
Grieco, & Jope, 2015). Furthermore, AKT promotes gluconeogenesis and lipogenesis by
regulating the Forkhead box (FOX) transcription factor (Oh, Han, Kim, & Koo, 2013).
Last but not least, mTOR pathway is regulated by AKT for protein synthesis (Ricciardi et
al., 2011). The above-mentioned insulin signaling cascades are commonly subjected to
alteration when insulin resistance occurs (Boucher et al., 2014).
I-3: Risk factors and metabolic basis
The most important risk factors for NAFLD are obesity, insulin resistance and
metabolic syndrome, especially type 2 diabetes (T2D). These conditions are also
identified as risk factors for steatohepatitis and hepatic fibrosis (Angulo, Keach, Batts, &
Lindor, 1999). NAFLD prevalence among the obese population is about 34% (Vernon,
Baranova, & Younossi, 2011). Recent research has indicated that increased abdominal
fat also increases the risk for NAFLD (Yu et al., 2015). While NAFLD is common in
the obese population, obesity in patients with NAFLD is even more prevalent. Globally,
the combined prevalence of obesity is estimated at 51% in patients with NAFLD
(Younossi et al., 2016). While overall obesity is closely tied to NAFLD, the distribution
of body fat is suggested to play an important role in NAFLD pathogenesis. Excess
visceral fat in particular, through its strong link with insulin resistance and also a source
of free fatty acids, appears to be a critical determinant in NAFLD pathogenesis (Cnop et
al., 2002). Nearly all patients with NAFLD show hepatic insulin resistance, which
4
significantly increases the risk for T2D (Marchesini et al., 2001). However, NAFLD is
associated with the insulin resistance in not only the liver, but also in the body adipose
tissue (Bugianesi et al., 2005). The impaired ability of insulin responsiveness in the
whole-body level causes 40 to 50 % reduction in glucose disposal, leading to systemic
hyperglycemia (Marchesini et al., 2001). It subsequently stimulates pancreatic β-cell to
secrete more insulin, causing hyperinsulinemia, in response to the increased blood
glucose. Under hyperinsulinemia condition, insulin induces the PI3K/AKT cascade to
suppress hepatic glucose production and increase de novo lipogenesis to convert blood
glucose into fat and ultimately hepatic lipid droplet in the liver. Furthermore, a defect
insulin suppression of free fatty acids in the insulin-resistant adipose tissue on NAFLD
patients, contributing excess free fatty acids flux to the liver. In support of the notion that
insulin resistance may be an intrinsic defect in NAFLD, individuals with alterations of
the insulin receptor or with different types of lipodystrophy are often subject to NAFLD
(Alkhouri, Dixon, & Feldstein, 2009; Graham, 2009).
I-4: Potential therapeutic targets
As the prevalence and clinical burden of NAFLD grow rapidly, the need for a
FDA‐approved treatment intensifies. With the increased understanding of the NAFLD
pathogenesis, novel therapeutic strategies for NAFLD targeting various pathways of the
disease have emerged. Here we review the latest pharmacological targets in drug
pipeline and clinical trials. Peroxisome proliferator‐activated receptors (PPARs) are a
group of nuclear receptor proteins that regulate metabolic processes (Rotman & Sanyal,
2017). PPARα is expressed in multiple organs and regulates lipid metabolism and energy
5
homeostasis (Grygiel-Gorniak, 2014). PPARα and δ is expressed in liver and other
metabolically active organs where it is important to regulate energy homeostasis and
drive the hepatic metabolism towards fatty acid oxidation (Rotman & Sanyal, 2017).
Elafibranor (Genfit Inc.) is a dual PPARα/δ agonist that improves glucose homeostasis,
increases insulin metabolism, and reduces inflammation. Elafibranor is showed to
decrease plasma triacylglycerol (TG) levels and low-density lipoprotein (LDL)
cholesterol while also demonstrated an insulin-sensitizing effect in short-term Phase 2
studies (Cariou & Staels, 2014). Farnesoid X receptor (FXR) is a bile acid-activated
nuclear receptor that plays a key role in bile acid and TG synthesis in liver. Obeticholic
Acid (OCA) (Intercept Pharmaceuticals Inc.) is a synthetic bile acid that promotes insulin
sensitivity and decreases gluconeogenesis and circulating TG when bound to FXR
(Neuschwander-Tetri et al., 2015). In the Phase 2 trail, administration of OCA has
showed to increase insulin sensitivity and reduce plasma TG/high-density lipoprotein
(HDL) cholesterol and liver inflammation markers in NAFLD and T2D patients
(Mudaliar et al., 2013). Thyroid hormone receptors (THRs) are thyroid hormone-
activated nuclear receptors known to reduce body weight, influence growth, modulate
lipid metabolism (Sandler et al., 2004). Patients with abnormally high thyroid hormone
level demonstrate clinical profiles of reduced body weight and atherogenic lipids
associated with cardiovascular disease (Sandler et al., 2004). MGL-3196 (Madrigal corp.)
is a THR-β selective agonist. In Phase 1 studies, it has demonstrated to reduce the
plasma lipids, LDL cholesterol and TG in test subjects (Sandler et al., 2004). It is under
the investigation of Phase 2 and 3 clinical programs for patients with dyslipidemia and
NASH.
6
Chapter II: NR3B1 - a potential therapeutic target for NAFLD/NASH
II-1: NR3B as a regulator for lipid homeostasis
In mammals, TG is the predominant form of energy storage that sustains the
homeostatic control of energy balance. It is largely found in the energy storage tissue
such as body fat, muscle and liver. In individuals with metabolic syndromes, excess lipid,
glucose and accompanying elevated insulin signals the lipid buildup in these tissues. In
the liver, TG can accumulate via different ways. Elevated de novo lipogenesis occurs
with hyperglycemia and hyperinsulinemia in obese and insulin resistant individuals.
Transportation of excess lipid into the liver is another way liver can accumulate TG. In
addition, decreased lipid degradation or export can also cause hepatic lipid to accumulate
in hepatocytes.
Several lines of evidence suggest that nuclear receptor 3 B (NR3B), also known
as the estrogen related receptor (ERR), plays a crucial role in lipid homeostasis. The
orphan nuclear receptor subfamily comprises three members referred to as NR3B1
(ERRα), NR3B2 (ERRβ), and NR3B3 (ERRγ). They display constitutive transcriptional
activity independent of natural estrogen ligands and do not directly take part in classic
estrogen signaling pathways (Zhang, Luo, Wu, & Xu, 2015). NR3B1 is abundantly
expressed in high oxidative organs and is recognized as a key regulator of adaptive
energy metabolism in response to environmental stimuli and energy demands (Villena &
Kralli, 2008). NR3B2 is found to have a vital role in cell fate determination and
pluripotency by interacting with OCT4-SOX2 complex and NANOG (van den Berg et
al., 2008). NR3B3 works to switch bioenergetics responses to hypertrophic stress
7
primarily in heart as forced expression of NR3B3 activates the heat-specific transcription
factor GATA4 to induce cardiac hypertrophy (Kwon et al., 2013). Both NR3B1 and
NR3B3 orchestrate mitochondrial functions with the coactivator, peroxisome proliferator-
activated receptor gamma coactivator 1-alpha (PGC-1α), either by directly activating
genes encoding proteins needed to maintain the mitochondrial components, or by
indirectly activating major transcription factors governing mitochondrial biogenesis
(Figure II-1) (Gaillard, Dwyer, & McDonnell, 2007; Kamei et al., 2003). These
transcriptional factors include nuclear respiratory fator-1 (NRF-1) and -2 (NRF-2) that
control key mitochondrial respiratory components as well as mitochondrial transcription
machinery including mitochondrial transcription factor A (TFAM), mitochondrial
transcription factor B1 and B2, (TFB1M and TFB2M) and mitochondrial RNA
polymerase (POLRMT) that act on mitochondria genome (Gleyzer, Vercauteren, &
Scarpulla, 2005; Virbasius & Scarpulla, 1994). A number of genes encoding proteins
involved in the TCA cycle, mitochondrial oxidative phosphorylation and respiratory
chain such as citrate synthase, succinate dehydrogenase, cytochrome c (CytC) and
NADH dehydrogenases etc. are also directly transcriptionally regulated by NR3B1.
8
Figure II-1. NR3B is the master regulator for mitochondrial functions and
biogenesis.
The expression of mitochondrial respiratory complexes and metabolic enzymes are
controlled by the NR3B in both nucleus and mitochondria. In nucleus, nuclear
transcription factors NR3B along with coactivator PGC-1α synergistically induce the
expression of genes that directly constitute the mitochondrial respiratory apparatus. In
mitochondria, nucleus-encoded but mitochondria-localized factors such as POLRMT,
TFAM, TFB1M, TFB2M and mitochondrial termination factor (MTERF), which are also
downstream of NR3B1, can activate the transcription of mtDNA.
PGC1- α
Nucleus genome
mtDNA
16kb
I
II
III
IV
V
OXPHOS
Complex I, II,
III, IV subunits
Cytochrome c
β-oxida on
Mitochondrial metabolic genes
TCA cycle
NR3B
Tfam TFB1/2M
POLRMT mTERF
Transcrip onal factors
NRF-1
NRF-2
9
As a transcriptional factor, NR3B contains two highly conserved zinc finger
motifs that preferentially bind to the consensus DNA sequence 5’-AAGGTCA-3’ (Figure
II-2), referred as the estrogen-related response element (ERRE) (Sladek, Bader, &
Giguere, 1997). This signature motif is found at the promoter region of genes encoding
for canitine-acrylcanitine carrier (SLC25A29), involving in net transport of fatty acyl
units into mitochondrial matrix where they are oxidized by the β-oxidation reaction;
medium chain acyl-CoA dehydrogenase (MCAD), a key enzyme in the initial step of β-
oxidation; and fatty acid binding protein 3 (FABP3), involving in fatty acid and bile acid
transportation in liver (Dufour et al., 2007). In fact, the expression of MCAD, FABPs
along with other fat metabolic genes, e.g. pancreatic lipase related protein and apoprotein
A, are reduced when NR3B1 is absent in adipocytes, intestinal epithelial and hepatocytes.
These characterizations of NR3B1’s targets strongly suggest an indispensable role of
NR3B1 in fatty acid -oxidation for lipid catabolism.
10
Figure II-2. The DNA binding motif sequence for the nuclear receptor NR3B.
Sequence logos of consensus DNA binding sites, AAGGTCA referred as the estrogen-
related receptor response element (ERRE), for NR3B. The consensus sequence of NR3B
is variable: a number of possible bases at certain positions in the motif, whereas other
positions have a fixed base. The height of the letter represents how frequently that
nucleotide is observed in that position. These logos were generated from information
obtained from the MEME database (Vernon et al., 2011).
11
Despite the identification of these transcriptional targets suggest loss of NR3B
function may cause lipid buildup due to lowered lipid degradation, loss of NR3B1 in
mice led to resistance rather than sensitivity to high fat diet (HFD)-induced obesity (J.
Luo et al., 2003). This observation highlights the complexity of the physiological
functions of NR3Bs in metabolic regulation. In fact, the chromatin immunoprecipitation
with DNA microarray (ChIP-on-chip) data reveal that NR3B1 may also positively
regulate lipogenesis by directly binding to the regulatory domain of lipogenic genes
(Charest-Marcotte et al., 2010). Analysis on the literature-curated data as shown in Table
II-1 shows the putative target lipogenic genes of NR3B1. In agreement with the putative
function of NR3B1 in lipogenesis, NR3B1 along with coactivator PGC1-α are found
required for adipogenic differentiation induced by glucocorticoid, cAMP and insulin
(Ijichi et al., 2007). In NR3B1 null mice, the prevention of HFD-induced NAFLD is
attributed to the suppression of de novo lipogenesis (B'Chir et al., 2018). Taken together,
these literatures and findings support the strong role of NR3B1 in promoting lipogenesis
for TG biosynthesis in the liver. Further discovery from independent studies indicates
distinct upregulation of NR3B1 in a spectrum of liver diseases with underlying hepatic
steatosis (Table II-2), implying that NR3B1’s possible role in advancing NAFLD/NASH
development.
12
Table II-1. NR3B1 directly targets the lipogenic genes and lipogenesis-related
transcriptional factors *
*: The table is generated from the Chip-on-chip data published on Genes & Dev. (2010).
**: DAG: diacylglycerol. PA: phosphatidic acid. TG: triacylglycerol. DHAP: dihydroxyacetone phosphate.
G3P: glycerol-3-phosphate.
Table II-2. NR3B1 active expression in human liver diseases*
*: The table is generated from the BasesSpace Correlation Engine (Illumina Inc.).
