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2009 37: 492 originally published online 22 April 2009 Toxicol Pathol
Atrayee Banerjee, Robert Rose, Greg A. Johnson, Robert C. Burghardt and Shashi K. Ramaiah
The Influence of Estrogen on Hepatobiliary Osteopontin (SPP1) Expression in a Female Rodent Model of
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The Influence of Estrogen on Hepatobiliary Osteopontin (SPP1)
Expression in a Female Rodent Model of Alcoholic Steatohepatitis
ATRAYEE BANERJEE,1ROBERT ROSE,1GREG A. JOHNSON,2ROBERT C. BURGHARDT,2AND SHASHI K. RAMAIAH1
1Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University,
College Station, TX 77843-4467, USA
2Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University,
College Station, TX 77843-4467, USA
Our recent studies suggest that higher neutrophil infiltration in females correlates with increased hepatobiliary expression of osteopontin (OPN)
in alcoholic steatohepatitis (ASH). The objective of this study was to understand the role of alcohol in altering estrogen levels in females by examin-
ing the effect of ethanol (EtOH) on the estrous cycle and then investigate the potential relationship between estradiol (E2) and hepatobiliary OPN
expression in a female rat ASH model. Ovariectomized (OVX) and E2-implanted OVX rats in the ASH group were evaluated for OPN mRNA and
protein expression. Low doses of E2 resulted in significant down-regulation of OPN protein and mRNA as compared to the OVX group. However,
with increasing doses of E2, there was up-regulation of both OPN mRNA and protein. Osteopontin was localized primarily to the biliary epithelium.
Liver injury assessed by serum ALT and histopathology revealed a pattern similar to OPN expression. In all groups, hepatic neutrophilic infiltration
correlated positively with OPN expression. Based on these data, we conclude that in our ASH model, low doses of E2 appear to be hepatoprotective,
whereas the protective effect appears to diminish with increasing doses of E2, although additional cause and effect studies are needed for
alcoholic steatohepatitis; estrogen; liver; inflammation; osteopontin.
Osteopontin (OPN, also known as secreted phosphoprotein
1 or SPP1) is an acidic member of the small integrin-binding
ligand N-linked glycoprotein (SIBLING) family of proteins
involved in cell-to-cell and cell-to-matrix communication
(Fisher et al. 2001). This protein, a monomer of 264–301 amino
acids with molecular mass ranging from 44 to 80 kDa, is
known to play a key role in a variety of inflammatory diseases
like glomerular nephritis (Denhardt et al. 2001; Giachelli and
Steitz 2000; O’Regan and Berman 2000), inflammation during
CC14-induced hepatotoxicity (Kawashima et al. 1999), puro-
mycin-induced toxicity (Denhardt et al. 2001), and nonalco-
holic steatohepatitis (Sahai et al. 2004). Recent studies from
our laboratory have shown that OPN also plays a significant
role in alcoholic steatohepatitis (ASH) (Apte et al. 2005;
Banerjee et al. 2006a; Banerjee et al. 2006b; Ramaiah and
Rittling 2007a; Ramaiah and Rittling 2007b). In a rodent model
of ASH, increased hepatic neutrophil infiltration and liver
injury have been reported to be mediated by the higher hepato-
biliary expression of OPN (Apte et al. 2005; Banerjee et al.
2006b; Ramaiah and Rittling 2007a; Ramaiah and Rittling
2007b). In addition, females with ASH are also reported to
have significantly higher hepatobiliary expression of OPN as
compared to males. Higher OPN expression was found to cor-
relate positively with higher neutrophilic infiltration and liver
injury in females (Banerjee et al. 2006b).
Although it is recognized that OPN influences hepatic
inflammation, the precise mechanism by which OPN is
up-regulated in the liver during inflammation is not well under-
stood. Osteopontin is known to be regulated by a variety of
hormones (estrogen, progesterone, vitamin D3), cytokines, and
growth factors (Craig and Denhardt 1991; Noda et al. 1988;
Prince and Butler 1997). Several inflammatory mediators and
growth factors like interlukin-1 (IL-1), tumor necrosis factor
a (TNF-a), platelet-derived growth factor (PDGF), and trans-
forming growth factor-b (TGF-b) are also known to stimulate
OPN gene transcription (Denhardt and Noda 1998). Osteopon-
tin expression in rat mammary cells has also been reported to
be enhanced by estrogen (El-Tanani et al. 2001), and this func-
tion is postulated to be mediated by ERa. Although an estro-
gen–response element is not present in the OPN promoter,
there are seven steroid factor-response element (SFRE)-like
sequences in this region (El-Tanani et al. 2001) that have been
shown to potentially bind ERa and transactivate the OPN gene.
