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Differential effect of ethanol and r-sulforaphane on regulation of heme oxygenase-1 in endothelial cells
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Differential effect of ethanol and r-sulforaphane on regulation of heme oxygenase-1 in endothelial cells
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i
DIFFERENTIAL EFFECT OF ETHANOL AND R-SULFORAPHANE ON
REGULATION OF HEME OXYGENASE-1 IN ENDOTHELIAL CELLS
by
Jo Lee
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2012
Copyright 2012 Jo Lee
ii
Acknowledgements
I would like to thank my mentor and PI, Dr. Vijay Kalra for the help, support and
encouragement that he has provided during my time in his lab. I would also like to thank
Dr. Stanley Tahara for his help and for patiently providing answers to my numerous
questions.
I also would like to extend my thanks to Dr. Zoltan Tokes, for agreeing to be part
of my committee as well as the moral support through the journey towards my degree.
Furthermore, I would like to thank my lab members, especially Dr. Caryn
Gonsalves and Chen Li, my family and friends for all the support and encouragement
through these years.
iii
Table of Contents
Acknowledgements ............................................................................................................. ii
Table of Figures ................................................................................................................. iv
Abstract ............................................................................................................................... v
Chapter 1: Introduction ....................................................................................................... 1
A) Pathophysiology of alcoholic liver disease (ALD) .................................................... 1
B) Metabolism of Alcohol within the liver. .................................................................... 2
C) Cytoprotective enzymes prevent oxidant-stress induced by consumption of alcohol.
......................................................................................................................................... 4
D) Role of HO-1 in ethanol mediated oxidative stress. .................................................. 4
E) Role of sulforaphane in the induction of phase II enzymes. ...................................... 6
F) MicroRNA (miRNA) and its roles in the cell............................................................. 8
Chapter 2: Materials and Methods .................................................................................... 12
Chapter 3: Results ............................................................................................................. 16
1. Ethanol and Sulforaphane induce HO-1 mRNA expression in HMEC. ................... 16
2. siRNA for Nrf2 decreased HO-1 mRNA levels. ....................................................... 19
3. The expression of luciferase reporter with HO-1 promoter constructs in response to
ethanol and sulforaphane. ............................................................................................. 20
4. MicroRNA (miRNA) expressions in HMEC in response to ethanol and sulforaphane
treatments. ..................................................................................................................... 21
Chapter 4: Discussion ....................................................................................................... 24
Bibliography ..................................................................................................................... 27
iv
Table of Figures
Figure 1: Schematics of the pathways in alcohol mediated liver injury ........................... 2
Figure 2: Chemical reactions involved in the heme oxygenase-1 mediated catalysis of
free heme. ............................................................................................................................ 6
Figure 3: Chemical Structures of A) Glucoraphanin and B) Sulforaphane ...................... 7
Figure 4: Schematics of the biogenesis of miRNA ........................................................... 9
Figure 5: Sulforaphane augments HO-1 expression in HMEC cells.. ............................. 17
Figure 6: Sulforaphane induced HO-1 mRNA and protein levels in HMEC cells.. ........ 18
Figure 7: Analysis of the HO-1 promoter. ....................................................................... 20
Figure 8: Sulforaphane induced luciferase expression in the -4.5kb HO-1 construct. .... 21
Figure 9: miRNA expression levels in HMEC cells.........................................................23
v
Abstract
Alcoholism is one of the leading causes of liver disease in the United States. The
liver metabolizes 80% of ethanol consumed. Ethanol and its metabolite, acetaldehyde and
reactive oxygen species (ROS) generated by the metabolism of ethanol contribute to liver
damage and injury, leading to release of inflammatory cytokines and fibrogenesis, which
contributes to cirrhosis of the liver. Heme oxygenase-1 (HO-1) is a phase II cyto-
protective enzyme which when induced, offers increased protection against various
oxidative stresses. It has been observed that HO-1 expression contributes to protection
against liver damage induced by several compounds such as acetaminophen and carbon
tetrachloride, suggesting a role for HO-1 as a prominent hepatoprotectant (Farombi &
Surh, 2006). Previous studies have shown increased expression of HO-1, when liver cells
are treated with ethanol (Yao et al., 2007). Isothiocyanates such as sulforaphane are
naturally occurring compounds found in brussel sprouts, broccoli and other cruciferous
vegetables.
Studies suggest that sulforaphane may protect cells from the side effects of
chronic ethanol consumption by increasing levels of HO-1. We hypothesized that
sulforaphane may induce HO-1 expression in endothelial cells and thereby contribute to
the protection of these cells from the oxidative stress generated by ethanol metabolism.
We also hypothesized that miRNAs may play a role in the regulation of HO-1 in
endothelial cells under the influence of both sulforaphane and ethanol. In the present
work, we showed that in the human dermal microvascular endothelial cells (HMEC),
HO-1 expression was induced with both ethanol and sulforaphane at both the mRNA and
protein levels. We also examined the possible role the HIF-1α binding sites within the
vi
HO-1 promoter may play in mediating HO-1 expression with sulforaphane. We observed
that sulforaphane activity mainly involved the ARE site approximately 4 kb upstream of
the transcriptional start site, but not the HIF-1α sites located within this region of the
promoter. Furthermore, miRNA 762, miRNA 518c*, miRNA 323-5p, and miRNA 33b*
were identified as potential miRNAs that may be involved in the post-transcriptional
regulation of HO-1 under the influence of ethanol or sulforaphane.