**: HCV: hepatitis C. HBV: hepatitis B.
p-value Site of action Gene Function**
1.80E-07 PROMOTER Dgkz DAG to PA
8.67E-04 PROMOTER Dgat1 DAG to TG
1.85E-11 PROMOTER Gyk DHAP to G3P
8.60E-08 PROMOTER Lpin1 Glycerol to G3P
6.21E-05 PROMOTER Gpd1 Glycerol to G3P
1.47E-04 PROMOTER Agpat2 LPA to PA
4.31E-05 PROMOTER Lpin2 PA to DAG
6.73E-12 PROMOTER Nr3b1 Transcription factor
2.29E-05 PROMOTER Cebpb Transcription factor
2.39E-04 PROMOTER Jun Transcription factor
1.21E-11 PROMOTER Ppara Transcription factor
6.36E-07 PROMOTER Pgc1b Transcription factor
1.86E-04 PROMOTER Sp1 Transcription factor
Public ID Study design** p-value Fold change
GSE54102 HCV infected cirrhosis late stage vs. early stage 0.0029 18.6
GSE19665 HBV infected cancerous liver vs. noncancerous tissue 0.0348 1.69
GSE62547 Steatosis induced in hepatic progenitors vs. control 0.0028 1.69
GSE12443 Regenerative cirrhotic nodules vs. normal liver 2.20E-11 1.59
GSE12443 High-grade dysplastic nodules vs. normal liver 6.30E-05 1.53
GSE33650 HCV infected liver Ishak fibrosis stage5 vs. stage0 0.0057 1.44
GSE84587 HCV infected hepatocytes vs. control 0.0458 1.43
GSE56140 Hepatocellular carcinoma tumor vs. adjacent cirrhosis 0.0196 1.35
GSE14323 HCV hepatocellular carcinoma vs. adjacent cirrhosis 5.50E-07 1.27
13
II-2: Design of a polyamide molecule to target NR3B DNA-binding activity
To address the role of NR3B in fatty liver disease in vivo, particularly in hepatic
lipid metabolism, we aimed to design a bioavailable inhibitor for NR3B. Pyrrole-
imidazole (Py–Im) polyamides were a class of synthetic, programmable DNA minor
groove-binding ligands. Their DNA sequence selectivity was achieved by recognizing
differences in the shape and hydrogen bonding pattern presented by the edges of the
Watson-Crick base pairs (Figure II-3) (Kielkopf et al., 1998). In a heterodimeric
polyamide molecule where two polyamide strands were linked antiparallely, side-by-side
Im/Py pair discriminated G/C base pair from C/G, whereas Py/Py pair recognized both
A/T and T/A base pair (Pilch et al., 1996). The binding between polyamides and DNA
was sufficient to compete for the binding of transcriptional factor and other DNA-binding
proteins, thus, allowed the activity of transcriptional factors to be modulated by
specifically designed polyamide sequences to affect the expression of genes regulated by
the transcriptional factors (Dervan & Edelson, 2003).
14
Figure II-3. The general binding model for a hairpin polyamide to Watson-Crick
base-paring DNA.
A simple hairpin polyamide ImPyPy-γ-PyPyPy where two polyamide strands are
covalently linked with γ-aminobutyric acid serving as a turn. Circles with dots represent
lone electron pairs of either N3 of adenine, O2 of thymine, or O2 of cytosine. Circles
containing an H represent the 2-amino hydrogen of guanine. Putative hydrogen bonds are
illustrated by dotted lines. Im, imidazole; Py, pyrrole, γ, γ-aminobutyric acid.
.
.
.
.
.
.
.
.
.
.
.
.
H
.
.
.
.
5 ’ 3 ’
A
T
T
G
.
.
.
.
T
15
To inhibit the function of NR3B, we sought to interfere the binding of NR3B to
DNA target motif. The most common DNA sequence in ERRE is 5’-AAGGTCA-3’
(Figure II-2). The Dervan group who developed the polyamide technique had previously
designed several polyamides to target the estrogen receptor element (ERE) which
contains a sequence of 5’-AAGGTCAnnnTGACCTT-3’. The ERRE sequence was
similar to this ERE sequence and shared homology with half of the ERE sequence. 4
polyamide sequences were designed to target the ERE, all of them had the half maximal
inhibitory concentration (IC50) at micromolar range in a ERE-driven luciferase reporter
assay (Nickols et al., 2013): among those, Polyamide 1 that targeted the sequence 5’-
AGGTCA-3’ had the lowest IC50 at 0.14 μM; additionally, Polyamide 2 that targeted the
sequence 5’-AGTTCA has IC50 reported at 0.51 μM.
16
Figure II-4. Structure of Polyamide 1 (NR3B-PA) to target DNA sequence 5’-
AAGGTCA-3’.
This Polyamide 1 (NR3B-PA) is consisted of two polyamide strands covalently linked by
a γ-aminobutyric acid (γ-turn) linker. Within this structure design, the Py/Im pair target
C/G; Im/Py pair target G/C; Py/Py target A/T and T/A and the γ-turn linker demonstrating
selectivity for A,T over G,C. Top, chemical structure of NR3B-PA. Bottom, ball-and-
stick models of polyamides. Solid circle, imidazole; Open circle, pyrrole. IPA: isophthalic
acid.
17
We screened these two compounds for their ability to bind ERRE and displace
NR3B1 in an in vitro gel shift assay (Figure II-5). In the gel shift assay, biotin-labeled
DNA fragment containing ERRE was incubated alone (lane 1), or with nuclear extract
from cultured hepatocytes (lane 2). A band migrating at higher molecular weight than the
probe alone (lane 1) was observed when nuclear extract was incubated with free probe
(lane 2), indicating the complex of ERRE with NR3B (NR3B+ERRE). When unlabeled
competitor (cold probe) was added in lane 3, the cold probe diminished the observed
NR3B+ERRE complex band, suggesting that the complex observed in lane 2 is indeed
complexed with ERRE. When monoclonal antibody for NR3B1 is added (lane 4), the
antibody binds to the complex of NR3B+ERRE causing further upwards shift of the
complex due to increased overall molecular weight, suggesting that the complex indeed
contained NR3B1. For lanes 5-10, increasing dose of Polyamide 1 (10 pM - 1 µM in log
scale) was added to the lysate and probe mixture. The data showed that incubation with
Polyamide 1 at 0.1 µM concentration and beyond completely blocked the binding of
NR3B to ERRE as demonstrated by the diminishing band of NR3B+ERRE in lanes 9 and
10. In contrast, a 10-fold higher dose of Polyamide 2 (1 µM) was needed before any loss
of complex formation was observed (lane 16). A band was still present in lane 15, which
was incubated with 0.1 µM Polyamide 2. This result suggested that Polyamide 1 was able
to disrupt the Polyamide-ERRE complex at therapeutic dose. We termed this Polyamide
1 as NR3B-PA. The Polyamide 2 that did not exhibit ability to block binding of NR3B to
ERRE at micromolar dosing was then served as a mismatched control compound (MM-
PA) for following experiments.
18
Figure II-5. The Polyamide 1 (NR3B-PA) reduces NR3B1 binding to the ERRE
motif.
Gel shift assay performed with the Polyamide 1 designed to target NR3B1 (NR3B-PA)
and Polyamide 2 as a sequence mismatched control (MM-PA). From left, lane 1, ERRE
dsDNA only; lane 2, ERRE dsDNA + cell extract containing overexpressed NR3B1; lane
3, unlabeled competitor added; lane 4, antibody for NR3B1 added; lanes 5-10, increasing
dose (10 pM - 1 µM in log scale) of Polyamide 1; lanes 11-15, increasing dose (10 pM - 1
µM in log scale) of Polyamide 2.
19
To further evaluate whether NR3B-PA could work as an inhibitor for NR3B
transcriptional activity, we utilized a dual-luciferase reporter assay where the
transcription of firefly luciferase could be regulated by NR3B1. A pGL4-CytC-Luc
construct containing a promoter from codon -686 to +55 of CytC was introduced to and
stably expressed in mouse hepatocyte. The CytC promoter contained the sequence 5’-
AAGGTCA-3’, which was the core consensus ERRE motif for NR3B recognition. CytC
was also previously reported to be under the transcriptional regulation of NR3B1
(Schreiber et al., 2004). In the luciferase reporter expressing cells, we found that 1 μM
NR3B-PA significantly inhibited the firefly luciferase expression, whereas 1 μM MM-PA
failed to alter levels of the luciferase activity (Figure II-6).
20
Figure II-6. NR3B-PA suppresses the NR3B1-dependent luciferase expression.
Polyamide NR3B-PA and MM-PA are selected for suppression on the NR3B1 responsive
CytC-driven luciferase in mouse PTEN-null (Pm) hepatocyte. NR3B-PA blocks NR3B1
from binding to CytC promoter, thus inhibits the luciferase expression, whereas MM-PA
did not.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Neg con Na ïve Con 0.1 µM 1 µM 0.1 µM 1 µM
Rela ve luciferase ac vity
NR3B-PA MM-PA
**
21
II-3: NR3B-PA as an effective NR3B1-targeting inhibitor
NR3Bs were initially characterized as a transcriptional activator that promoted
mitochondria biogenesis and respiration in collaboration with the PGC1-α (C. Luo et al.,
2017). Thus, inhibiting NR3B1 function in hepatocytes was expected to decrease basal
and maximal mitochondrial respiration. Accordingly, we explored if modulating NR3B
DNA binding activity with NR3B-PA could affect the mitochondrial respiratory function
of NR3B. In this experiment, the human hepatocyte cell line Huh7 was used for the
measurements of oxygen consumption rate (OCR). Two siRNAs for NR3B1 were
introduced to Huh7 cells via Lipofectamine-based transfection 24 hours prior to
measurement of OCR. As expected, siRNA targeted inhibition of NR3B1 resulted in 70%
and 60% reduction of basal and maximal respiration respectively (Figure II-7B). To
assess the effect of NR3B-PA on this NR3B regulated mitochondrial respiration, Huh7
cells were pre-treated with NR3B-PA, MM-PA as well as XCT-790 (a potent NR3B1
inverse agonist) for 24 hours before measurements of OCR. Similar to siRNA treatment,
NR3B-PA treatment at 0.2 μM led to 52% and 54% reduction in basal and maximal
respiration; 1 μM led to 70% and 72% reduction in basal and maximal respiration (Figure
II-7B). This effect is also similar to that induced by treatment with XCT-790 (Figure II-
7C) (Eskiocak, Ali, & White, 2014). XCT-790 was reported as a selective NR3B1
inverse agonist that inhibits NR3B1 constitutive activity with an IC50 of ≈400 nM in
various cell-based and biochemical assays (Willy et al., 2004).
22
A
B
C
23
Figure II-7. NR3B-PA suppresses NR3B1 function and reduces mitochondria
respiration similar to siNR3B1-KD and NR3B1 inverse agonist XCT.
A. Study design for the mitochondrial respiration analysis.
B. In mouse PTEN-null hepatocyte, NR3B-PA dose dependently inhibits the overall and
basal OCR (Top), similar to siNR3B1 (Bottom). n=3, p<0.05.
C. In the human hepatocellular carcinoma Huh7, NR3B-PA (1 μM) is capable of reducing
OCR. n=3, p<0.05. Mismatched polyamide (MM-PA, 1 μM) did not inhibit OCR at
the same dose. XCT (2 μM) is an inverse agonist for NR3B used as a positive control.
NR3B-PA induced similar inhibition on the overall and basal OCR like XCT.
24
Additionally, we compared the effect of NR3B-PA on the suppression of NR3B1
regulated gene transcription. In the mouse hepatocyte Pm and human hepatocellular
carcinoma SNU398, NR3B-PA treatment resulted in downregulation of endogenous CytC
expression in a similar fashion as NR3B1 knockdown by siRNA (Figure II-8A and B).
These data strongly supported the biological function of NR3B-PA as an inhibitor of
NR3B. Taken together, our data suggested NR3B-PA could not only block NR3B1 from
binding to target gene CytC but also suppress the CytC gene expressions as well as the
mitochondrial respiration.
25
A
B
Figure II-8. NR3B-PA suppresses NR3B1-depdent CytC expression.
A. siNR3B1 against NR3B1 reduced expression of CytC in Pm mouse hepatocytes as
well as SNU398 human hepatocellular carcinoma cell line.
B. NR3B-PA (1 μM) treatment reduced the expression of CytC in Pm as well as SNU398.
26
II-4: NR3B-PA’s specificity toward NR3B1 downstream gene targets
Finally, we sought to evaluate the potential off-target effects of the NR3B-PA. We
used an unbiased genome-wide RNA-sequencing analysis to compare the global gene
expression in Huh7 cells introduced with siNR3B1 or treated with NR3B-PA at 1 μM or
0.1 μM concentrations. Unsupervised clustering analysis showed that siNR3B1 and
NR3B-PA treatments resulted in comparable changes in global gene expression patterns
as opposed to the control group (Figure II-9. top). Compared to the vehicle treated cells,
8,051 genes were found to be differentially expressed (with fold change ≥1.2; false
discover rate <0.05) when siRNA was used to inhibit NR3B function (Figure II-9.
bottom). With NR3B-PA treatment, 6,151 and 6,160 genes were found to be
differentially expressed with 0.1 and 1 μM NR3B-PA treatments respectively. Comparing
of these differentially expressed genes induced by siRNA vs. NR3B-PA, we found that
92.86% of the genes (5,720 out of 6,160 genes) altered by NR3B-PA treatment (0.1 μM)
were also altered by siNR3B1. 10.4% (640 out of 6,151 genes) were exclusively affected
in 1 μM NR3B-PA group possibly due to the off-target effect by the high concentration of
polyamide. This data was indicative of a high specificity and low off-target effect for
NR3B-PA against NR3B1 function.
27
Figure II-9. NR3B-PA and siNR3B1 share common downstream gene targets in
Huh7 cells.