Clearly, understanding the influence of estrogen on the
previously reported hepatobiliary OPN expression during ASH
is noteworthy, since estrogen has been shown to play an
important role in higher susceptibility of females to alcoholic
liver disease. Studies by Iimuro et al. (1997) and Nanji et al.
Address correspondence to: Shashi K. Ramaiah, D.V.M., Ph.D., Diplomate
ACVP, DABT, Department of Pathobiology, College of Veterinary Medicine,
Texas A&M University, MS-4467, College Station TX 77843-4467; e-mail:
Competing interests: The authors have not declared any competing interests.
Abbreviations: ALT, alanine aminotransferase; ASH, alcoholic steato-
hepatitis; BE, biliary epithelium; E2, estradiol; EtOH, ethanol; LPS, lipo-
polysaccharide; OPN, osteopontin; OVX, ovariectomized; SPP1, secreted
Toxicologic Pathology, 37: 492-501, 2009
Copyright # 2009 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
(2001) have shown that female rats fed EtOH have higher
levels of endotoxin in plasma than their male counterparts.
Because estrogen receptors exist in the intestinal epithelium,
estrogen has been reported to affect the permeability of the gut,
leading to increased absorption of gut microflora or endotoxin
in the blood in females (Kono et al. 2000; Yin et al. 2000),
which initiates a cytokine cascade, leading to macrophage acti-
vation via membrane CD14 (Schumann et al. 1990) and greater
liver injury. Because higher OPN expression in female rodent
models of ALD contribute to more severe liver injury, it was
reasonable to test the hypothesis that higher estrogen in females
also contributes to higher hepatic OPN expression in females.
In this study, we report that (1) alcohol alters estrogen levels
in females, as indicated by the estrous cycle; and (2) estradiol
has a protective effect on liver injury at lower doses, and the
protection appear to decrease with increasing doses of estro-
gen, and the extent of OPN expression caused by estradiol is
also dose dependent.
MATERIALS AND METHODS
Assessment of Estrous Cycle
To determine the effect of ethanol on the estrous cycle, daily
cytological vaginal smears of animals were performed before
and after feeding of EtOH. Briefly, vaginal samples were taken
in the morning (between 9:00 and 11:00 AM) using a small cot-
ton swab moistened with saline. The vaginal swabs were then
smeared onto a glass slide and stained with Diff-Quik stain.
The different stages of the estrous cycle were determined by
microscopic visualization of vaginal cell types, as follows:
(a) proestrus, determined by the presence of clusters of round,
nucleated epithelial cells; (b) estrus, characterized by kerati-
nized cornified cells; (c) metestrus, defined as the presence
of noncornified epithelial cells with a few leukocytes; and (d)
diestrus, with a predominance of leukocytes and few round
epithelial cells (Goldman et al. 2007; Long and Evans 1922).