We hope to continue our miRNA studies by using miRNA antagonists or mimics
to study their effects on HO-1 expression as well as site-directed mutagenesis of the
binding sites for these miRNAs in the 3’ UTR of HO-1.
1
Chapter 1: Introduction
A) Pathophysiology of alcoholic liver disease (ALD)
Chronic consumption of alcohol or alcoholism is one of the leading causes of
liver disease in the United States, therefore making alcoholism a serious health issue.
About 80% of the alcohol consumed is metabolized through the liver. The
pathopysiology of alcoholic liver disease (ALD) involves the release of pro-inflammatory
chemokines such as TNF-α, IL-6 and IL-8, oxidative stress, lipid peroxidation and
acetaldehyde toxicity which contribute to inflammation, apoptosis and fibrogenesis of the
liver, a characteristic of cirrhosis (H., 1996)(Figure 1).
The liver comprises four major cell types: the sinusoidal endothelial cells
(LSECs) , Kupffer cells, which are hepatic macrophages, hepatic stellate cells (HSCs)
and a subset of natural killer cells called pit cells, in addition to hepatocytes and bile duct
endothelial cells (Okanoue, Mori, Sakamoto, & Itoh, 1995; Wisse E, 1999). These cells
are adversely affected by ethanol metabolism and the metabolic product acetaldehyde or
its protein adduct, causing liver dysfunction (Duryee et al., 2003; French, 2001; Xu et al.,
1998).
Studies have indicated that the feeding of ethanol to rats resulted in increased
polymorphonuclear leukocyte (PMN) vascular infiltration in the liver, causing increased
adhesion of these cells to the endothelium. Increased leukocytosis in peripheral blood and
neutrophil infiltration in the liver are characteristics of alcoholic hepatitis (AH). The
transmigration of neutrophils across the LSECs occurs in response to increased
chemokine expression, which is induced by ethanol.
2
Figure 1: Schematics of the pathways in alcohol mediated liver injury (Gramenzi et al.,
2006).
B) Metabolism of Alcohol within the liver.
There are three major pathways involved in the metabolism of alcohol within
different subsets of the liver cells or hepatocytes namely: 1) the alcohol dehydrogenase
(ADH) pathway that functions within the cytosol of the hepatocyte, 2) the microsomal
ethanol-oxidizing system (MEOS)/ cytochrome P450 (CYP2E1) pathway in the smooth
endoplasmic reticulum, and 3) through catalase which is present in the peroxisomes.
1) The ADH pathway:
The ADH pathway is the major pathway involved in the metabolism of ethanol
within the liver. The ADH enzyme is a dimeric zinc metalloprotease which catalyses the
conversion of ethanol into acetaldehyde. There are three isoforms of the ADH enzymes in
3
humans. ADH-1, which is found in the liver has a low Km for ethanol (Km< 5mM) and
therefore has a major role in the metabolism of alcohol. ADH-2 is also present in the
liver, but has a higher Km for ethanol (5-10 mM)(Crabb, Matsumoto, Chang, & You,
2004).
The ADH mediated oxidation of alcohol requires the transfer of hydrogen from
alcohol to the co-factor, nicotinamide adenine dinucleotide (NAD
+
), therefore reducing it
to NADH and converting alcohol to acetaldehyde. The acetaldehyde thus produced is
then converted to acetate by acetaldehyde dehydrogenase, present in the mitochondria.
NAD
+
functions as a co-factor for this step and is reduced to NADH. The NADH thus
generated is utilized as a source of energy by the electron transport chain. The acetate
released by these reactions is then converted to CO
2
and water in the peripheral tissues .
The rate at which ADH and ALDH can metabolize ethanol may be critical in determining
toxicity, as the reaction intermediates of this pathway are toxic (Dwabb C, 2009).
2) The Microsomal ethanol oxidizing system (MEOS)/cytochrome P450 pathway:
The cytochrome P450 isozymes, CYP2E1, and CYP 1A2 and 3A4 are present
predominantly in the microsomes of the endoplasmic reticulum (Lieber, 1999; Salmela,
Kessova, Tsyrlov, & Lieber, 1998). CYP2E1 activity is induced by elevated levels of
alcohol and has a high Km for ethanol (8-10 mM), thus playing an important role in the
oxidation of ethanol to actaldehyde at high concentrations of ethanol (Lieber, 2004). The
reaction also produces reactive oxygen species (ROS) such as superoxide anions and
hydroxyl radicals that increase the risk of tissue damage (Nagy, 2004).
4
3) The Catalase pathway:
The catalase mediated ethanol metabolism is dependent on hydrogen peroxide.
Under normal physiological conditions, this pathway plays only a minor role in
metabolizing ethanol to acetaldehyde (Lieber, 1999, 2004; Quertemont, 2004).
C) Cytoprotective enzymes prevent oxidant-stress induced by consumption of
alcohol.
Reactive oxygen species (ROS) such as hydroxyl radicals and superoxide anions
are generated during ethanol metabolism via the CYP2E1 pathway in hepatocytes and are
thought to contribute to liver injury (Nagy, 2004) .