More than 90% of the genes altered by NR3B-PA treatment were also targeted by
siNR3B1. Top, unsupervised cluster of all genes altered by siNR3B1-1 and NR3B-PA
treatment in Huh7. Bottom, Venn diagram of genes altered by NR3B-PA vs. those
regulated by siNR3B1-1 shows 90% overlap of these altered by NR3B-PA by those
altered by siNR3B1.
28
II-5: Conclusion
NR3B1 is a pivotal transcriptional factor that coordinates mitochondrial and lipid
homeostasis by mediating the expression of genes for mitochondrial components and for
metabolic enzymes in lipid metabolism. Here, we designed a high potency and low off-
target inhibitor, NR3B-PA, for targeting NR3B. Our data indicate that NR3B-PA
treatment is equally effective as siRNA knockdown at blocking the binding of NR3B to
ERRE as well as inhibiting the biological effects governed by NR3B such as
mitochondrial function and downstream gene expression. These findings suggest that
NR3B-PA is a potent NR3B1 inhibitor and that it may be used for studying mitochondrial
abnormality, particularly the NR3B1-associated diseases.
29
Chapter III: Inhibition of NR3B1 blocks hepatic steatosis caused by
dietary and genetic induction
Insulin resistance is closely associated with metabolic syndromes such as obesity,
T2D, and dyslipidemia and attributes to the development of NAFLD/NASH (Buzzetti,
Pinzani, & Tsochatzis, 2016). In the insulin-resistant state, hyperinsulinemia and
hyperglycemia leads to increased hepatic de novo lipogenesis and decreased inhibition of
lipolysis in peripheral adipose tissue (Azzout-Marniche et al., 2000; Browning & Horton,
2004). Studies have demonstrated that insulin signal regulates this de novo lipogenesis
through the activation of PI3K. Binding of insulin to its receptor induces PI3K leading to
activation of insulin receptor substrate (IRS) 1 and 2 and subsequent AKT
phosphorylation cascades. PTEN, or phosphatase and tensin homolog deleted on
chromosome 10, serves as a negative regulator of PI3K/AKT signaling pathway. PTEN
antagonizes the insulin/PI3K/AKT signal by dephosphorylating phophoinositides (PIP)
and modulates metabolic homeostasis through this action (Figure III-10).
30
Figure III-10. The insulin/PI3K/AKT signaling pathway
AKT
mTOR GSK3 FOXO
PI3K
Insulin receptor
PIP2
PIP3
IRS1/2
Insulin
Protein synthesis
Gluconeogenesis
Glycogenesis
MAF1
Lipogenesis
SREBP1c
31
During homeostasis, increased PI3K/AKT activation by insulin signal is required
for insulin to reduce plasma glucose. In adipose tissue and muscle, this insulin signal
stimulates uptake of glucose to maintain normoglycemia. In the liver, elevated insulin
signal shuts down gluconeogenesis to reduce glucose output to maintain normoglycemia.
During insulin resistant state, the inability to inhibit hepatic output or increase
adipocyte/muscle uptake of glucose leads to chronic hyperinsulinemia. In addition to
inhibiting gluconeogenesis, sustained hepatic hyperinsulinemia as observed with insulin
resistance can also lead to de novo lipogenesis, resulting in NAFLD and NASH.
Activation of AKT is found to be both necessary and sufficient to stimulate de novo
lipogenesis via FOXO (Gross, van den Heuvel, & Birnbaum, 2008), MAF1 (Palian et al.,
2014) and mammalian target of rapamycin complex 1 (mTORC1) (Laplante & Sabatini,
2010) to regulate the sterol regulatory element-binding protein (SREBP1c) for inducing
lipogenesis (Figure III-10).
III-1: PTEN-null model for NAFLD/NASH
The NAFLD/NASH histological phenotype is observed in a model where
uncontrolled activation of PI3K/AKT signal is produced due to loss of hepatic PTEN
(Alb-Cre
+
Pten
loxP/loxP
). In mice engineered to delete PTEN specifically in the liver,
chronic hyperinsulinemia, i.e. sustained activation of PI3K/AKT signal, is observed.
These mice develop NAFLD very early at 1 month of age (Figure III-11)(Stiles et al.,
2004). The NAFLD condition progresses to NASH with inflammatory cell infiltration
become a common observation at 6 months of age. Fibrosis is also observed starting at 6
months and advancing, as the mice get older. The mice develop HCC starting at 7 month
of age and all mice develop spontaneous tumors after 12 months of age. Thus, the
32
hepatic PTEN-loss model recapitulates a NAFLD/NASH model that will progress to the
most severe outcome, liver cancer. In addition, analysis of liver samples from healthy
donor and NASH patients shows a significant reduction of PTEN expression in NASH
samples (Figure III-12 top), indicating that the model is relevant to human NASH.
Similar to the insulin resistant state, NAFLD/NASH development in the hepatic
PTEN loss model is due to elevated de novo lipogenesis resulting from hyperactivated
PI3K/AKT signal (Stiles et al., 2004). In addition, activation of PI3K/AKT singling also
results in increased production of mitochondrial reactive oxygen species and scavenger
enzyme associated with enhanced mitochondrial function (Abid et al., 2013; Galicia et
al., 2010). This enhanced mitochondria function was the result of active mitochondrial
biogenesis due to increased activity of NR3B1 (Li et al., 2013), the predominant NR3B
isoform found elevated in the Pten deleted liver (Figure III-13). Analysis of available
Oncomine datasets for live cancer patient samples also demonstrated a negative
correlation of PTEN level to NR3B1, indicating that up-regulation NR3B1 may be
implicated in the development of liver disease (Figure III-12 bottom).
33
Figure III-11. The liver specific PTEN deletion mouse model
Figure III-12. Down-regulation of PTEN correlated with the up-regulation of
NR3B1 is associated with human liver diseases.
Top, PTEN mRNA level is significantly reduced in NASH patients. Bottom, NR3B1
expression is negatively correlated with PTEN in liver cancer patients.
NAFLD
Liver Cancer
Alb-Cre
+
Pten
loxP/loxP
model
Age: 1M 3M 6M 9M 12M
NASH
Lung
metastasis
34
Figure III-13. NR3B1 is robustly expressed in Pm hepatocyte from the
NAFLD/NASH mouse model.
Among the different isoforms in NR3Bs family, only the NR3B1, known as ERRα, was
up-regulated in response to the PTEN deficiency in the Pten-mutated hepatocyte (Pm)
compared to the wild-type control (Wt).
35
III-2: Inhibiting NR3B1 with NR3B-PA prevents in vivo NAFLD development
To test if NR3B1 inhibition could improve early NAFLD development, we treated
1.5-month old PTEN-null mice with 25 nmole NR3B-PA once every three days for 1
month via intraperitoneal injection route. As previously reported, morphology analysis of
liver section from Pten genotype control livers (Pten-Wt) displays proper architecture of
liver parenchyma without bulky lipid droplets, whereas macrovesicular fatty liver and
massive lipid deposition were found in the PTEN-null livers (Pm-Veh) (Figure III-14A
top and middle). Strikingly, 1-month NR3B-PA treatment in PTEN-null livers (Pm-
NR3B-PA) completely precluded the fatty liver phenotype, as evidenced by the restored
structure of liver lobule in H&E (hematoxylin and eosin) histological sections (Figure III-
14A bottom) and the dramatically reduced lipid deposition confirmed by Oil Red O
staining in the Pm-NR3B-PA group. The body weight of NR3B-PA-treated PTEN-null
mice (Pm-NR3B-PA) did not significantly deviate from the vehicle treated mice (Pm-
Veh), indicating a limited toxicity of NR3B-PA to the PTEN-null mice (Figure III-14B).
Consistent with the morphological analysis, hepatic TG quantification showed that
NR3B-PA treatment significantly reduced the TG buildup in PTEN-null livers, to a level
that was even lower than that of Pten genotype control group (Figure III-14B right).
Thus, targeting NR3B1 by NR3B-PA could effectively lower the excessive hepatic lipid
accumulation and improve fatty liver phenotype induced by PTEN loss.
36
A
B
Figure III-14. NR3B1 inhibition prevents the PTEN loss-induced NAFLD
development.
A. Histology of livers from the Pten wild-type control mice (Pten-Wt), Liver-specific
Pten deleted mice treated with vehicle (Pm-Veh) or with NR3B-PA (Pm-NR3B-PA).
H&E, Hemotoxylin&Eosin (H&E) staining to show liver microstructure; Oil Red O,
staining for lipid showing massive lipid droplets in the vehicle treated PTEN-null
liver. This phenotype development is blocked by NR3B-PA treatment.
B. Body weight and quantification of liver triglyceride (TG) content. Body weight is
lower in Pm mice.
37
III-3: NR3B1 inhibition leads to suppressed de novo lipogenesis and lipid oxidation
Interferences in lipid oxidation and/or de novo lipogenesis are metabolic changes
that may contribute to the reduced liver lipid deposition. The medium chain acetyl
decarboxylase gene Mcad encoded the enzyme that was essential for fatty acid oxidation
to metabolize medium-chain fatty acids and also a known transcriptional target of
NR3B1(G. Liu, Sun, Dong, & Sehouli, 2018). Expressions of NR3B1’s transcriptional
targets, CytC and MCAD, were both found higher in the PTEN-null liver tissues and
reduced by treatment of NR3B-PA (Figure III-15A), implying that there were the
suppression of lipid oxidation when NR3B1 was inhibited. Thus, enhanced degradation
of lipid is unlikely to contribute to the reduced NAFLD observed with NR3B inhibition
by NR3B-PA.
Next, we evaluated the expression of genes regulating de novo lipogenesis under
the NR3B-PA treatment, as de novo lipogenesis was how PTEN loss causes steatosis. In
the PTEN-null livers (Pm-Veh), de novo lipogenesis was induced concurrent with the
increased expression of the key lipogenic enzyme, FAS and acetyl-coA carboxylase
(ACC), as opposed to their expression levels in the Pten control group (Pten-Wt) (Figure
III-15A). When compared with vehicle treated PTEN-null mice, expression of ACC was
reduced by more than 30% while FAS expression was reduced by approximately 15% in
the NR3B-PA treated (Pm-NR3B-PA) group (Figure III-15A). These data suggested that
suppressed de novo lipogenesis was the rational mechanism by which inhibiting NR3B1
activity attenuated the early hepatic steatosis development in the PTEN-null mice.
Corroborated with the observation from the early NAFLD model, the results from gene
38
expression analysis on siNR3B1-treated Huh7 confirmed that NR3B1-KD downregulated
target CytC and Mcad expression, and reduced expression of the lipogenic genes, Fas and
Acc, expression (Figure III-15B).
39
A
B
Figure III-15. NR3B1 inhibition prevents fatty liver development by reducing the de
novo lipogenesis.
A. Expression of two rate-limiting enzymes in de novo lipogenesis, FAS and ACC are
both significantly reduced as a result of NR3B-PA treatment. Expression of MCAD,
a rate-limiting enzyme in lipid oxidation is also significantly reduced. Cytochrome c
(CytC) expression is a target gene of NR3B1 used as control.
B. Introduction of siNR3B1 to inhibit NR3B1 led to similar inhibition of FAS, ACC and
MCAD as well as CytC mRNA levels in Huh7 cells.
40
III-4: Inhibiting NR3B1 with NR3B-PA reverses the hepatic steatosis induced by
dietary treatment or PTEN deletion
To this point, we demonstrated that NR3B-PA was a potent DNA blocking agent
against NR3B1. It could be used to inhibit NR3B1 transcriptional activity and prevent
hepatic steatosis induced by Pten deletion. Analysis in the PTEN-null mice indicated that
de novo lipogenesis was the likely mechanism regulated by NR3B1. To support this
observation, we employed a high-carbohydrate diet (HCD) where 78% calories came
from carbohydrates to stimulate hepatic de novo lipogenesis. We confirmed that HCD
feeding promoted liver TG content by 2-fold in 2 days and approximate 3-fold after 10
days (Figure III-16A). In the histological analysis, the HCD-fed mice treated with
vehicle injection (Veh) showed moderate macrovesicular steatosis in comparison to the
normal chow group (Figure III-16B). Subsequently, we administered NR3B-PA and
MM-PA to two separate groups of HCD-fed mice at day 1, 4 and 7. After 10 days, the
results indicated that NR3B-PA receiving group had low lipid accumulation in livers and
the histological appearance resembling the livers from normal chow group. On the other
hand, the MM-PA group remained distinct macrovesicular structure, which was similar to
the livers from vehicle group (Figure III-16B). Quantification of TG levels affirmed that
NR3B-PA treatment reduced hepatic TG to a level that was comparable to that of the
normal chow fed mice (Figure III-16C). MM-PA treatment did not show the same level
of effect on reducing hepatic TG content, confirming that the effect of NR3B-PA was
specific on inhibiting NR3B1 rather than the polyamide treatment itself.
41
A B
C
Figure III-16. NR3B-PA blocks hepatic lipogenesis induced by high carbohydrate
diet (HCD).
A. Use of HCD to induce rapid TG accumulation in wild-type C57/B6 mice in 10 days.
B. Treatment of NR3B-PA to block fatty liver induced by HCD. MM-PA was used as the
drug control. HCD-fed Veh-treated mice as the control to the normal chow group.