Rodent ASH Model
The rat ASH model was based on previous studies from our
laboratory involving administration of endotoxin (LPS) after
six weeks of oral EtOH in the Lieber De-Carli diet (Apte
et al. 2005; Banerjee, Apte et al. 2006). Female Sprague-
Dawley (SD) rats, eight weeks old and approximately 220–
250 g in weight were purchased from Harlan Sprague-Dawley
(Houston, TX, USA), and housed individually in cages (area:
144.5 square inches) in a temperature- and humidity-controlled
(temperature: 65?F–80?F; humidity: 45%–50%) animal facility
with a twelve-hour light-dark cycle. Rats were entered into the
study after a one-week acclimatization period. Age- and
weight-matched female SD rats were divided into control,
EtOH, controlþLPS, and EtOHþLPS (n¼ 4 each) groups. The
experimental rats were fed with EtOH-containing (EtOH ¼
35.5% of total calories, Apte et al. 2005) Lieber-DeCarli liquid
diet for a period of six weeks. The control rats were pair-fed
with isocaloric maltose-dextrin diet. After six weeks of
feeding, the rats were injected with a single dose of LPS or sal-
ine (E. coli 0111:B4: 500,000 EU/mg, 10 mg/kg body weight,
intraperitoneally (i.p.) in saline, Sigma Diagnostics, St. Louis,
MO, USA), and sacrificed twelve hours later by CO2
In the case of the ovariectomized (OVX) rats, they were
allowed to recover for three weeks after surgery, and then they
were divided into the following experimental groups (n ¼ 4 in
each group): EtOHþLPS (Ovx), EtOHþLPSþ0.18 mg E2
(Ovx), EtOHþLPSþ0.36 mg E2 (Ovx), EtOHþLPSþ0.72 mg
E2 (Ovx), EtOHþLPSþ1.7 mg E2 (Ovx), controlþLPS (Ovx),
controlþLPSþ0.18 mg E2 (Ovx), controlþLPSþ0.36 mg
E2 (Ovx), controlþLPSþ0.72 mg E2 (Ovx), and con-
trolþLPSþ1.7 mg E2 (Ovx). The animals in the experimental
and the control groups were fed EtOH-containing Lieber-
DeCarli diet and isocaloric maltose dextrin diet for a period
of six weeks, as mentioned previously. After six weeks of feed-
ing, the rats were injected with a single dose of LPS (E. coli
0111:B4, 10 mg/kg, i.p. in saline, Sigma Diagnostics, St.
Louis, MO, USA). The animals in the neutralizing OPN
(nOPN) antibody-treated group were injected i.p. with the
two doses of nOPN (diluted in PBS, 200 mg/kg body weight,
R&D Systems, Minneapolis, MN, USA) antibody, six hours
apart, before the LPS injection (last dose of nOPN antibody
was injected at the same time as LPS). The rats were then
sacrificed by CO2asphyxiation twelve hours after the LPS
injection. All rats were weighed on the day the study was
initiated and weekly thereafter. Animals were provided
humane care in compliance with the institutional guidelines
(ULACC; University Laboratory Animal Care Committee) of
Texas A&M University.
Ovariectomy and Estradiol Supplementation
This surgical procedure was followed to assess the affects of
estradiol on ethanol-mediated liver injury in the ASH model.
Female Sprague-Dawley rats were anesthetized with ketamine
(87 mg/kg body weight) and xylazine (13 mg/kg body weight),
and bilateral OVX was performed using a dorsal midline inci-
sion ventral to the spine and caudal to the last rib (Jezerski and
Sohrabji 2000). In some of the animals (n ¼ 8 in each group), a
sixty-day, time-release 17-b estradiol pellet (0.18, 0.36, 0.72,
and 1.7 mg; Innovative Research, Clearwater, FL, USA) was
inserted subcutaneously prior to closing the surgical incision.
The estradiol doses employed in this study were based on the
therapeutic and supratherapeutic concentration achieved in
blood (Nordell et al. 2003). Based on this study, the 0.18- and
0.36-mg doses were considered to be therapeutic low doses,
whereas 0.72 and 1.7 mg were considered higher doses.
Sample Collection and Processing
After CO2asphyxiation, blood from the different experi-
mental groups was collected from the dorsal aorta and placed
in heparinized tubes. Liver transaminase activities were esti-
mated from a fraction of heparinized plasma (about 0.5 mL),
and the remaining plasma was snap-frozen in liquid N2and
Vol. 37, No. 4, 2009 ESTROGEN AND OSTEOPONTIN RELATIONSHIP IN ALCOHOLIC STEATOHEPATITIS493
stored at ?80?C. Livers were harvested, weighed, and divided
into two parts. Slices of the left and median lobes were fixed in
10% neutral buffered formalin for histopathologic evaluation,
whereas remaining liver tissue was snap-frozen in liquid N2
and stored at ?70?C for OPN mRNA and protein expression
Evaluation of Liver Injury
Liver injury was assessed by plasma transaminase activities
(alanine aminotransferase; ALT) and histopathology of hema-
toxylin and eosin (H&E)-stained liver sections, as described
previously (Apte et al. 2005; Banerjee et al. 2006a).
Histological evaluation of inflammation mediated by
ethanol was evaluated on paraffin-embedded liver sections
(one section each from the left and median lobe was examined).