Kupffer cells play an important role in mediating liver injury as ethanol causes
increased permeability of the gut to endotoxins from Gram-negative bacteria, thereby
causing the activation of Kupffer cells, generating ROS and TNF-α (Pritchard & Nagy,
2005; Vidali, Stewart, & Albano, 2008). NADPH-oxidase derived oxidants in these cells
contribute to cytotoxic TNF-α production. Knock-out mice , lacking p47
phox
, a critical
subunit of NADPH-oxidase, showed decreased levels of ROS and TNF-α production in
Kupffer cells, leading to attenuated liver injury (Kono et al., 2000). Ethanol-mediated
oxidative stress also plays a crucial role in liver injury (De Minicis & Brenner, 2008; D.
Wu & Cederbaum, 2009).
D) Role of HO-1 in ethanol mediated oxidative stress.
The harmful effects of oxidative stress are countered by antioxidant proteins and
phase II detoxifying enzymes such as hemeoxygenase-1 (HO-1) and NADPH-quinone
5
oxidoreductase-1 (NQO-1). The induction of these enzymes renders cells more resistant
to the challenges of oxidative stress. HO-1 mediated anti-oxidant and anti-inflammatory
effects have been well characterized in vivo and in vitro (Rushworth, MacEwan, &
O'Connell, 2008; Ryter, Alam, & Choi, 2006) and have been corroborated in both HO-1
deficient patients and gene knock-out mice (Kapturczak et al., 2004; Poss & Tonegawa,
1997).
Three isoforms of the heme-oxygenase protein (HO) protein are encoded by three
separate genes (Willis, Moore, Frederick, & Willoughby, 1996). HO-1 is a 33 kDa
enzyme that is inducible by stresses (Keyse & Tyrrell, 1989), such as oxidative stress and
hypoxia, whereas HO-2 and HO-3 are constitutively active(Maines, Trakshel, & Kutty,
1986; Trakshel, Kutty, & Maines, 1986). Both HO-1 and HO-2 have catalytic activity
and their respective C-terminal hydrophobic regions bind to the microsomal membranes.
HO-3 however, does not have any catalytic activity (Maines, 1988) .
The HO isozymes catalyze the initial step in the oxidative degradation of heme,
yielding equal amounts of biliverdin IXa, carbon monoxide (CO) and free iron (Figure 2).
HO-1 is thought to protect the cell from oxidative stress by either controlling the
intracellular levels of “free” heme, which is a pro-oxidant, or by producing biliverdin,
which functions as an anti-oxidant (Bauer & Bauer, 2002).
The transcriptional regulation of redox-modulated gene products such as HO-1
(Kobayashi & Yamamoto, 2005; Taylor, Acquaah-Mensah, Singhal, Malhotra, & Biswal,
2008; Wasserman & Fahl, 1997) involves the cis-acting antioxidant response element
(ARE). Studies have shown that Nuclear factor erythroid 2-related factor (Nrf2) binding
to the ARE sites within the promoters of the cytoprotective phase II genes, regulates their
6
expression (Hayes & McMahon, 2009; Kensler, Wakabayash, & Biswal, 2007;
Kobayashi & Yamamoto, 2005; I. T. Lee et al., 2008; Nguyen, Sherratt, & Pickett, 2003;
Taylor et al., 2008). Within the cell cytosol, Nrf2 is bound to its inhibitor Keap1. Under
oxidative stress, Keap1 and Nrf2 dissociate and Nrf2 translocates into the nucleus where
it dimerizes with Maf, Jun, Fos, ATF4 and/ or CBP, followed by binding to the ARE sites
within the promoters of target genes, therefore coordinately regulating gene expression
(Hayes & McMahon, 2009; Kensler et al., 2007).
E) Role of sulforaphane in the induction of phase II enzymes.
Sulforaphane is a naturally occurring isothiocyanate found in cruciferous
vegetables such as broccoli, Brussel sprouts, kale and cauliflower (Zhang, Talalay, Cho,
& Posner, 1992). Isothiocyanates are cytoprotective compounds, formed by the
enzymatic hydrolysis of their parent compounds, glucosinolates. The glucosinolate
precursor of sulforaphane, glucoraphanin, is abundant in broccoli, cauliflower and
cabbage with the highest concentrations found in Brussels sprouts and broccoli.
Hydrolysis of glucoraphanin to sulforaphane is catalyzed by the enzyme myrosinase
Figure 2
Figure 2: Chemical reactions involved in the heme oxygenase-1 mediated
catalysis of free heme.
7
which is released from the plant during consumption as well as present within the gut
(Clarke et al., 2008)(Figure 3).
Sulforaphane’s cytoprotective activity is thought to occur via either the inhibition
of phase I enzymes or the induction of phase II enzymes (Maheo et al., 1997).
Sulforaphane is, also, the most potent of the isothiocyanate inducers of the phase II
enzymes (Brooks, Paton, & Vidanes, 2001). Sulforaphane can inhibit cytochrome P450
enzyme activity or modulate their transcript levels within the cell. Sulforaphane inhibits
CYP1A1 and CYP1B1/2 in rat hepatocytes and CYP3A4 in human hepatocytes (Maheo
et al., 1997), therefore directly influencing the metabolism of ethanol via the MEOS
system.