C. Top, histology of livers from the various groups of wild-type mice. HCD, mice on
high carbohydrate diet for 10 days. Veh, vehicle treated mice; NR3B-PA, NR3B-PA
treated mice; MM-PA, MM-PA treated mice. Bottom, quantification of liver
triglyceride (TG) content in the various groups of mice.
42
III-5: NR3B-PA treatment ameliorates NASH phenotype
In a NAFLD/NASH model where insulin/PI3K signaling was constitutively
activated by the deletion of the negative regulator PTEN, we found prominent
pathological features such as infiltration of inflammatory cells and hepatic fibrosis,
concurrently with a robust induction of NR3B1 (Debebe et al., 2017). To address if
NR3B1 inhibition could reduce macrophage infiltration in the liver in addition to hepatic
steatosis, we treated the 7-month old PTEN-null mice manifesting strong pathological
feature of NAFLD/NASH with NR3B-PA for two months. By the age of 9-month, in the
histological analysis, we found that NR3B-PA treatment greatly improved the hepatocyte
ballooning and reduced macrovesicular steatosis (Figure III-17A). Moreover, with the
pan-macrophage marker for CD68
+
macrophages, we observed drastically decreased
presence of infiltrating macrophages in the livers from NR3B-PA treatment group (Figure
III-17B). Liver TG content was also significantly dropped by NR3B-PA treatment
(Figure III-17C). Taken together, these data suggested that NR3B-PA not only prevented
lipid droplet accumulation in early-developed hepatic steatosis, but also alleviated
NAFLD/NASH pathology in reducing inflammatory macrophages after the disease
phenotype was already established.
43
A
B
44
C
Figure III-17. NR3B-PA reverses the hepatic steatosis and reduces macrophage
infiltration in the NASH mice induced by PTEN loss.
A. Histology of livers from the various groups of mice. Veh, vehicle treated PTEN-null
mice. NR3B-PA, NR3B-PA treated PTEN-null mice.
B. Representative immunofluorescence staining of panCK and CD68 on PTEN-null
NASH mouse treated with vehicle or NR3B-PA. The number of infiltrated CD68
+
macrophages were lower in the livers treated with NR3B-PA compared to those
treated with vehicle (Veh). Immunofluorescence staining of DAPI (blue), panCK
(green) (pan cytokeratin) and CD68 (red).
C. Quantification of liver triglyceride (TG) content in the various groups of mice. The
TG level was significantly reduced in the Pm mice received NR3B-PA treatment (Pm-
NR3B-PA) compared to the Pm mice received vehicle injection (Pm-Veh).
45
III-6: Conclusion
Beyond its role in mitochondrial metabolism, NR3B1 functions to regulate lipid
homeostasis. Early studies have shown that the NR3B1 knockout mice exhibit decreased
peripheral fat deposition and are resistance to high fat diet-induced obesity (J. Luo et al.,
2003). Histological analysis shows that adipocyte cell size is smaller in NR3B1 null
adipose tissues, suggesting reduced lipid accumulation in these adipocytes. Concurrently,
microarray analysis showed significantly reduced expression of genes involved in fatty
acid synthesis, including FAS, stearoyl coenzyme A desaturase 2 (SCD2), elongation of
very long chain fatty acids-like 3 (ELOVL3), and others in mice lacking NR3B1(J. Luo
et al., 2003). Moreover, it’s been shown that NR3B1 induces adipogenesis via regulating
expressions of lipogenic nuclear co-activators and transcriptional factors such as SREBP-
1c and PGC-1β (Ferber et al., 2012). In the Alb-Cre
+
Pten
flox/flox
mice, hepatomegaly and
fatty liver phenotype were developed as early as age of 1-month together with a
significant induction of NR3B1 expression (Galicia et al., 2010; Li et al., 2013). Using
the hepatic PTEN-null mice as well as mice fed HCD diet to mimic physiological
NAFLD development, we show here that treatment with NR3B1-PA that targets NR3B1
inhibit the fatty liver phenotype together with reduced triglyceride content and lipogenic
enzyme expression. These liver-specific PTEN deleted mice also develop NASH-like
pathological features such as steatosis, Mallory bodies, pericellular fibrosis, and the
presence of inflammatory cells (Matteoni et al., 1999). Our results further demonstrated
that NR3B-PA rescues the steatosis and reverses NASH in these mice. Thus, controlled
blocking of NR3B1 acting downstream of PI3K by NR3B-PA may provide therapeutic
benefit to patients predisposed to NASH.
46
Chapter IV: Mechanistic study of NR3B1 in triacylglycerol biosynthesis
In a normal physiological condition, the low steady-state hepatic TG
concentration is sustained by the various intricate metabolic functions, including the de
novo lipogenesis, fatty acid uptake for TG biosynthesis along with very-low-density
lipoprotein (VLDL) secretion and catabolism of fatty acid by the β-oxidation (Figure
IV-18) (Gluchowski, Becuwe, Walther, & Farese, 2017). Thereby, pathological
accumulation of TG in liver csn be multifactorial, such as increased uptake of fatty
acid from peripheral and/or elevated de novo lipogenesis. In non-diabetic NAFLD
patients, despite the insulin resistance, insulin signal continues to stimulate de novo
lipogenesis via SREBP-1c pathway (Greco et al., 2008), contributing to 25% of
hepatic TG production in NAFLD patients, whereas only 5% in healthy individuals
(Fabbrini, Sullivan, & Klein, 2010). Similar to this finding, hepatic PTEN deletion,
which elevates hepatic insulin signal enhances the rate of de novo fatty acid synthesis
2.5-fold higher and increases plasma TG levels, with no significant alterations in fatty
acids uptake. On the other hand, dyslipidemia and insulin resistance also promote the
lipid uptake in peripheral tissues and liver in patients (Toledo, Sniderman, & Kelley,
2006).
47
Figure IV-18. Hepatic lipid droplet accumulation results from an imbalance in lipid
synthesis, storage, secretion and fatty acid oxidation.
Hepatic steatosis can result from different processes: increased fatty acid (FA)
uptake, de novo lipogenesis and triglyceride synthesis combined with lipid droplet (LD)
growth; decreased fatty acid oxidation), or impaired very-low-density lipoprotein
(VLDL) secretion.
48
IV-1: Downregulation of glycerolipid biosynthesis genes by NR3B1-KD
To further explore the metabolic changes associated with reduction of liver
steatosis by targeting the NR3B1, we performed the Gene Set Enrichment Analysis
(GSEA) on the RNA-sequencing data from siNR3B1-treated Huh7 cells (Figure IV-
19A). We found that knocking down NR3B1 activity led to overall downregulation of
the expression of multiple key enzymes for the TG biosynthesis, also known as the de
novo glycerolipid biosynthesis pathway (Figure IV-19B). Liver TG is produced from the
sequential esterification of glycerol-3-phosphate (G3P) by each of the three esterification
enzymes: glycerophosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate
acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT). GPAT catalyzes
the rate-limiting and first committed step for TG biosynthesis by esterification of long-
chain fatty acids to glycerol 3-phosphate, forming lysophosphatidic acid (LPA). There are
four GPAT isoforms encoded by separate genes in mammals: GPAT1 - 4. Mice lacking
GPAT1 (also known as GPAM) is reported to have lower hepatic TG content and
resistance to high fat diet-induced insulin resistance (Neschen et al., 2005). GPAT2 is
suggested to esterify both G3P and lysoglycerol-3-phosphate in testis (Cattaneo et al.,
2012). GPAT3 is mainly expressed in white adipose tissue (Cao et al., 2014). GPAT4 is
first called as acylglycerol-3-phosphate acyltransferase isoform 6 (AGPAT6) due to its
similarity to AGPAT1 and -2. GPAT4 is the major GPAT isoform in liver and mammary
gland where it is responsible for the deposition of DAG and TG in milk (Beigneux et al.,
2006; Nagle et al., 2008). DGAT catalyzes the final step in TG synthesis. DGAT1 and
DGAT2 are both expressed broadly in all tissues including the liver. DGAT1 is found to
be upregulated for hepatic TG production in patients with NAFLD (Q. Liu, Siloto,
49
Lehner, Stone, & Weselake, 2012). As a substitute, DGAT2 is considered to compensate
DGAT1 for TG storage when DGAT is knocked down in adipose tissue (Chitraju,
Walther, & Farese, 2019).
We further validated each of the gene expression by quantitative PCR and
confirmed that GPAM, GPAT4, DGAT1 and DGAT2 were significantly downregulated
when NR3B1 was knocked down in Huh 7 cells (Figure IV-19C). Consistent with the
cell-based experiment, expressions of these glycerolipid biosynthesis genes were shown
to be downregulated in the NR3B-PA treatment in both the HCD- and hepatic PTEN
deletion-induced mice with NAFLD (Figure IV-19D). Taken together, by targeting
NR3B1 with NR3B-PA, we showed that NR3B1 inhibition reduced both de novo
lipogenesis and glycerolipid biosynthesis, which may both prevent and reverse the
hepatic lipid droplet accumulation in NAFLD/NASH mice.
50
A
B
51
C
52
D
Figure IV-19. NR3B1 inhibition downregulates the glycerolipid biosynthesis.
A. Immunoblotting for NR3B1 protein that are knocked down by siNR3B1 in Huh7
cells.
B. Top, heatmap showing inhibition of key enzymes in the glycerolipid biosynthesis.
Bottom, the glycerolipid biosynthesis pathway. NES: normalized enrichment score.
FDR: false discover rate.
C. Quantitative PCR analysis in Huh7 cells to confirm that siNR3B1 introduction leads
to inhibition of genes in the glycerolipid biosynthesis pathway.
D. NR3B-PA treatment in vivo downregulates genes that are involved in the glycerolipid
biosynthesis pathway. Top, HCD mice liver. Bottom, Pm mice liver.
53
IV-2: The effect of NR3B1 inhibition is partially mediated via CEBPb
To deduce the mechanism of how glycerolipid biosynthesis genes were regulated
by NR3B1 directly and indirectly, we cross-matched genes whose expressions were
altered by siNR3B1 with genes that were recognized by NR3B1 at their promoters based
on the ChIP-on-chip date (Charest-Marcotte et al., 2010). In Table 2.2, we listed the
glycerolipid biosynthesis genes and transcriptional factors that were shown to be under
the NR3B1 direct transcription regulation. Among those, Dgat1 gene was directly
regulated by NR3B1. This was corroborated with DGAT1 downregulation when we
knocked down NR3B1 in cell-based and animal experiments. Next, we considered the
indirect regulation by NR3B1, so we screened for downstream transcription factors
whose expressions were altered by the introduction of siNR3B. 31 transcriptional factors
and co-factors were identified that were known to participate in the regulation of
glycerolipid biosynthesis (Table IV-3). Three out of the 31 listed transcriptional factors
were downregulated due to introduction of siNR3B1: NR3B1, CEBPb and PPARGC1B
(Table IV-4). NR3B1 expression was reduced due to siRNA knockdown through its self-
regulation mechanics (D. Liu, Zhang, & Teng, 2005). PPARGC1B (PGC-1β) and CEBPb,
known as CCAAT enhancer binding protein beta, both stimulated the activities of other
nuclear receptors, such as estrogen receptor alpha, nuclear respiratory factor 1 and
glucocorticoid receptor. They were shown involved in fatty acid oxidation, non-oxidative
glucose metabolism, and the regulation of energy expenditure in various tissues.
Noteworthy, PGC-1β and CEBPb were both prominently expressed in liver tissue (Figure
IV-20), suggesting their functional relevance in modulating hepatic gene expressions.
54
Table IV-3. TG biosynthesis-related transcription factors that are modulated by
siNR3B1*.
CEBPA CEBPB HIF1A HNF1A HNF1B HNF4A JUN
MARS MEF2A ChREBP NF1 NFYB NR1H2 NR1H3
NR1H4 NR3B1 NR3C1 NRF1 PPARA PPARG PPARGC1B
RXRA RXRB SP1 SP4 SREBF1 SREBF2 TFAP4
USF1 USF3 XBP1
*: The table is generated the RNA-seq data in this study and Chip-on-chip data from Genes & Dev. (2010).
Table IV-4. Transcription factors that are under NR3B1 transcriptional control.
Upregulated SP1 PPARA JUN
Downregulated CEBPB NR3B1 PPARGC1B
*: The table is generated the RNA-seq data in this study.
55
Figure IV-20. Candidate transcription factors, CEBPb and PGC-1β, are highly
expressed in liver.
The normalized gene expressions body atlas is produced using BaseSpace Correlation
Engine. Yellow broken line is the median expression level in whole body. Top, CEBPb
expression across body tissues. Bottom, PGC-1β expression across body tissues.
CEBPB
PGC1b
56
To investigate how PGC-1β and CEBPb might affect glycerolipid biosynthesis
genes, we knocked down PGC-1β and CEBPb individually by siRNA experiments.
Knocking down PGC-1β in Huh7 did not have evident change on the glycerolipid
biosynthesis genes (Figure IV-21). On the other hand, siCEBPb attenuated the expression
of three glycerolipid biosynthetic genes (Figure IV-22A). Conversely, over expression of
CEBPb induced GPAM and DGAT2 expressions and partially rescued the NR3B-PA
inhibitory effect on TG biosynthesis gene DGAT2 but not DGAT1 (Figure IV-22B).
These results indicated that NR3B1 regulated GPAM and DGAT2 expression dependent
on CEBPb level. In contrast, NR3B1 appeared to regulate DGAT4 and DGAT1
independent of CEBPb and PGC-1β.