Hematoxylin and eosin staining was employed to identify the
neutrophils, based on the segmented morphology of the nucleus
followed by quantification with naphthol AS-D chloroacetate
esterase staining (Sigma Diagnostics, St. Louis, MO, USA),
as described previously (Banerjee, Apte, et al. 2006). Briefly,
4-mm-thick, formalin-fixed, paraffin-embedded liver sections
were deparaffinized and incubated in the substrate solution
(40 mL warm distilled water, 1 mL sodium nitrate, 1 mL fast
blue violet LB, 5 mL of trizma 6.3 buffer concentrate, 1 mL
naphthol AS-D) for thirty minutes in a 37?C water bath. The
slides were kept in the dark during the incubation period, after
which they were washed in distilled water and counterstained
with Gill’s hematoxylin for forty-five seconds, followed by
rinsing in tap water four times. The slides were then dipped
three times in 70% alcohol, 100% alcohol, and xylene. The
dehydrated sections were then mounted in cytoseal and exam-
ined. The red-colored cytoplasmic staining was specific for
neutrophils. A blood-smeared slide was used as a positive con-
trol for the experiment. To quantify the degree of neutrophilic
inflammation (inflammation score), the neutrophilic foci per
five high power fields (40?, fields were randomly selected)
was counted. The neutrophilic foci (defined as an aggregate
of four neutrophils) were quantitated per five 40? fields.
Analysis of OPN mRNA Localization and Expression by
In Situ Hybridization
Osteopontin mRNA expression in liver sections was
localized by in situ hybridization as previously described
(Johnson et al. 1999). Briefly, deparaffinized, rehydrated, and
deproteinated liver cross-sections (5 mm) were hybridized with
[35S]-radiolabeled antisense or sense OPN cRNA probes. The
sense probe was used as a negative control to define nonspeci-
fic hybridization. Following washes and RNAaseA digestion,
the slides were dipped in Kodak NTB-2 liquid photographic
emulsion (Kodak, Rochester, NY, USA), stored at 4?C for five
days, developed in Kodak D-19 developer, counterstained with
Harris modified hematoxylin (Fisher Scientific, Fairlawn, NJ,
USA), dehydrated, and protected with cover slips. Digital
photomicrographs of representative bright and dark field
images were evaluated with a Zeiss Axioplan2 microscope
(Carl Zeiss, Thornwood, NY, USA) fitted with an Axiocam
sequentially with AxioVision 4.3 software and in the Zeiss
Vision Image (ZVI) file format, and they were subsequently
converted to tagged image file (TIF) format. All the figures
were assembled in Adobe Photoshop 7.0.1 (Adobe Systems,
Inc., San Jose, CA, USA).
Osteopontin Protein Expression
Western Blot Analysis: Liver cell lysates from control, intact,
and OVX ASH groups were prepared in lysis buffer (1% Tri-
ton-X-100, 50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM
EGTA, 2 mM Na vanadate, 0.2 mM PMSF, 1 mM HEPES,
1 mg/mL leupeptin, and 1 mg/mL aprotinin) and protein concen-
tration was estimated using a Bio-Rad protein assay kit
(BioRad, Hercules, CA, USA) according to the manufacturer’s
protocol. Briefly, 100 mg of cell lysate was resolved by electro-
phoresis on a 12% sodium dodecyl sulfate (SDS) polyacryla-
mide gel (100 v, 1.5 hours) in a running gel buffer containing
25 mM Tris, pH 8.3, 162 mM glycine, and 0.1% SDS. The
samples were transferred to nylon membrane for three hours
at 500 mA. The membranes were incubated overnight in a mix-
ture ofT-TBS with 0.1% tween and 2% milkand OPN antibody
(rabbit polyclonal to OPN, 1:1000 dilution, Abcam, Inc.,
Cambridge, MA, USA). Subsequently, the membrane was
incubated in anti-goat secondary antibody for one hour at room
temperature. The OPN antibody recognizes both the native
(uncleaved) form of OPN (*66 KD), and the cleaved form
of OPN (32 KD; Rittling and Feng 1998). Visualization was
carried out with the enhanced chemiluminescence kit (Pierce,
Rockford, IL). Glyceraldehyde-3-phosphate dehydrogenase
was used as an internal control to ensure equal loading of
proteins per well.