The induction of the phase II genes such as HO-1 and NQO-1 by sulforaphane
occurs via the antioxidant response element (ARE) and Nrf2 (Brooks et al., 2001) .The
Figure 3.
Figure 3: Chemical Structures of A) Glucoraphanin and B) Sulforaphane. (Clarke,
Dashwood, & Ho, 2008)
8
latter is sequestered in the cell cytosol by binding to Keap1. It is not completely clear
how Keap1 regulates Nrf2 expression. It is thought that two cysteine residues, C273 and
C288, are essential for the dissociation of Keap1 from Nrf2. Sulforaphane is able to bind
to the thiol groups of Keap1, forming thionacyl adducts that result in dissociation of Nrf2
from Keap1 leading to expression of HO-1 (Brooks et al., 2001). Sulforaphane plays a
role in protecting cells from mutagenesis and is beneficial in the prevention of cancers
(Cheung & Kong, 2010). By inhibiting cytochrome P450 enzymes and by increasing
levels of cyto-protective enzymes, sulforaphane may also be beneficial in reversing the
adverse effects of ALD (McCarty, 2001).
F) MicroRNA (miRNA) and its roles in the cell.
miRNAs are a class of single stranded , non-coding RNAs, which average about
22 nucleotides in length (Bartel, 2004). miRNAs are involved in gene regulation at the
post-transcriptional level by either targeting mRNAs for degradation, destabilization or
translational repression (Filipowicz, Bhattacharyya, & Sonenberg, 2008);(Meister et al.,
2004).
1) miRNA biogenesis
miRNAs are usually transcribed by RNA polymerase II and are initially
transcribed as an ~80- nucleotide long, double stranded RNA stem-loop that is part of a
larger precursor called the primary miRNAs or pri-miRNA (Lee Y . EMBO J 2004).
Within the nucleus, the pri-miRNA forms a hair-pin shaped stem-loop secondary
structure and enters a microprocessor complex, composed of an RNAse III endonuclease,
9
Drosha and a cofactor DGCR8/Pasha (Y . Lee et al., 2003) . Within the complex,
DGCR8/Pasha recognizes the stem-loop structures and binds to the pri-miRNAs. Drosha
cleaves both strands of the stem loop, asymmetrically at sites at the base of the stem,
releasing a 60-70 nt pre-miRNA (Han et al., 2004); (Landthaler, Yalcin, & Tuschl, 2004).
The pre-miRNA binds to RAN-GTP and exportin 5 to translocate
from the nucleus into cytoplasm (Bohnsack et al., 2004; Yi et al., 2003). In the
cytoplasm, the RNase III endonuclease, Dicer cleaves the pre-miRNA hairpin, yielding
the miRNA-miRNA* duplex, with a 5' phosphate and a 3' 2 nt overhang from the end of
the hairpin structure stem, which is about 20-25 nucleotides in length (Hutvagner et al.,
Figure 4.
Figure 4: Schematics of the biogenesis of miRNA. (Esquela-Kerscher &
Slack, 2006)
10
2001; Ketting et al., 2001). The duplex is then loaded into the miRISC complex where
the anti-miRNA strand is released and degraded in the cytoplasm (Esquela-Kerscher &
Slack, 2006; Rana, 2007).
2) The RISC complex
The RNA-induced silencing complex (RISC) is a protein complex that interacts
with miRNAs and siRNAs to regulate mRNA levels in the cell et al., 2005; Maniataki &
Mourelatos, 2005). Through the RISC complex , the miRNA can induce translational
repression or degradation of mRNA by binding to perfect or nearly perfect
complementary binding sites on the 3'untranslated region (3'UTR) of the target mRNA.
Perfect binding between the miRNA and the mRNA is thought to bring about cleavage of
the mRNA and its subsequent degradation, whereas imperfect binding is thought to bring
about translational repression (Bartel, 2004). It has also been suggested that miRNA
binding hampers the movement of ribosomes along the mRNA, thereby causing
translational repression (Carrington & Ambros, 2003). miRNAs may also bring about the
rapid deadenylation of the poly(A) tail of mRNAs, accelerating mRNA degradation
(Giraldez et al., 2006; Rana, 2007; L. G. Wu, Fan, & Belasco, 2006).
3) Cellular functions of miRNA
miRNAs have been shown to play a role in a number of cellular processes such as
cell differentiation, proliferation, differentiation and development of tissues in animals
(Calin et al., 2004; Karp & Ambros, 2005; Miska, 2005). They are also involved in
mediating cellular responses to stimuli such as hypoxia (Kulshreshtha et al., 2008). Loss
11
of miRNAs may lead to oncogenesis, suggesting a role for certain miRNA as tumor
suppressors. Over-expression of certain miRNAs could also contribute to oncogenesis,
also suggesting a role for miRNAs as oncogenes (McManus, 2003).
In the present study, we examined the transcriptional and post-translational
regulation by ethanol and sulphorophane of heme oxygenase.
12
Chapter 2: Materials and Methods
Endothelial cell culture
The immortalized human dermal microvascular endothelial cell line (HMEC-1),
originally developed by Dr. Edwin Ades and Francisco J. Candall of the CDC and Dr.