57
Figure IV-21. The glycerolipid biosynthetic genes are independent of PGC-1β in
Huh7 cells.
Introduction of siPGC1b in human HCC, Huh7, does not cause significant changes on the
mRNA level of GPATs, AGPATs and DGATs.
58
A
B
Figure IV-22.The glycerolipid genes under the regulation of NR3B1 were dependent
on CEBPb expression.
A. CEBPb knockdown reduces glycerolipid gene expressions. CEBPb protein level was
reduced by siCEBPb transfection in Huh7. siRNA against CEBPb was transfected
into Huh7 using Lipofectamine 2000 in low-serum media for 16 hours following
25mM glucose and 0.1mM insulin stimulation for 5 hours. Cell total RNA are
collected after the glucose/insulin stimulation and subjected to qPCR analysis.
B. Overexpression of CEBPb increases the expression of GPAM and DGAT2 genes and
blocked the ability of NR3B-PA to inhibit DGAT2, but does not affect its effect on
GPAM, GPAT4 and DGAT1.
59
IV-3: GPAT4 is a novel target of NR3B1
As GPAT4 and DGAT1 were identified to be under NR3B’s regulation, they also
appeared to be resistant to CEBPb downregulation (Figure IV-22). We speculated that
DGAT1 and GPAT4 gene expressions were likely directly governed by NR3B1 at their
transcriptional level that had not been reported before. To ascertain the theorized
interaction between NR3B1 protein and target gene promoters, we began with an in-silico
approach to investigate NR3B1 occupancy in relation to glycerolipid gene transcriptions
using the public genomic references from Encode database (Figure IV-23A). To track a
promoter location for NR3B1 binding which was often prior to transcription initiation
site, histone marks such as histone H3, lysine 4 trimethylation (H3K4me3), lysine 27
acetylation (H3K27ac) and lysine 9 acetylation (H3K9ac), were used because they were
closely associated with open chromatic structure during gene transcription (Nishida et al.,
2006). TATA-box binding protein (TBP), E1A binding protein (P300) and RNA
polymerase II (Pol2) were essential components required for transcription initiation.
Together they were regularly found proximal to the transcription start site. Notably,
transcription activation by NR3B1 highly depends upon the PGC-1α, which was
conjointly activated by deacetylation (Rodgers, Lerin, Gerhart-Hines, & Puigserver,
2008). Therefore, it was expected to see the colocalization of recruited NR3B1 protein
together with histone deacetylation marks H3K27ac/H3K9ac nearby actively transcribed
loci.
60
A
Nr3b1
Dgat1
GGTCA
DGAT1
NR3B1
NR3B1
GGTCA GGTCA
NR3B1
61
Gpat4
Agpat1
GGTCA
GPAT4
NR3B1
62
Agpat3
B
63
C
Figure IV-23. NR3B1 binds to the Dgat1 and Gpat4 promoter regions and regulates
their expression.
A. Visualization of genomic DNA sequences, transcriptional factors and histone markers for
Nr3b1, Dgat1, Gpat4, Agpat1 and Agpat3 at their promoters. The core 5’-GGTCA-3’
consensus sequence for NR3B1 to recognize is highlighted in blue and black vertical
lines. The black lines indicate the region of interests where the ChIP analyses are
performed.
B. ChIP analysis with NR3B1 antibody on Huh7. Top, the sequence motif 5’-GGTCA-3’ is
identified and further targeted using primer pairs for Nr3b1, Dgat1 and Gpat4
respectively. Bottom, use of PCR to probe NR3B1-bound DNA motifs in Nr3b1, Dgat1
and Gpat4. Input and IgG group serve as a positive and negative control. The physical
interaction of NR3B1 and DNA are detected in the control and MM-PA treatment;
simultaneous use of NR3B-PA to block the interaction hence minimal DNA is detected.
C. DGAT1 and GPAT4 expressions are reduced by NR3B-PA (1 µM) but not MM-PA (1
µM). Together they indicate that NR3B1 directly binding to the promoters is correlated
with gene transcriptions for DGAT1 and GPAT4.
DGAT1
GPAT4
0.0
0.5
1.0
1.5
Fold change
Con NR3B-PA MM-PA
**
*
64
NR3B1 protein preferentially recognizes the ERRE motif, which contains the
high frequency of 5’-GGTCA-3’ (Figure II-1). For this reason, we screened for the
“GGTCA” common motif and spotted several motifs in the promoters for Nr3b1, Dgat1,
Gpat4, Agpat1 and Agpat3 (Figure IV-23A). As we expected, peaks of active histone
marks and components for transcription initiation were aligned with the peak of NR3B1
at “GGTCA” motif for Nr3b1, Dgat1 and Gpat4 genes, but not Agpat1 and Agpat3
(vertical lines in Figure 4.6A). This suggests that NR3B1 was actively recruited to the
proximity of transcription initiation sites, which were flanked by the active histone marks
H3K27ac, K3K9ac and H3K4me3 (top 3 plots in each gene, Figure IV-23A). It was
worth noting that the basic-leucine zipper transcription factor CEBPb, which we
previously showed it only partially regulate glycerolipid biosynthesis gene DGAT1 but
not GPAT4, was peaked nearby Dgat1 promoter, but only minimally presented at Gpat4,
supporting the notion that CEBPb was unlikely to contribute to Gpat4 gene transcription
(4th plot from the bottom in Gpat4, Figure IV-23A). These analyses, thus, identified
Gpat4 as a potential novel direct transcriptional target of NR3B1.
To probe the NR3B1-DNA interaction within the natural chromatin context of the
cell, we performed the chromatin immunoprecipitation (ChIP) assay on NR3B1 followed
by detecting the NR3B1-bound DNA segments using PCR. The Dgat1 promoter is
reported to interact with NR3B1 (Table II-1); the Gpat4 gene, on the other hand, is a
novel NR3B1 target gene in this study and has not be reported previously. We then
identified the possible NR3B1-binding sites in Dgat1 and Gpat4 (Figure IV-23A and B).
With the primer pairs designed to detect the NR3B1-binding sites in both genes (Figure
IV-23B top), we detected and amplified the NR3B1-bound DNA segments that were
65
pulled down by NR3B1 monoclonal antibody in DGAT1 and GPAT4 groups (Figure IV-
23B bottom). Concurrently, we obstructed NR3B1 protein from binding to the gene
promoters using NR3B-PA, hence minimal PCR product were amplified; using the
sequence mismatched control, MM-PA, we observed visible PCR product instead (Figure
IV-23B bottom). As a result, it showed that, in addition to Dgat1, Gpat4 gene was a
NR3B1 direct target and physically bound by NR3B1 at the promoter region we
identified.
To address the protein-DNA binding activity and functional relevance of NR3B1,
we attempted to blocked NR3B1 transcriptional activity for glycerolipid gene induction
using NR3B-PA. Corroborated with the ChIP analysis, when NR3B1 was blocked from
binding to target gene promoters by NR3B-PA, the Dgat1 and Gpat4 mRNA expressions
were significantly reduced, compared to the control group and MM-PA treatment that was
unable to block the NR3B1-DNA binding (Figure IV-23C). In summary, these results
indicated that NR3B1 served as a transcriptional regulator to promote Dgat1 and Gpat4
gene expressions.
66
IV-4: Conclusion
In this study, we have elucidated novel targets in the glycerolipid biosynthesis
pathway that are regulated by NR3B1. Blocking NR3B1 transcriptional activity
downregulates both de novo lipogenesis and glycerolipid biosynthesis in human
hepatocytes and in vivo NAFLD/NASH model (Figure IV-24). In spite of the effect on
reducing MCAD, the rate-limiting enzyme for fatty acid β-oxidation, our data
demonstrates that NR3B1 inhibition directly reduces multiple lipogenic enzyme
expressions, namely FAS for de novo lipogenesis, as well as GPAT4 and DGAT1 for
glycerolipid biosynthesis (G. Liu et al., 2018). GPAT4 catalyzes the first step of
glycerolipid synthesis and is required for the expansion of lipid droplet where it is
delocalized and stably associated with (Wilfling et al., 2013). Here, we report a novel
finding that GPAT4 is a direct target regulated by NR3B1 transcriptional activity. In
addition, NR3B1 also indirectly regulates the glycerolipid synthesis dependent on other
transcription factors and CEBPb, which can subsequently modulate GPAM, DGAT1 and
DGAT2 for glycerolipid synthesis. Some of these glycerolipid biosynthesis enzymes, e.g.
DGAT, has been link to de novo lipogenesis and hepatic steatosis in mice challenged
with high-fat diet (Wilfling et al., 2013). Our results that both de novo lipogenesis and
glycerolipid biosynthesis genes are downregulated by inhibition of NR3B1 suggests that
NR3B1, in addition to promoting mitochondrial biogenesis/bioenergetics, is a primary
regulator of lipid biosynthesis.
Despite the recent growing understanding in NAFLD and NASH, currently there
is a lack of pharmacological therapies beyond weight loss and lifestyle changes. In
patients with NAFLD/NASH, the fatty acids derived from de novo lipogenesis are often
67
found increased for esterification into liver triacylglycerol production through the
glycerolipid biosynthesis (Yamamoto et al., 2010). Noteworthy, the improvement of
steatohepatitis almost never occurs without resolution of fatty liver (Sanyal et al., 2015).
Therefore, our finding for the role of NR3B1 as a master regulator of lipid biosynthesis
together with the small molecule polyamide that can target NR3B1 present a potential
novel therapeutic strategy for NAFLD/NASH treatment.
68
Figure IV-24. Master regulator of NR3B1 in hepatic triacylglycerol biosynthesis
In patients with fatty liver diseases, increased free fatty acids from de novo lipogenesis
and/or dietary fat are converted into triacylglycerol (TG) through the glycerolipid
biosynthesis carrying out by GPATs, AGPATs, PAPs, and DGATs enzymes. By knocking
down NR3B1 with siRNA or NR3B-PA polyamide, we have shown that it suppresses the
de novo lipogenesis and glycerolipid biosynthesis that are NR3B1directly- and indirectly-
regulated. Through the downstream transcriptional factor CEBPb, NR3B1 indirectly
regulate part of the glycerolipid genes Gpam, Dgat1 and Dgat2; independent of CEBPb,
NR3B1 directly binds to the promoters of Gpat4 and Dgat1 and induces gene
expressions.
69
Chapter V: Materials and Methods
Animals
Pten
loxP/loxP
; Alb-Cre
+
(PTEN-null) and Pten
loxP/loxP
; Alb-Cre
-
(PTEN-wt) mice were
reported previously (Stiles et al., 2004). Male animals of C57BL/6 and J129svj
background were used for all experiments. Experiments were conducted according to
Institutional. All experimental procedures were conducted according to Institutional
Animal Care and Use Committee guidelines of University of Southern California. For
diet-induced NAFLD model, mice were fed with high-carbohydrate diet (TD.99252,
Envigo) for 10 days.
All experimental procedures were conducted according to the Institutional Animal Care
and Use Committee guidelines of the University of Southern California.
Cell culture
Mouse hepatocytes were isolated from mouse livers as previously described (Zeng et al.,
2011). Mouse and human hepatocytes (SNU398 and Huh7 cells purchased from ATCC)
were cultured in Dulbecco’s Modified Eagle’s Medium (Mediatech) supplemented with
10% FBS (Atlas Biologicals), 5 μg/ml insulin (Sigma-Aldrich), and 10 ng/ml epidermal
growth factor (Invitrogen). C4-2b human prostate cancer cell lines was cultured in
RPMI-1640 medium (Mediatech) supplemented with 10% FBS. All cell culture was
supplemented with 1% of penicillin-streptomycin and incubated at 37°C with 85%
relative humidity/5% CO2.
70
Electromobility shift assay (EMSA)
Oligonucleotide containing the 5’-AGGTCA-3’ consensus sequence corresponding to the
ERRE binding site was synthesized. NE-PER Nuclear and Cytoplasmic Extraction
Reagents, Biotin 3’ End DNA Labeling and LightShift Chemiluminescent EMSA kits
were purchased from Thermo Fisher Scientific. In brief, two complementary
oligonucleotides were labeled separately with biotin according to manufacturer’s
instruction and allowed to re-anneal. For binding and competition reactions, 20 fmole
biotin-labeled ERRE dsDNA, were incubated with 10 µg nuclear protein extract from
NR3B1 overexpressing mouse hepatocytes with or without unlabeled ERRE dsDNA,
NR3B1 antibody, and indicated concentrations of two different polyamides. The mixture
was then separated on polyacrylamide gel following incubation and DNA transferred to
membrane. Biotin-labeled DNA-protein complexes were detect using X-ray film
followed the instruction of EMSA kit.
Western blot
Cell lysates were prepared in lysis buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 150
mM NaCl, 1% NP40, 5mM NaF, 0.25% Na deoxycholate and 2 mM NaVO3)
supplemented with phosphatase inhibitors and protease inhibitors (Roche). Protein
electrophoresis and Western blotting were performed using Mini Trans-Bolt Cell system
(Bio-Rad). Blots were probed with anti-NR3B1 (ab76228, Abcam) and anti-β-actin
(a2228, Sigma-Aldrich) antibodies. The protein blot membranes were visualized with
ECL Western Blotting Substrate (Thermo Fisher Scientific) and images were taken with
X-ray film or ChemiDoc Imaging System (Bio-Rad).