Group comparisons were performed using one-way analysis
of variance (ANOVA) test and by the Tukey post hoc test.
Statistical analyses were made using Graph Pad Prism 5.01 soft-
ware (GraphPad Software, Inc., La Jolla, CA, version 5.01.).
Statistical significance was set at p < .05.
Role of Ethanol in Influencing Estrous Cycle
Consistent with Long and Evans (1922), the estrous cycles
in rats fed control diet were found to be about 4.5 days long and
regular in about 88% of the animals studied. Only animals
cycling for about four days were fed EtOH in Lieber DeCarli
diet for six weeks. After the initiation of EtOH feeding, the ani-
mals were found to have prolonged estrous cycles (about
5.7 days) due to increased length of the diestrous phase (about
three days). The diestrous phase was marked by the
494 BANERJEE ET AL.TOXICOLOGIC PATHOLOGY
predominance of leukocytes and nucleated epithelial cells in
the vaginal smear, indicating that EtOH was interfering with
the estrous cycle in these rats, leading to alteration of the estro-
Effect of Ovariectomy on Ethanol-mediated Liver
Both the intact and the ovariectomized females had little or no
increase in plasma transaminase (ALT) activity following treat-
nase activity was noted in both the intact and the OVX group
following EtOHþLPS treatment as compared to the respective
control, EtOH alone, and LPS alone groups (Figure 1A).
When the liver injury was compared between OVX and
intact groups, the OVX females in the EtOHþLPS-treated
group had approximately 1.5-fold higher plasma transaminase
activity as compared to the intact females in the same group.
The findings of the H&E-stained liver sections were consistent
with the plasma transaminase data, wherein increased
multifocal necrosis and neutrophilic infiltration in the OVX
EtOHþLPS-treated animals (approximately two-fold) was
noted as compared to the intact EtOHþLPS-treated animals
(Figures 1B and 2). The animals in the control group that were
implanted with estrogen pellets experienced no liver damage
(data not shown).
Dose-dependent Effect of Estradiol on Liver Injury and
To assess the effect of estradiol on hepatic injury, plasma
ALT transaminase activity and liver histopathology were also
evaluated in OVXþE2-implanted rats fed EtOHþLPS. Com-
pared to the OVX group, all animals in the estrogen-implanted
group had a significant decrease in plasma transaminase activ-
ity. However, a biphasic response to ASH was observed in the
OVXþE2-implanted rats. Low doses of E2 (0.18 mg, 0.36 mg)
resulted in a significant decrease in plasma transaminase activ-
ity as compared to the OVX group. However, the highest E2
dose employed (1.7 mg) resulted in decreased protection as
compared to the 0.36-mg and 0.72-mg doses of E2, suggesting
a mild elevation of plasma transaminase activity (Figure 3A).
Rats treated with the 0.36-mg dose of E2 seemed to have the
least liver injury in these groups (Figures 3A, 3B, and 4).
The plasma transaminase activity was further confirmed with
H&E-stained liver sections, where minimal or no neutrophilic
infiltration and lack of multifocal coagulative necrosis was
observedin animalstreatedwith 0.36 mgestrogen
FIGURE 1.—(A) ALT activities in plasma; (B) representative photomi-
crographs of hematoxylin and eosin-stained liver sections of intact and
ovariectomized rats fed either control or EtOH-containing Lieber
DeCarli diet for six weeks followed by a single LPS injection and
sacrificed twelve hours post-LPS injection, as described in the Meth-
ods section. *Values significantly different from the respective con-
trols (intact and ovariectomized control rats).!Values significantly
different from the intact EtOHþLPS-treated females. Data are
expressed as mean + standard error, p ? .05. Circles indicate necrosis
and neutrophil infiltration. EtOH, ethanol; LPS, lipopolysaccharide.
FIGURE 2.—Inflammation scores in the livers of intact and ovariecto-
mized rats fed either control or EtOH-containing Lieber DeCarli diet
for six weeks followed by a single LPS injection and sacrificed twelve
hours post-LPS injection, as described in the Methods section. *Val-
ues significantly different from the respective controls (intact and
ovariectomized control rats).!Values significantly different from the
intact EtOHþLPS-treated females. Data are expressed as mean +
SE, p ? .05. EtOH, ethanol; LPS, lipopolysaccharide.