Thomas Lawley of Emory University, was obtained from Centers for Disease Control and
Prevention (CDC, Atlanta). These cells were cultured in RPMI-1640 supplemented
with10% FBS, 5 mM HEPES buffer, 1 mM sodium pyruvate, 1 mM glutamine, MEM
vitamins and non-essential amino acids (1X), heparin (20 units/mL), and 50 μg/mL
endothelial cell mitogen (Biomedical Technologies, Stoughton, MA). Cells were
incubated in serum-free media for two hours prior to stimulation.
Reagents
Primary antibody for heme oxygenase-1 (HO-1) and HRP conjugated secondary
antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The β-
actin conjugated HRP antibody was purchased from Sigma-Aldrich (St. Louis, MO).
Unless otherwise specified, all other reagents were purchased from Sigma-Aldrich (St.
Louis, MO). MicroRNA mimics and anti-microRNAs were purchased from GenePharma
(Shanghai, China). The human -4.5kb- HO-1-pGL3 and the -4.5kb-HO-1-pGL3 promoter
plasmid with the ARE enhancer region deleted (E1) were generously provided by Dr.
Anupam Agarwal (University of Alabama, Birmingham).
13
Isolation of RNA and qRT-PCR
HMEC-1 cells were treated with ethanol (100 mM) and sulphorophane (50 µM)
for indicated time periods followed by total RNA extraction using TriZol (Invitrogen,
Carlsbad, CA). Real-time quantitative PCR of HO-1 and GAPDH was performed using
the iScript SYBR Green One-Step RT-PCR Kit (Bio-Rad, Hercules,CA), using specific
primers as follows: HO-1 (S):5'-CGACAGTTGCTGTAGGGCTT-3', (AS) 5'-
ACCGGACAAAGTTCATGGC-3' and GAPDH: (S) 5’-GTGCTGAGTATGTCGTGGA-
3’ and (AS)5’- ACAGTCTTCTGGGTGGCAGT-3’. Real time PCR analyses were
performed at the Analytical-Metabolic-Instrumentation Core of the USC Research Center
for Liver Disease (NIH grant P30 DK048522). PCR amplification was performed using
100 ng of RNA was under the following conditions: cDNA synthesis at 50°C for 10 min,
iScript reverse transcriptase inactivation at 95°C for 5 min, PCR cycling and detection at
95°C for 10s, and followed by elongation at 60°C for 45s, utilizing the ABI 7900 HT
sequencing detection system (Life Technologies, Carlsbad, CA). Values were expressed
as relative expression levels of mRNA normalized to housekeeping GAPDH or β-actin
mRNA levels. Relative quantitative (RQ) levels of mRNA expression were calculated by
the comparative Ct or the 2
-ΔΔCt
method, where -ΔΔCt = (Ct (target mRNA of treated
sample) – Ct (reference gene of treated sample)) – (Ct (target mRNA of control sample) –
Ct( reference gene of control sample).
Isolation and quantification of microRNAs (miRNAs)
Total miRNA was isolated from HMEC-1 using the miRVana miRNA isolation kit
(Ambion-Applied Biosystems by Life Technologies, Carlsbad, CA). miRNA levels were
14
determined and quantified utilizing specific miRNAs primers (Ambion-Applied
Biosystems by Life Technologies, Carlsbad, CA). cDNA was prepared from 50 ng of
isolated miRNA using the TaqMan microRNA assay kit (Life Technologies, Carlsbad,
CA), according to the manufacturer's protocol. Isolated miRNA was reverse transcribed
at 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. qRT-PCR was performed
using the following conditions: 95°C for 15s and 60°C for 60s, utilizing ABI 7900 HT
sequencing detection system (Life Technologies, Carlsbad, CA). miRNA expression was
normalized to reference gene U6 small nuclear RNA (snU6).
Transient transfections with luciferase promoter constructs, miRNA mimics and
inhibitors.
HMEC-1 (approximately 2 x 10
6
cells) were re-suspended in 100 µl of phosphate
buffered saline (PBS), containing 1 µg of luciferase reporter plasmid and 1 µg of SV40
Renilla plasmid or 90 pmol/1 x 10
6
cells of miRNA mimic or inhibitors. Cells were
transfected using the Y-001 program (Amaxa Nucleofector II) in accordance with the
manufacturer's protocol. The transfected cells were incubated for 24 hours in RPMI-1640
with 10% FBS, followed by over-night incubation in serum free media and treatment
with ethanol or sulforaphane for the indicated time periods. The cells were lysed and
analyzed for luciferase activity using a luminometer (Lumat LB 950, Berthold,
Badwildbad, Germany) and Dual-Glo luciferase assay system (Promega, Madison, WI).
15
Protein extraction and western blots
Total protein was extracted from HMEC-1 as previously described ((Patel, et al.,
2009). Protein concentrations were determined using the Bradford method (Bradford,
1976). The protein extracts were subjected to SDS-PAGE gel electrophoresis, followed
by transfer to nitrocellulose membranes. Membranes were probed with HO-1(dilution of
1:250), followed by incubation with goat secondary antibody (1:500). The membranes
were stripped with stripping buffer (Bioland, Paramount, CA) and re-probed with an
antibody to β-actin (1:25000) to demonstrate equal loading of the protein.