71
Polyamide synthesis and treatment
The polyamides 1 and 2 were synthesized by and courtesy Dr. Peter Dervan (California
Institute of Technology) as described (Nickols et al., 2013). The NR3B-PA targeted the
sequence 5’-WGGWCW-3’ and MM-PA targeted 5’-WGWWCW-3’ as an off-target
control. Experimental group received PA-GGWC (1 mg/kg) diluted in PBS supplemented
with approximate 5% DMSO by intraperitoneal injection in every 3 days for 60 days.
Control group received 100 μl PBS supplemented with 5% DMSO. Drug regimen:
Polyamide (1 mg/kg) was dissolved in PBS supplemented with 5% DMSO and
administered by intraperitoneal injection on Day 1, 4, and 7 of HCD. Control group
received 100 μl PBS supplemented with 5% DMSO.
Cell culture
Mouse hepatocytes were isolated from mouse livers as previously described (Zeng et al.,
2011). Mouse and human hepatocytes (SNU398 and Huh7 cells purchased from ATCC)
were cultured in Dulbecco’s Modified Eagle’s Medium (Mediatech) supplemented with
10% FBS (Atlas Biologicals), 5 μg/ml insulin (Sigma-Aldrich), and 10 ng/ml epidermal
growth factor (Invitrogen). C4-2b human prostate cancer cell lines was cultured in
RPMI-1640 medium (Mediatech) supplemented with 10% FBS. All cell culture was
supplemented with 1% of penicillin-streptomycin and incubated at 37°C with 85%
relative humidity/ 5% CO2.
RNA interference, RNA isolation and quantitative PCR (qPCR)
siRNAs for NR3B1 were purchased from Santa Cruz Biotechnology (sc-44707,
72
siNR3B1-1; sc-44706) and OriGene (SR414015, siNR3B1-2). Control siRNA was from
Santa Cruz Biotechnology (sc-37007). Plasmids and siRNA were delivered using
Lipofectamine 2000 according to the manufacturer’s instruction. 4 μg of DNA or 100
pmol siRNA and 10 μL of Lipofectamine 2000 were added to cells growing at 70 - 90%
confluence in 6-well plates in triplicates and incubated for 24 hours. Total RNA was
isolated using TRIzol reagent (Invitrogen) following manufacturer’s instruction. Total
RNA (2 µg) was used for first-strand synthesis of complementary DNA (Promega).
Quantitative PCR was performed using the 7900 HT fast real-time PCR system (Applied
Biosystems) and Maxima SYBR Green quantitative polymerase chain reaction Master
Mix (Bioland). Gene expression level was determined using the delta delta Ct method.
Gene-specific primers used for qPCR: mouse Acc forward 5’-
ATGGGCGGAATGGTCTCTTTC-3’ and reverse 5’-
TGGGGACCTTGTCTTCATCAT-3’. Mouse Fas forward 5’-
GGAGGTGGTGATAGCCGGTAT-3’ and reverse 5’-
TGGGTAATCCATAGAGCCCAG-3’. Mouse Mcad forward 5’-
AGGGTTTAGTTTTGAGTTGACGG-3’ and reverse 5’-
CCCCGCTTTTGTCATATTCCG-3’. Mouse Cebpb forward 5’- CGC CTT ATA AAC
CTC CCG CTC-3’and reverse 5’- CAGTCGGGCTCGTAGTAGAAG-3’. Mouse Gpam
forward 5’- ACAGTTGGCACAATAGACGTTT-3’ and reverse 5’-
CCTTCCATTTCAGTGTTGCAGA-3’. Mouse Gpat4 forward 5’-
TCAGTTTGGTGACGCCTTCT-3’ and reverse 5’- TTCAATCCACCGTCCCACAG-3’.
Mouse Agpat1 forward 5’-TAAGATGGCCTTCTACAACGGC-3’and reverse 5’-
CCATACAGGTATTTGACGTGGAG-3’. Mouse Agpat3 forward 5’- CTG CTT GCC
73
TAC CTG AAG ACC-3’ and reverse 5’- CTGCTTGCCTACCTGAAGACC-3’. Mouse
Dgat1 forward 5’- TCCGTCCAGGGTGGTAGTG-3’ and reverse 5’-
TGAACAAAGAATCTTGCAGACGA-3’. Mouse Dgat2 forward 5’-
CGAGACACCATAGACTACTTGCT-3’ and reverse 5’-
GCGGTTCTTCAGGGTGACTG-3’. Mouse CytC forward 5’-
CCAGTGCCACACCGTTGAA-3’ and reverse 5’-TCCCCAGATGATGCCTTTGTT-3’.
Mouse Gapdh: forward 5’-GCACAGTCAAGGCCGAGAAT-3’ and reverse 5’-
GCCTTCTCCATGGTGGTGAA-3’. Mouse Nr3b1 forward 5’-
CTCAGCTCTCTACCCAAACGC-3’ and reverse 5’-CCGCTTGGTGATCTCACACTC-
3’.
Human Dgat1 forward 5’-TATTGCGGCCAATGTCTTTGC-3’ and reverse 5’-
CACTGGAGTGATAGACTCAACCA-3’. Human Gpat4 forward 5’-
TCTTTGGGTTTGCGGAATGTT-3’ and reverse 5’-
ATGCACATCTCGCTCTTGAATAA-3’. Human Gpam forward 5’-
TCTTTGGGTTTGCGGAATGTT-3’ and reverse 5’-
ATGCACATCTCGCTCTTGAATAA-3’. Human Dgat2 forward 5’-
GAATGGGAGTGGCAATGCTAT-3’ and reverse 5’-
CCTCGAAGATCACCTGCTTGT-3’. Human Agpat1 forward 5’-
AGGACGCAACGTCGAGAAC-3’ and reverse 5’-
GCAGTACCTCCATCATCCCAAG-3’. Human Agpat3 forward 5’-
CTGCTGGTCGGCTTTGTCTT-3’ and reverse 5’-TCCAGAGTGAGTAGGCGAGG-3’.
Human Mcad forward 5’- ACAGGGGTTCAGACTGCTATT-3’ and reverse 5’-
TCCTCCGTTGGTTATCCACAT-3’. Human Acc forward 5’-
74
TCACACCTGAAGACCTTAAAGCC-3’ and reverse 5’-
AGCCCACACTGCTTGTACTG-3’. Human Fas forward 5’-
ACAGCGGGGAATGGGTACT-3’ and reverse 5’- GACTGGTACAACGAGCGGAT-3’.
Human CytC forward 5’-TCAGGCCCCTGGATACTCTT-3’ and reverse 5’-
GCTATTAAGTCTGCCCTTTCTTCC-3’. Human Gapdh 5’-
GAAGGTGAAGGTCGGAGTC-3’ and reverse 5’-GAAGATGGTGATGGGATTTC-3’.
Human Nr3b1 forward 5’-GAGATCACCAAGCGGAGACG-3’and reverse 5’-
ATGAGACACCAGTGCATTCAC-3’
Luciferase reporter assay
For the luciferase reporter plasmid, the -686 to +55 human genomic sequences, relative to
the transcription initiation site of CytC, containing ERRE (Schreiber, Emter et al. 2004)
was amplified and cloned upstream of the luciferase coding sequences of pGL4
(Promega). Subsequently, the pGL4-CytC (-686/+55) reporter plasmid was introduced to
mouse hepatocytes using Lipofectamine 2000 (Invitrogen) and selected with puromycin.
Prior to polyamide treatment, the mouse hepatocytes stably expressing pGL4-CytC
luciferase were seeded in 6-well plates (1×10
5
cells per well) and allowed for attachment
for 24 hours. These cells were then treated with the indicated concentration of
polyamides for another 24 hours. All experiment was repeated at least three times. For
cell lysate preparation and luminescence detection, follow the technical manual of Dual-
Luciferase Reporter Assay System (Promega).
75
Seahorse oxygen consumption rate (OCR) assay
Cells treated with polyamide or transfected with siNR3B1 were harvested and replated (2
- 4×10
4
cells per well) on XF24 Cell Assay Plate (Agilent technologies). After being
allowed for attachment for 16 hours, cells were washed twice with Mito Assay Medium
and pre-incubation in a non-CO2 incubator for 1 hour before the assay. During the
Seahorse OCR assay, four baseline respiration rates were recorded for each condition
followed by sequential injections of the four mitochondrial inhibitors, oligomycin (1
μM), FCCP (1 μM) and rotenone/antimycin A (1 μM) to measure OCR.
Immunohistochemistry
Liver sections were stained with H&E and Oil Red O for morphology analysis. Six
sections per group were stained.
RNA-Sequencing
RNA isolated from Huh7 was analyzed using the Illumina Human gene chip (Illumina,
San Diego, CA).
Data analysis was conducted using Partek Flow software (St. Louis,
MO) with 1.2-fold or greater change in expression and false discovery rate < 0.05
considered to be different.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed according to the protocol (Carey, Peterson, & Smale,
2009) from Cold Harbor Spring Laboratory.
76
Statistical analysis
Data in this study were presented as mean ± SEM. Differences between individual groups
were analyzed by student t-test, with 2-tailed P value and less than 0.05 was considered
statistically significant.
77
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84
Supplement
Table S-5. Genes regulated by 1 μM NR3B-PA (>2 fold)
AC005077.14 CAV1 EPO IGDCC4 NEXN RORA SLC2A4 UQCRFS1P1
AC005562.1 CCAT1 EPOR IGFBP1 NFATC4
RP11-
216L13.19 SLC39A10 URB2
AC009014.3 CCDC183 EPPK1 INHBE NLGN2
RP11-
267M23.1 SLC51A UTP20
ACSS2 CCDC86 ERO1A INSIG2
NME1-
NME2
RP11-
274H2.5 SLC51B UTP4
ADAMTS6 CCNG2 ESPN INTS6L NOC3L
RP11-
284F21.10 SLC6A8 WDR4
ADAMTS9 CD68 ESPNP JAG1 NOP2
RP11-
327E2.5 SLC7A5 WDR77
ADGRD1 CDC25A F2RL2 KCCAT211 NOTCH3
RP11-
34P13.15 SMAD6