Vol. 37, No. 4, 2009ESTROGEN AND OSTEOPONTIN RELATIONSHIP IN ALCOHOLIC STEATOHEPATITIS 495
(Figures 3B and4). Inanimals treated with 0.72 mgand 1.7 mg
E2, an increase in neutrophilic infiltration and focal areas of
necrosis was observed.
Localization and Expression of OPN mRNA by In Situ
Because we have shown previously that OPN is the likely
mediator of hepatic neutrophil infiltration and liver injury
in the intact rat ASH model (Banerjee et al. 2006a), we assessed
the effect of estradiol on OPN expression and hepatic neutrophil
infiltration.Insituhybridization was carriedout todetermine the
hepatic source of OPN expression in OVX and E2-supplemen-
had a higher OPN mRNA signal (Figure 5A) as compared to the
low-dose, estradiol-supplemented (0.18 mg, 0.36 mg), ovariec-
tomized rats (Figures 6A and 6B). However, as compared to the
low doses of E2 (0.18 mg, 0.36 mg), a significantly higher OPN
FIGURE 3.—(A) ALT activities in plasma of ovariectomized and ovariectomizedþestrogen-implanted female rats; (B) representative photomicro-
graphs of H&E-stained liver sections of ovariectomizedþestrogen-implanted female rats fed either control or EtOH-containing Lieber DeCarli
diet for six weeks followed by a single LPS injection and sacrificed twelve hours post-LPS injection, as described in the Methods section. *Values
significantly different from the ovariectomized EtOHþLPS-treated group. Data are expressed as mean + SE, p ? .05. Circles indicate necrosis
and neutrophil infiltration. EtoHþLPS, ethanolþlipopolysaccharide; O, ovariectomized.
496BANERJEE ET AL.TOXICOLOGIC PATHOLOGY
mRNA signal was observed in the animals treated with increas-
ing doses (0.72 mg and 1.7 mg) of estrogen (Figures 6C and
6D).Thelowest OPNmRNAsignalwas observedinthe animals
show any nonspecific hybridization (Figure 5B). The localiza-
tion of OPN was mostly within the biliary epithelium.
Dose-response Effects of Estradiol on Hepatobiliary
Western blotting illustrated a minimaldecreaseinthe levelof
OPN protein in the OVX animals supplemented with E2 in
the ASH model (i.e., EtOHþLPSþE2) as compared to the
EtOHþLPS alone group, with the exception of the highest dose
of E2 (Figure 7). The animals treated with the highest E2 dose
Induction of hepatic OPN protein was also supported by
immunohistochemistry (data not shown), where animals treated
with the 0.18-mg and 0.36-mg doses of E2 had lower expression
of OPN, with maximum expression observed at the highest dose
of E2 employed in this study. As previously reported, biliary
epithelial cells are the predominant OPN-producing cells in the
liver in the rat model of alcoholic liver disease employed in this
study (data not shown).
Effect of Neutralizing OPN (nOPN) Antibody on Liver
Injury and Neutrophil Infiltration
An nOPN experiment was carried out to assess the role
E2and OPNin liver
nOPN (ovariectomizedþnOPNþEtOHþLPSþ1.7 mg
had a significant decrease in liver injury as determined by
ofinjury. Ratstreated with
plasma ALT, when compared to the untreated group
(ovariectomizedþEtOHþLPSþ1.7 mg E2; Figure 8A). In
addition, the nOPN antibody-treated group also had a signifi-
cant decrease (approximately 85%) in neutrophilic infiltration
in the hepatic parenchyma (Figure 8B), indicating that OPN
was involved in the severe liver injury and neutrophilic infiltra-
tion experienced by the ovariectomized rats implanted with
1.7 mg E2 in the EtOHþLPS-treated group.