Statistical analysis
Data are presented as mean ± S.D. Student's t-test was used to determine the
significance between treated and untreated samples. The significance in difference
between multiple groups was determined using Anova followed by Tukey-Kramer test.
Analysis was performed using the Instat 2 software (Graphpad, San Diego, CA). Values
of p < 0.05 were considered statistically significant.
16
Chapter 3: Results
1. Ethanol and Sulforaphane induce HO-1 mRNA expression in HMEC.
Previous studies have shown that sulforaphane induces HO-1 activity in human
hepatocellular carcinoma cells (Prawan et al., 2008). As shown in figure 5A, in
comparison to untreated HMEC, sulforaphane treated HMEC show ~ 4-fold increase in
HO-1 mRNA expression after 4 hours of treatment, as determined by qRT-PCR. Ethanol,
which is known to induce HO-1 expression in endothelial cells, showed similar levels of
HO-1 mRNA expression as sulforaphane, after 4 hours of treatment (Figure 5A).
Sulforaphane treatment of HMEC cells also increased HO-1 protein expression by ~1.3
fold when compared to untreated cells (Figure 5B). Additionally, ethanol treatment also
increased HO-1 expression by ~1.6 fold, as shown in western blots (Figure 5B, lane 2). β-
actin was used as a loading control.
17
Figure 5: Sulforaphane augments HO-1 expression in HMEC cells. A) mRNA levels
of HO-1 in ethanol (EtOH) and sulforaphane (R-Sul) treated cells. B) HO-1 protein
expression levels in cells treated with EtOH and sulforaphane.
To determine the optimal concentration of sulforaphane that would induce HO-1
expression, we treated HMEC-1 cells with 25 µm, 50 µm, 75 µm and 100 µm of
sulforaphane (Figure 6A). 25 and 50 µm sulforaphane showed the maximum induction
of HO-1 mRNA levels, when compared to untreated cells. Additionally, HMEC cells
treated with varying amounts of sulforaphane, showed increased levels of HO-1 protein,
after 4 hours of treatment (Figure 6B). 25 µm of sulforaphane induced HO-1 protein
expression activity by 1.8 fold, while 50 µm of sulforaphane increased HO-1 protein
levels by 1.5 fold. Samples treated with 75 µm and 100 µm also showed a 1.5 fold
induction of HO-1 protein levels.
18
At the beginning of this project, we conducted a literature search that suggested
50 µM of sulforaphane was sufficient for physiological activity. We did a comparison
study using 50 and 100 µM concentrations of sulforaphane and noticed that samples
treated with 50 µM sulforaphane had about the same induction ratio as the samples
treated with 100 µM of sulforaphane. Thus, we used 50 µM of sulforaphane throughout
the project. However, a literature search conducted closer to completion of the project
showed researchers using 25 µM or even lower concentrations of sulforphane. This might
be related to the improved purification methods for sulforaphane from various sources.
Therefore, we did a titration using 25, 50, 75 and 100 µM of sulforaphane treatment
(Figure 6A and B). However, higher concentrations (50 µm versus 25 µM) also showed
induction of HO-1 at both the mRNA and the protein levels.
Figure 6: Sulforaphane induced HO-1 mRNA and protein levels in HMEC cells. A)
HO-1 mRNA levels in HMEC cells treated with various concentrations of sulforaphane.
19
B) HO-1 protein levels in HMEC cells treated with varying concentrations of
sulforaphane.
2. siRNA for Nrf2 decreased HO-1 mRNA levels.
As shown in the schematic in Figure 7A, the HO-1 promoter region has multiple
HIF-1α binding sites that are characterized by RCGTG or RGCAC. In addition, a single
ARE binding site characterized by TGCTGAGTCA at nt positions -3936 to -3927 is
present within 4.5 kb of the transcriptional start site. We therefore determined if HIF-1α
may play a role in HO-1 expression, when treated with sulforaphane. siRNA for HIF-1α
did not show significant reduction in HO-1 levels when treated with sulforaphane, when
compared to the cells treated with sulforaphane only. However, the siRNA for Nrf2
significantly reduced sulforaphane induced HO-1 levels. Control scrambled siRNA
(designated as sc) did not affect HO-1 levels in response to sulforaphane. Therefore, these
results indicate that sulforaphane induced HO-1 expression directly requires the activity
of Nrf2 but not that of HIF-1α. This result is unique as previous studies (Yeligar et al.,
2010) have shown the involvement of both Nrf2 and HIF-1α in ethanol induced HO-1
expression.
20
Figure 6: Analysis of the HO-1 promoter. A) Schematics of the HO-1 promoter. B)
Transfection results with siRNA for HIF-1α and Nrf2. (SFN: Sulforaphane)
3. The expression of luciferase reporter with HO-1 promoter constructs in response
to ethanol and sulforaphane.