XXbac-
BPG252P9.9
ADRM1 CDKN1A FABP3 KLHL24 NYNRIN
RP11-
386G11.10 SMURF2P1
XXbac-
BPG32J3.22
AEN CHORDC1 FABP5P7 KRT18 ODF3B
RP11-
399B17.1 SNHG4 YPEL2
AGTR1 CHST3 FAM131C KRT8 OLFM3
RP11-
424C20.2 SORBS1 ZDHHC19
AIM1 CIDEC FAM13A LDHD ORAI3
RP11-
449H3.3 SPAG4 ZG16
AK7 CKB FAM214B LGALS1 P3H2
RP11-
458D21.1 SPINT1 ZNF204P
AKAP7 CLDN19 FAM3C2 LGI2 P4HA1
RP11-
465B22.3 SPSB4 ZNF395
ALDOC CLSPN FAXDC2 LGI4 PALMD
RP11-
603J24.9 SPTSSB ZSWIM4
ALOX15B COL11A1 FBXL16 LGSN PBXIP1
RP11-
65G9.1 SRCIN1
ALPK3 COL12A1 FER1L4 LMTK3 PCSK2
RP11-
683L23.7 SRXN1
ANGPTL3 COL6A2 FGF11 LOX PDF
RP11-
70C1.3 SURF2
ANKRD1 COL7A1 FGF19 LRRC3 PDGFRB
RP11-
717K11.2 SYNGR3
ANKRD33 CP FSBP LRRC37BP1 PDK1
RP11-
798K3.2 SYT7
ANKRD37 CRABP2 FST LYAR PFKFB4
RP11-
89K21.1 TAGLN
ANO9 CTGF FSTL3 MAP1A PHEX
RP11-
903H12.5 TAX1BP3
ANXA1 CTPS1 FUT11 MEIS3 PHKA2
RP13-
1032I1.10 TBX15
ANXA3 CTRL FXYD3 MELTF PIGZ RPL32P29 TBX2
ANXA4 CXCL1 GATA2 MIR210HG PINX1 RPP40 TCP11L2
AP001626.1 CXCL5 GATA6-AS1 MIR34A PITX2 RRP9 THBS1
APOA4 CXCL8 GCNT1 MOGAT3 PIWIL2 RRS1 TIGAR
APOBEC3C CYR61 GOLGA6L4 MPZL2 PKLR RSC1A1 TLE2
APOC3 DCHS1 GPATCH4
MRPL23-
AS1 PLB1 RTN1 TMC8
AQP10 DDIT4 GPR137B MST1 PLEKHA2 RTN2 TMCC1
AQP7 DEPTOR GPR157 MST1L PLG RTN4RL2 TMEM141
ARG1 DGKK GPR160 MST1P2 PLOD2 RUNX1 TMEM151A
ARRDC2 DHX37 GPR75 MT1E PNCK S1PR3 TMEM198
ATAD3B DKC1 H3F3AP4 MT1F PNO1 SAFB2 TMEM45B
ATF5 DLG4 HAL MT1G PNRC1 SATB1
TMEM51-
AS1
BBC3 DLX1 HAVCR1 MT1M PODN SCN9A TMPRSS9
BCYRN1 DMTN HDAC11 MT1X PPFIA4 SEMA3G TNFRSF14
BEX4 DNAJA1 HECA MT2A PPP1R13L SEMA6C TNS1
BHLHE40 DPH2 HEY1 MUC13 PPP1R3C SERINC2 TOMM40
BIRC7 DRAIC HGH1 MUC3A PPP1R3G SERPINA11 TP53INP1
BMF DUOX2 HIST1H2AC MVP PRAP1 SERPINA12 TP53INP2
85
BNIP3 EDN1 HIST1H2BJ MXI1 PRR36 SERPINE1 TRPV2
BNIP3L EEF1A2 HIST1H2BK MYBBP1A PSMC1P1 SEZ6L2 TRPV3
BOP1 EFNA1 HIST1H4I MYC PTPRH SFXN3 TUBA1A
BORCS7-
ASMT EFNA3 HLA-DQB1 MYL9 RARB SH2D4A TUBB8
BZW1P2 EIF1AXP1 HPN MYO15B RASD1 SH3D21 TUBB8P12
C4orf3 ELF5 HSD17B14 NACAD RBM43 SLC15A1 TUBB8P7
C8orf58 ENO2 HSP90AA1 NAV2-AS1 RHOF SLC16A3 TXLNB
C9orf72 ENPP3 HSPA8 NDRG1 RIT1 SLC1A3 TXNIP
CALCOCO1 ENTPD2 HSPH1 NECAB1 RNF19A SLC2A10 UNC93A
CAPN12 EPHB3 HYAL1 NEDD9 ROCK1P1 SLC2A3 UNKL
86
Table S-6. Genes regulated by 0.1 μM NR3B-PA (>2 fold)
ABC7-
42404400C24.1 CCNG2 FAM3C2 LGSN PKLR SH3D21 VAMP4
AC005077.14 CD3EAP FAXDC2 LMTK3 PLEKHA2 SLC15A1 WDR4
AC009014.3 CD68 FBXL16 LOX PLG SLC16A3
XXbac-
BPG32J3.22
ACSS2 CDC25A FER1L4 LSM12P1 PLOD2 SLC1A3 YPEL2
ADAMTS6 CDKN1A FGF11 LYAR PNCK SLC29A3 ZDHHC19
ADAMTS9 CHORDC1 FSBP MAP1A PNO1 SLC2A4 ZG16
ADGRD1 CHST3 FST MEIS3 PNRC1 SLC30A1 ZNF204P
ADHFE1 CIDEC FSTL3 MELTF PPFIA4 SLC39A10 ZNF395
ADRM1 CKB FUT11 MIR210HG PPP1R13L SLC51A ZSWIM4
AEN CLDN19 FXYD3 MIR34A PPP1R3G SLC51B
AIM1 CLSPN GATA2 MPZL2 PRAP1 SLC6A8
AIMP2 COL11A1 GATA6-AS1 MRTO4 PRR36 SLC7A5
AKAP7 COL12A1 GIPR MST1L PSMC1P1 SMAD6
ALDOC COL6A2 GOLGA6L4 MST1P2 PTPRH SNHG4
ALOX15B CP GPATCH4 MT1E RARB SORBS1
ALS2CL CRABP2 GPR157 MT1G RASA4 SPAG4
ANGPTL3 CTGF GPR75 MT1M RIT1 SPSB4
ANKRD1 CTPS1 H2BFS MT1X ROCK1P1 SRCIN1
ANKRD33 CXCL5 H3F3AP4 MT2A RORA SYT7
ANKRD37 CXCL8 HABP2 MUC3A RP11-134F2.8 TAGLN
ANO9 CYR61 HAL MVP
RP11-
267M23.1 TAX1BP3
ANXA1 DCHS1 HAVCR1 MXI1
RP11-
284F21.10 TBX2
ANXA3 DDIT4 HECA MYBBP1A
RP11-
386G11.10 TCEA1P2
AP001626.1 DEPTOR HEY1 MYC RP11-449H3.3 TCP11L2
APOA4 DGKK HGH1 MYL9
RP11-
458D21.1 THBS1
APOBEC3C DHX37 HIST1H2AC MYO15B
RP11-
465B22.3 THBS3
APOC3 DMTN HIST1H2BJ NDRG1 RP11-54O7.3 TLE2
AQP10 DNAJA1 HIST1H2BK NECAB1
RP11-
683L23.7 TMC8
AQP7 DOK3 HIST1H4I NEDD9 RP11-70C1.3 TMCC1
ARG1 DRAIC HIST2H4B NEXN
RP11-
717K11.2 TMEM141
ARMCX1 EDN1 HLA-DQB1 NFATC4
RP11-
903H12.5 TMEM151A
ARRDC2 EEF1A2 HNRNPA3P6 NIPAL1
RP13-
1032I1.10 TMEM45B
ATAD3B EFNA3 HPN NLGN2 RPL32P29 TMEM51-AS1
ATF5 EIF1AXP1 HSD17B14 NOP2 RPSAP58 TMPRSS9
ATP8B3 ELF5 HSP90AA1 NOTCH3 RRP12 TNFRSF14
BBC3 ENO2 HSPA8 NYNRIN RRS1 TNFRSF21
BEX4 ENPP3 HSPH1 ODF3B RSC1A1 TNS1
BHLHE40 ENTPD2 IGDCC4 OLFML3 RTN2 TOMM40
BIRC7 EPHB3 IGFBP1 ORAI3 RTN4RL2 TP53INP1
BMF EPO INSIG2 OSGIN1 RUNDC3B TP53INP2
BNIP3 EPOR INTS6L P3H2 RUNX1 TRPV2
BNIP3L EPPK1 JAG1 P4HA1 SATB1 TUBB8
BOP1 ERO1A KCCAT211 PALMD SCN9A TUBB8P12
BZW1P2 ESPNP KLHL24 PBXIP1 SEMA6C TXLNB
C8orf58 F2RL2 KRT18 PDGFRB SERINC2 TXNIP
C9orf72 FABP3 KRT23 PDK1 SERPINA12 UNC93A
CALCOCO1 FABP5P7 KRT8 PFKFB4 SERPINE1 UQCRFS1P1
CAPN12 FAM131C LDHD PHEX SEZ6L2 URB2
CCDC183 FAM13A LGI2 PIGZ SFXN3 UTP20
CCDC86 FAM214B LGI4 PITX2 SH2D4A UTP4
87
Table S-7. Genes regulated by siNR3B1 (>2 fold)
AAGAB ALPK3
ATP6V0A
2 C8orf58 CEP55 CXCL5 DTYMK EXOSC2 FTSJ3
ABC7-
42404400C24
.1 ALS2CL ATP8B3 C9orf72 CERS1 CXCL8 DUOX2 EXOSC3 FUT11
ABCA5 ALYREF ATR CA11 CFB CXXC4 DUSP1 EZH2 FXN
ABCE1 AMDHD1 AUNIP CA2
CH507-
513H4.3 CYP19A1 DYNLL1 F10 FXYD3
ABCF2 AMIGO1 BACH1 CACNB3
CH507-
513H4.4 CYP4F3 DYNLL2 F2RL1 GABRA2
ABHD5 ANAPC7 BAG2 CACYBP
CH507-
513H4.6 CYR61 E2F1 F2RL2
GADD45
B
ABLIM3 ANG BAZ1A
CALCOCO
1
CH507-
9B2.5 DBN1 E2F2 FABP1 GALNT10
ABTB1 ANGPTL4 BAZ1B CALCR CHAC2 DCHS1 E2F4 FABP3 GALNT6
AC004057.1 ANK3 BBC3 CAPN12 CHAF1A DCTPP1 E2F7 FABP5 GART
AC005077.14 ANKRD33 BCCIP CASKIN1 CHAMP1 DDIAS E2F8 FABP5P7 GAS2L1
AC005562.1 ANKRD37 BCYRN1 CASP16P CHML DDIT3 EBF4 FADS6 GATA2
AC006116.27 ANKRD52 BEX4 CAV1 CHN1 DDIT4
EBNA1BP
2 FAIM
GATA6-
AS1
AC009014.3 ANKZF1 BHLHE40 CCAT1
CHORDC
1 DDX10 ECM2 FAM111B GBE1
AC010970.2 ANO4 BHLHE41 CCDC137 CHUK DDX20 EDN1
FAM114A
1 GCLM
AC062029.1 ANO9 BHMT2 CCDC138 CIAPIN1 DDX21 EED FAM118B GCNT1
ACAD11 ANXA1 BIRC7 CCDC18 CIDEB DDX46 EEF1A2 FAM131C GDF15
ACER3 ANXA4 BLMH CCDC183 CIDEC DDX52 EEF1E1 FAM132A GEMIN5
ACP5 AP000648.5 BMF
CCDC183-
AS1 CKB DDX56 EEF2KMT FAM134B GEN1
ACSL5 AP001626.1 BMP8B CCDC86 CLDN19 DENND3 EFNA1 FAM13A GFM1
ACSS2
AP003419.1
1 BNIP3 CCNB1 CLIP2 DEPTOR EFNA3 FAM162A GGT1
ACTN3 AP1S3 BNIP3L CCND3 CLRN3 DGKZ EGR1 FAM173B GINS1
ADAMTS13 AP4E1 BOP1 CCNE2 CLSPN DHFRP1 EIF1AXP1 FAM214B GINS2
ADAMTS6 APAF1
BORCS7-
ASMT CCNG2 CLSTN3 DHX29 EIF2B3 FAM222A GINS3
ADAMTS9 APITD1 BRIX1 CCNJ CLUH DHX33 EIF2S1
FAM47E-
STBD1 GIPR
ADAT1 APOA4 BRMS1 CCT2 CNDP2 DHX37 EIF3B FAM53B GNB1L
ADGRD1
APOBEC3
C BRSK1 CCT3 COA7 DHX9 EIF3J FAM58A GNL3L
ADHFE1
APOBEC3
D BTBD16 CCT5 COL11A1 DIRC2 EIF4G1 FAM86C1
GOLGA8
A
ADI1 APOC3 BTBD8 CCT6A COL6A1 DKC1 EIF5A FAM99A
GOLGA8
S
ADORA2B AQP10 BTG2 CD320 COL6A2 DLEU2 EIF5B FARSB GOT2
ADRM1 AQP7 BTN2A2 CD3EAP COL7A1 DLG4 ELAC2 FAXDC2 GPATCH4
AEN ARFGEF3 BTN3A1 CD68 COL9A2 DLX1 ELF3 FBLN7 GPR137B
AF011889.5 ARG1 BYSL CDC20 COPS3 DMTN ELMO2 FBXL16 GPR157
AFG3L1P
ARHGAP4
5 BZW1 CDC25A COTL1 DNAH1 EMC8 FDX2 GPR160
AGAP2 ARHGEF25 BZW1P2 CDC42EP5 CP DNAJA1 EMP3 FECH GPR75
AGMAT ARMC6 C10orf2 CDC45 CPEB4 DNAJB11 ENO2 FEN1 GPSM1
AGTR1 ARMCX1 C11orf98 CDC6 CPSF2 DNAJB6 ENPP3 FER1L4 GPT
AGXT ARMCX3 C14orf169 CDC7 CPT1A DNAJC2 ENTPD2 FGF11
GRAMD1
A
AHSA1 ARNTL2 C16orf59 CDCA7 CRABP2 DNAJC9 EPB41L1 FGF19 GRAMD3
AIFM2 ARPC5L C17orf51 CDH1 CREB5
DNASE1L
1 EPHB3 FKBP4 GRWD1
AIM1 ARRDC2 C17orf89 CDH17 CREBRF DNMT1 EPO FKBP5 GSN
AIMP2 ASF1A C1QBP CDK18 CRELD2 DNTTIP2 EPOR FLAD1 GSPT1
AJ006995.3 ASGR1 C1RL CDK2AP1 CRYL1 DOHH EPPK1 FMO5 GTF2A2
AK7 ASPHD1 C1orf210 CDKN1A CSE1L DOK3 ERBB2 FNDC4 GTF2IP7
AKAP12 ATAD3A C1orf228 CEACAM1 CSTF2 DOLPP1 ERO1A FOS GTSE1
AKAP7 ATAD3B C20orf24 CELSR3 CSTF3 DPH2 ERRFI1 FOSL2 GUCY2C
AKNA ATF3 C2orf54 CENPF CTC- DRAIC ESCO2 FOXM1 H2AFX
88
260E6.4
AL513122.2 ATF5 C3 CENPM CTPS1 DSCC1 ESPN FOXO4 H2BFS
ALDH1B1 ATIC C4B CENPN CTRL DSP ESPNP FSBP H3F3AP4
ALDOC ATP2A3 C4orf3 CENPO CTU2 DTL ETF1 FST H6PD
ALOX15B ATP2B4 C7orf26 CEP170B CUL7 DTX3 EXO1 FSTL3 HAL
89
Table S-7. Gene regulated by 0.1 μM siNR3B (>2 fold) cont.