The promoter region of the HO-1 genes has potential binding sites for HIF-1α, as
depicted in the schematic (Figure 7A). Because sulforaphane mediated HO-1 expression
was attenuated by siRNA for Nrf2 but not that for HIF-1α, we determined if sulforaphane
mediated HO-1 expression involved the ARE sites present within the promoter region of
HO-1. There are two ARE sites within the full length HO-1 promoter, at ~ -4 kb and ~ -
9.5 kb from the transcription start site and are designated as E1 and E2, respectively
(Hill-Kapturczak et al., 2003). As shown in figure 8, both the -9.5 kb (1.5 fold) and the
serially deleted, -4.5 kb promoter construct (2.3 fold) showed induction of promoter
activity in response to sulforaphane, when compared to cells transfected with the
21
promoter-less pGL3 vector. (Figure 8). This suggests that the -4.5 kb promoter construct
is sufficient to induce promoter activity, as previously shown (Yeligar, et al). In contrast,
sulforaphane treated cells that were transfected with the -4.5kb- construct, with a deletion
of the first ARE site (E1) (-4.5kb ΔE1) showed reduced luciferase expression.
Figure 7: Sulforaphane induced luciferase expression in the -4.5kb HO-1 construct.
Luciferase activity was inhibited by the deletion of the ARE site (∆E1).
4. MicroRNA (miRNA) expressions in HMEC in response to ethanol and
sulforaphane treatments.
MicroRNAs play an important role in post-transcriptional gene regulation. To
date, the microRNAs involved in the post-transcriptional regulation of HO-1 have not
been studied. We used a bio-informatics approach to identify the miRNAs which may
play a role in the regulation of HO-1. We identified a number of potential microRNA
22
candidates using the Microcosm web tool (http://www.ebi.ac.uk/enright-
srv/microcosm/htdocs/targets/v5/), as depicted in figure 9A.
As shown in figure 9B, four of 12 possible candidate miRNAs that were tested
showed reduced expression levels in ethanol treated cells when compared to un-treated
cells: miR762 (87%), miR518c (87%), miR33b* (78%), and miR323-5p (78%), while 8
candidates had either ca. 50% reduction (miR671, miR642, miR667, miR505, miR125a-
3p, and miR1251-5p) or their expression levels were too low to be detected by qRT-PCR
(miR673 and miR125b).
We then determined expression levels of the miRNAs in sulforaphane treated cells
(Figure 9C). Of the four miRNAs that showed the lowest expression levels in ethanol
treated cells, sulforaphane reduced miR762 by 41% , miR518c by 32%, miR 33b* by
70% and miR323-5p by 41% when compared to untreated cells.
23
Figure 8: miRNA expression levels in HMEC cells. A) Schematics of the potential
miRNA binding sites within the HO-1 3’ UTR, as determined by bio-informatics. B)
miRNA expression in HMEC cells treated with ethanol. C) miRNA expression profiles in
HMEC cells treated with sulforaphane.
24
Chapter 4: Discussion
Chronic consumption of alcohol is the leading cause of liver disease.
Acetaldehyde and reactive oxygen species (ROS), both by-products of ethanol
metabolism, contribute to the induction of inflammatory cytokines leading to liver
damage (D. Wu & Cederbaum, 2009). The oxidative stress generated by ethanol
metabolism is countered by the induction of phase II detoxifying enzymes such as HO-1
and NQO-1. These enzymes protect the cell from the effects of chronic alcohol
consumption mainly by countering increased ROS levels.
Previous studies have shown a relationship between the expression of phase II
detoxifying enzymes such as HO-1 in the liver and their stimulation by ethanol
(Drechsler et al., 2006). It is also known that sulforaphane induces these genes via the
antioxidant response element (ARE), which is bound by Nrf2 within the promoter regions
(Lin et al., 2008). However, the effect of sulforaphane on HO-1 expression in endothelial
cells has not been determined previously. In this study we hypothesize that sulforaphane
also induces HO-1 expression in endothelial cells and that transcription factors HIF-1α
and Nrf2 may play a role. Previous studies have shown ethanol treatment induces HO-1
expression in endothelial cells (Yeligar et al., 2010), thus we used ethanol as a positive
control.
We showed that sulfurophane induced HO-1 expression at both the mRNA level
and protein levels, as shown in Figure 6. The levels of induced HO-1 expression by
sulforaphane were similar to that seen with ethanol.
The expression of HO-1 has been shown to be regulated by Nrf2 (Martin et al.,
2004) and HIF-1α (Dawn & Bolli, 2005). It has been suggested that sulforaphane induces
25
HO-1 and NQO-1 via Nrf2, through formation of thiol adducts with Keap1, the binding
partner that inactivates Nrf2 activity within the cell (Prawan et al., 2008). We used small
interfering RNA (siRNA) to knock down both Nrf2 and HIF-1α in order to examine the
relationship between Nrf2 and HIF-1α in sulforaphane mediated HO-1 induction. siRNA
for Nrf2 abrogated HO-1 expression upon sulforaphane treatment; however, knocking
down HIF-1α with siRNA did not have any effect on HO-1 expression after sulforaphane
treatments. Thus, HIF-1α does not play a role in HO-1 induction by sulforaphane.
We used luciferase reporter constructs with the HO-1 promoter to further examine
the role of Nrf2 in mediating HO-1 expression. Comparison between the -4.5kb and -
9.2kb promoter constructs, showed the -4.5kb construct was more highly stimulated by
sulforaphane (2.25 fold) when compared to that seen with the -9.2kb promoter construct
(1.75 fold). This may be due to possible repressor sites present in the -9.2kb region of the
promoter that are absent in the -4.5 kb promoter construct. The -4.5kb-ΔE1, with deletion
of the ARE site at position -3936 to -3927 bp reduced HO-1 promoter activity
significantly following sulforaphane treatment. This indicated that the first ARE site (E1)
is the key binding site for Nrf2 and likely contributes to HO-1 induction in response to
sulforaphane.