HAUS8 HYAL1 KLHL25 MCM10 MUC13 NOL11 PDCL3 PODN PSMD3
HAVCR1 ICAM5 KNOP1 MCM3 MUC3A NOL12 PDE4C POLA1 PSMD6
HBEGF ICMT KPNA1 MCM4 MVP NOL6 PDF POLA2 PSME3
HBP1 IDE KPNA2 MCM5 MXI1 NOL9 PDGFRL POLD3 PSME4
HCAR2 IDH3A KPNA3 MCM6 MYBBP1A NOLC1 PDK1 POLD4 PSMG1
HCAR3 IDS KPNA4 MCM7 MYBL2 NOM1 PDLIM2 POLE2 PSPC1
HCN3 IFRD2 KPNB1 MCM8 MYC NOP14 PEG10 POLR1A PTPRH
HCN4 IGDCC4 KRT18 MCMBP MYCBP NOP16 PFKFB4 POLR1B PTPRM
HDAC11 IGF2-AS KRT8 MED27 MYL9 NOP2 PGAM5 POLR2L PUM3
HEATR3 IGFBP1 L3MBTL2 MEIS3 MYO15B NOP56 PGK1 POLR3G PUS7
HECA IGFBP3 LANCL2 MELTF MYOM1 NOP58 PHEX POLR3H PUSL1
HELLS IL1RAP LBP METTL1 N4BP2L1 NOS3 PHF19 POLR3K PYCRL
HEY1 IL22RA1 LCN15 MFAP3L NAA15 NOTCH3 PHF21A POP1 PYGB
HGH1 IL2RG LDHD MGAT5 NAA25 NPLOC4 PHF5A PPAN PYURF
HILPDA ILKAP LETM1 MICAL2 NAA50 NPM2 PHKA2 PPARA RAB17
HIST1H1C INHBE LGALS1 MIR210HG NACAD NR0B2 PHLDA2 PPARGC1B RAB35
HIST1H2AC INSIG1 LGI2 MIR34A NAV2 NSUN2 PI15 PPFIA3 RAB3B
HIST1H2BD INSIG2 LGI4 MKI67
NAV2-
AS1 NUDC PIGW PPFIA4 RANBP1
HIST1H2BJ INSR LGR5 MLXIPL NCAPD3 NUFIP1 PIGZ PPIL1 RANGAP1
HIST1H2BK INTS6L LGSN MMS22L NCAPG NUP88 PIK3CD PPP1R13L RASA4
HIST1H4H INTS7 LIMCH1 MOB3A NCL NUP93 PIK3IP1 PPP1R3C RASD1
HIST1H4I IPO13 LIMD1-AS1 MOCOS NCLN NYNRIN PIK3R3 PPP1R3G RBBP8
HIST2H2AA3 IPO4 LINC00641 MOGAT3 NCOA5 OBSL1 PINX1 PPP2CA RBM14
HKDC1 IQSEC3 LINC01314 MON1A NDOR1 OCEL1 PIR PPRC1 RBM24
HLA-A IRS2 LINGO1 MPI NDRG1 ODC1 PITX1 PRAP1 RBM38
HLA-B ITGAE LMBR1L MPP2 NDUFAF4 ODF3B PITX2 PRELID2 RBM43
HLA-DQB1 ITIH3 LMNB1 MPV17L2 NDUFAF5 OGFOD1 PIWIL2 PRKAB2 RBM45
HLA-E ITM2B LMNTD2 MPZL2 NECAB1 OLFM3 PKHD1 PRODH RCBTB2
HLA-F-AS1 ITPR3 LMTK3 MRM2 NECTIN1 OPTN PKI55 PRPF4 RCC2
HMGB1 JAG1 LOX MRPL12 NEDD9 ORAI3 PKIB PRR36 RECQL4
HMGB1P5 JAML LPIN3
MRPL23-
AS1 NEIL3 ORC1 PKLR PRSS16 RFWD3
HMGCL JPH2 LRR1 MRPL35 NETO2 ORC5 PKMYT1 PSAT1 RGL3
HMGCS2 KBTBD6 LRRC3 MRPS11 NEU1 OTUD6B PLA1A PSEN2 RGS10
HNRNPA3P6 KBTBD8 LRRC37BP1 MRTO4 NEURL3 P3H2 PLAGL2 PSMA4 RHBDD3
HNRNPAB KCCAT211 LRRC45 MST1 NEXN P4HA1 PLB1 PSMA7 RHOF
HNRNPM KCNMB3 LRWD1 MST1L NFATC4 P4HA2 PLCD1 PSMB2 RIMKLA
HOXA13 KCTD11 LSG1 MST1P2 NFE2L2 PA2G4 PLD6 PSMB8 RIN2
HPN KDF1 LSM10 MT-RNR2 NFKB1 PAK1IP1 PLEKHA2 PSMC1 RIOK1
HRAS KDM3A LTV1 MT1E NGRN PALM PLEKHA4 PSMC1P1 RIT1
HSD17B14 KEAP1 LYAR MT1F NIFK PALMD PLG PSMC2 RNASE4
HSF4 KIAA1524 MAF MT1G NIP7 PAM PLK1 PSMC3 RNASEH1
HSP90AA1 KIF18B MAGED2 MT1M NIPA2 PARP1 PLOD2 PSMC3IP RND1
HSPA14 KIF23 MAK16 MT1X NIPAL1 PBK PLSCR4 PSMC4 RNF103
HSPA1A KIF3C MAP1A MT2A NKX3-1 PBXIP1 PLTP PSMC5 RNF19A
HSPA1B KIF5A MAPK13 MTA2 NLGN2 PCNA PLXNA3 PSMD1 ROCK1P1
HSPA4 KIF7 MARCH3 MTERF3 NME1 PCOLCE PMM1 PSMD11 RORA
HSPA8 KLC4 MARS MTHFD1
NME1-
NME2 PCSK4 PNCK PSMD12 ROS1
HSPBP1 KLHL18 MARS2 MTHFD1L NOC2L PDCD11 PNO1 PSMD13
RP1-
60O19.1
HSPE1 KLHL21 MAT2A MTMR7 NOC3L PDCD2L PNPT1 PSMD14
RP11-
1024P17.1
HSPH1 KLHL24 MCC MTMR9LP NOC4L PDCD4 PNRC1 PSMD2
RP11-
1100L3.7
90
Table S-7. Gene regulated by 0.1 μM siNR3B (>2 fold) cont.
RP11-1143G9.4 RRP12 SLC16A3 SRXN1 TM6SF2 TUBB2B WDR76
RP11-119D9.1 RRP1B SLC19A1 SSB TMC8 TUBB6 WDR77
RP11-135L13.4 RRP36 SLC19A3 ST3GAL1 TMCC1 TUBB8 WFS1
RP11-159D12.8 RRP7A SLC1A1 STARD4 TMEM138 TUBB8P12 WHRN
RP11-16P6.1 RRP9 SLC1A3 STAT2 TMEM140 TUBB8P7 WWC1
RP11-216L13.19 RRS1 SLC1A7 STEAP3 TMEM141 TUBGCP5 XPO4
RP11-234B24.6 RSC1A1 SLC25A10 STIP1 TMEM144 TWF2 XPO5
RP11-23D24.2 RSU1 SLC25A19 STON1 TMEM150B TXLNB XRCC2
RP11-267M23.1 RTEL1 SLC26A3 STRA13 TMEM151A TXNDC9 XRCC5
RP11-274H2.5 RTN1 SLC27A4 SULT1C2 TMEM198 TXNIP XXYLT1
RP11-284F21.10 RTN2 SLC2A1 SULT1C4 TMEM201 TXNRD1 XXbac-BPG252P9.9
RP11-303E16.2 RTN4RL1 SLC2A10 SUPT16H TMEM37 UACA XXbac-BPG32J3.22
RP11-343C2.11 RTN4RL2 SLC2A14 SURF2 TMEM45B UBASH3B YPEL1
RP11-348P10.2 RUNDC3B SLC2A3 SV2A
TMEM51-
AS1 UBE2F YPEL2
RP11-399B17.1 RUNX1 SLC2A4 SYNE4 TMEM70 UBE2G2 YPEL3
RP11-424C20.2 RUVBL1 SLC30A1 SYNGAP1 TMEM74B UBE2L6 YPEL5
RP11-438J1.1 S1PR3 SLC38A3 SYNGR3 TMEM87B UBE2SP2 YRDC
RP11-442H21.2 SAFB2 SLC39A10 SYT11 TMPRSS9 UCHL1 YWHAH
RP11-449H3.3 SAMHD1 SLC39A13 SYT7 TNFRSF14 UCK2 ZC3H18
RP11-458D21.1 SAT2 SLC39A3 TACC3 TNFRSF21 UFD1L ZC3H6
RP11-465B22.3 SATB1 SLC39A5 TAF15 TNFRSF25 UHRF1 ZDHHC19
RP11-496I9.1 SBDSP1 SLC51A TAGLN TNS1 UNC13C ZDHHC23
RP11-512M8.5 SCGN SLC52A2 TAL1 TOE1 UNC93A ZFP36
RP11-525K10.3 SCTR SLC5A6 TAX1BP3 TOMM22 UNG ZFYVE1
RP11-552M11.4 SDF2L1 SLC6A8 TBC1D30 TOMM40 UNKL ZG16
RP11-566E18.1 SEC14L2 SLC7A2 TBX15 TOMM40L UQCRFS1P1 ZHX2
RP11-603J24.9 SEMA3B SLC7A5 TBX2 TOMM5 URB2 ZMPSTE24
RP11-683L23.7 SEMA3G SLC7A6 TCEAL8 TONSL USB1 ZMYND19
RP11-70C1.3 SEMA6A SLCO1A2 TCF19 TOR3A USH1C ZNF142
RP11-717K11.2 SEMA6C SMAD6 TCHP TP53INP1 USP39 ZNF160
RP11-77P16.4 SENP3 SMG1P4 TCIRG1 TP53INP2 UTP11 ZNF204P
RP11-798K3.2 SERINC2 SMURF2P1 TCOF1 TRIM21 UTP14A ZNF35
RP11-798M19.6 SERPINA11 SNAI1 TCP1 TRIM26 UTP15 ZNF395
RP11-80H18.3 SERPINA12 SNHG17 TCP11L2 TRIM35 UTP20 ZNF581
RP11-848P1.9 SERPINC1 SNHG4 TESK2 TRIP13 UTP4 ZNF786
RP11-89K21.1 SERPIND1 SNRNP25 TESMIN TRIQK VAMP4 ZNHIT2
RP11-903H12.5 SERPINE1 SNRPD1 TFB2M TRMT6 VARS ZSWIM4
RP13-1032I1.10 SEZ6L2 SNX10 TFDP1 TRMT61A VCP ZWINT
RP4-539M6.19 SF3B4 SORBS1 TFR2 TRPV2 VEGFB
RP5-875O13.7 SFPQ SORL1 THBS1 TRPV3 VPS37D
RPF1 SFXN3 SPAG4 THBS3 TSEN15 VRK1
RPL32P29 SGPP2
SPECC1L-
ADORA2A THRIL TSEN2 VWA7
RPP40 SH2D4A SPINT1 TIGAR
TSPEAR-
AS1 WARS
RPS26 SH3D21 SPRTN TIMM10 TSPO WDR3
RPS6KL1 SHCBP1 SPSB4 TIMM23 TSR1 WDR34
RPUSD1 SHH SRCIN1 TIMM8A TSSC4 WDR4
RRAGD SIPA1L2 SRM TINAGL1 TTC13 WDR43
RRAS SKA3 SRPK1 TIPIN TTC21A WDR54
91
RRM2 SLBP SRPRB TLE2 TTLL12 WDR60
RRP1 SLC15A1 SRSF2 TM4SF4 TUBA1A WDR62
Abstract (if available)
Abstract
The nuclear receptor NR3B1 plays an important role in energy homeostasis especially the lipid metabolism. Using a liver-conditional PTEN deletion model where the activation of its downstream PI3K/AKT signaling pathway led to fatty liver and steatohepatitis, we reported previously that the NAFLD/NASH development was accompanied by elevated mitochondrial bioenergetics and dramatic induction of NR3B1. In this project, a polyamide (NR3B-PA) that recognized the promoters in NR3B target genes was designed. It was shown that NR3B-PA blocked the NR3B1 binding to the estrogen-related orphan receptor response element (ERRE) and inhibited NR3B1’s transcriptional activity. In the mouse models, we discovered that inhibiting NR3B1 not only prevented the early fatty liver development but also reversed the steatosis in NASH. We further demonstrated that NR3B-PA inhibited NAFLD/NASH by suppressing the key metabolic pathways for de novo lipogenesis and glycerolipid biosynthesis. Mechanistically, NR3B1 directly regulated the transcription of fatty acid synthase (Fas), acetyl-CoA carboxylase (Acc), and diacylglycerol acyltransferase (Dgat) genes
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Asset Metadata
Creator
Chen, Chien-Yu
(author)
Core Title
The role of estrogen-related receptor alpha (NR3B1) in nonalcoholic fatty liver diseases
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology and Toxicology
Publication Date
08/05/2019
Defense Date
05/31/2019
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
liver,mitochondria,NAFLD,NR3B1,OAI-PMH Harvest,triacylglycerol
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Stiles, Bangyan Li (
committee chair
), Cadenas, Enrique (
committee member
), Shih, Jean Chen (
committee member
), Stolz, Andrew Abba (
committee member
)
Creator Email
chen539@usc.edu,rabultrapi@yahoo.com.tw
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-205029
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UC11662776
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etd-ChenChienY-7734.pdf
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205029
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Dissertation
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Chen, Chien-Yu
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University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
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University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
mitochondria
NAFLD
NR3B1
triacylglycerol