We also identified potential miRNA targets that potentially play a role in the post-
transcriptional regulation of HO-1 by ethanol or sulforaphane. We used Microcosm
Targets (www.ebi.ac.uk/enright-srv/microcosm) to identify potential miRNA which target
the HO-1 3’-UTR region. The 12 candidates were chosen because of their high –ΔG
values and high conservation among multiple mammalian species. Of the potential 12
miRNAs, we saw significant reduction in the levels of 4 miRNAs in response to
26
sulphorophane, viz. miR-762, miR-518c, miR- 33b* and miR-323-5p when compared to
untreated cells. However after sulforaphane treatment, miR-323 showed the lowest levels
of expression and may potentially play a central role in HO-1 regulation.
Overall, these studies showed that sulforaphane induces HO-1 expression and that
Nrf2, but not HIF-1α plays a role, in endothelial cells. We also indentified miR-762, miR
518c, miR-33b* and miR-323-5p as potential miRNAs that may be involved in the post-
transcriptional regulation of HO-1.
Future Studies.
We have identified potential miRNAs that could play a role in the post-
transcriptional regulation of HO-1 by both ethanol and sulforaphane. Future studies
would involve the use of miRNA mimics and inhibitors which would knock-down or
induce theHO-1mRNA, and therefore lead to identification of miRNAs that may play a
role in post-transcriptionalregulation of HO-1. Additionally, we need to perform both the
western blot assay to show the effects of the miRNAs and miRNA inhibitors on HO-1
protein expression. Further studies are needed to identify the potential target sites within
the 3’UTR region of HO-1 for the binding of putative miRNAs. These results would then
need to be validated by site-directed mutagenesis of miRNA binding sites within the HO-
1 3’ UTR. By these approaches it would be possible to identify miRNAs that play a role
in both ethanol and sulforophane mediated post-transcriptional regulation of HO-1
expression.
27
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Abstract (if available)
Abstract
Alcoholism is one of the leading causes of liver disease in the United States. The liver metabolizes 80% of ethanol consumed. Ethanol and its metabolite, acetaldehyde and reactive oxygen species (ROS) generated by the metabolism of ethanol contribute to liver damage and injury, leading to release of inflammatory cytokines and fibrogenesis, which contributes to cirrhosis of the liver. Heme oxygenase-1 (HO-1) is a phase II cyto-protective enzyme which when induced, offers increased protection against various oxidative stresses. It has been observed that HO-1 expression contributes to protection against liver damage induced by several compounds such as acetaminophen and carbon tetrachloride, suggesting a role for HO-1 as a prominent hepatoprotectant (Farombi & Surh, 2006). Previous studies have shown increased expression of HO-1, when liver cells are treated with ethanol (Yao et al., 2007). Isothiocyanates such as sulforaphane are naturally occurring compounds found in brussel sprouts, broccoli and other cruciferous vegetables. ❧ Studies suggest that sulforaphane may protect cells from the side effects of chronic ethanol consumption by increasing levels of HO-1. We hypothesized that sulforaphane may induce HO-1 expression in endothelial cells and thereby contribute to the protection of these cells from the oxidative stress generated by ethanol metabolism. We also hypothesized that miRNAs may play a role in the regulation of HO-1 in endothelial cells under the influence of both sulforaphane and ethanol. In the present work, we showed that in the human dermal microvascular endothelial cells (HMEC), HO-1 expression was induced with both ethanol and sulforaphane at both the mRNA and protein levels. We also examined the possible role the HIF-1ɑ binding sites within the HO-1 promoter may play in mediating HO-1 expression with sulforaphane. We observed that sulforaphane activity mainly involved the ARE site approximately 4 kb upstream of the transcriptional start site, but not the HIF-1ɑ sites located within this region of the promoter. Furthermore, miRNA 762, miRNA 518c*, miRNA 323-5p, and miRNA 33b* were identified as potential miRNAs that may be involved in the post-transcriptional regulation of HO-1 under the influence of ethanol or sulforaphane. ❧ We hope to continue our miRNA studies by using miRNA antagonists or mimics to study their effects on HO-1 expression as well as site-directed mutagenesis of the binding sites for these miRNAs in the 3' UTR of HO-1.
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Lee, Jo
(author)
Core Title
Differential effect of ethanol and r-sulforaphane on regulation of heme oxygenase-1 in endothelial cells
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
11/28/2014
Defense Date
10/22/2012
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
heme oxygenase-1,OAI-PMH Harvest,r-sulforaphane
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Kalra, Vijay K. (
committee chair
), Tahara, Stanley M. (
committee member
), Tokes, Zoltan A. (
committee member
)
Creator Email
jolee@usc.edu,roelee12@yahoo.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-121882
Unique identifier
UC11291474
Identifier
usctheses-c3-121882 (legacy record id)
Legacy Identifier
etd-LeeJo-1364.pdf
Dmrecord
121882
Document Type
Thesis
Rights
Lee, Jo
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
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University of Southern California Digital Library
Repository Location
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Tags
heme oxygenase-1
r-sulforaphane