We have previously reported that female rats in the ASH
model experience significantly more severe liver injury than
the males. In this model, severe liver injury is accompanied
by increased neutrophil infiltration, which appears to be
OPN mediated (Banerjee et al. 2006a). However, the precise
mechanism by which OPN is increased in the rat ASH
model, especially in females, is unknown. Osteopontin
expression is known to be regulated by E2 (Lessey 2002;
White et al. 2005; White et al. 2006). In addition, estrogen
has also been shown to increase susceptibility of females to
alcoholic liver disease. However, nothing is known about
the role of estrogen in the regulation of OPN expression
in alcoholic liver disease, which is the basis for this inves-
tigation. In this study, we found that estradiol has a poten-
tial protective effect on ethanol-mediated liver injury,
although the observed protection seemed to diminish with
increasing doses of estrogen.
Alteration of the estrous cycle following alcohol consump-
tion has been previously reported by Rettori et al. (1987) and
Emanuele et al. (2001). Rats fed an alcohol diet consistently
exhibit a prolonged diestrous phase. The alteration in the
estrous cycle in the former study was attributed to alcohol-
induced depression in serum luteinizing hormone (LH) and
consequent diminished release of LHRH from the hypothala-
mus. Our results are consistent with these studies, where rats
experienced a prolonged diestrous phase following alcohol
consumption, suggesting that alcohol is altering the estrogen
level in our model. Also, previous studies from this laboratory
have reported that females in ALD have higher expression of
OPN (Banerjee et al. 2006a), suggesting that expression of
OPN is possibly regulated by estrogen.
Although ethanol consumption alters circulating levels of
sex hormones, estrogen has been implicated in severe liver
injury in females in alcoholic liver disease (Yin et al. 2000).
These results were obtained using a continuous intragastric
feeding model, in which OVX females with alcoholic liver dis-
ease had significantly lower liver injury and inflammation as
compared to intact females, and this condition could be
reversed with estrogen replacement. This study is somewhat
contradictory to results of the present study, where OVX
females experienced significantly greater liver injury and
inflammation as compared to intact females. Factors such as
the type of alcoholic liver disease model used, species of rats
employed, and dose of estradiol may have contributed to the
observed contradiction. In fact, in this study we showed that
FIGURE 4.—Inflammation scores in the liver of ovariectomized and
ovariectomizedþestrogen-implanted female rats fed either control or
EtOH-containing Lieber DeCarli diet for six weeks followed by a sin-
gle LPS injection and sacrificed twelve hours post-LPS injection, as
described in the Methods section. *Values significantly different
from the ovariectomized EtOHþLPS-treated group. Data are
expressed as mean + standard error, p ? .05. EtoHþLPS, etha-
nolþlipopolysaccharide; O, ovariectomized.
Vol. 37, No. 4, 2009ESTROGEN AND OSTEOPONTIN RELATIONSHIP IN ALCOHOLIC STEATOHEPATITIS 497
higher E2 doses increased hepatic injury and neutrophil infil-
tration compared to the lower doses. In our model, the level
of estrogen in the blood of animals treated with the highest dose
of E2 were similar to the levels achieved in the studies by Yin
et al. (2000; 224 pg/mL).
In concordance with our studies supporting the protective
effect of estradiol, other researchers have shown that estradiol
mental model of autoimmune encephalomyelitis and an animal
model of multiple sclerosis, low-dose estrogen therapy was
shown to prevent the clinical signs and histopathological lesions
of the disease (Bebo et al. 2001; Jansson et al. 1994; Rosette and
Karin 1995). Also, other inflammatory conditions such as wound
2002), ischemia (Dubal et al. 2001), uveitis (Miyamoto et al.
1999), and leukodystrophy (Matsuda et al. 2001) were shown
to be significantly influenced by estradiol, where estradiol
decreased disease susceptibility and severity of damage.
The precise mechanism by which estradiol is protective dur-
ing inflammation is worthy of discussion. Studies by Ghisletti
et al. (2005), have shown that estrogen blocks inflammation by
its action on the transcription factor p65/relA of the NF-kB
family. In macrophages, estrogen was reported to block lipopo-
lysaccharide-induced DNA binding and transcriptional activity
of p65 by preventing its nuclear translocation, without altering
the Ik-B kinase (IKK) activity. This has been suggested to be
mediated by ERa through a nongenomic mechanism, indicat-
ing the role of the estrogen-ERa signaling pathway in
mediating early inflammatory response (Ghisletti et al. 2005).
Certainly, these mechanisms need to be investigated in future
studies to ascertain the mechanistic basis for estrogen
In the literature, OPN expression is known to be regulated
by estrogen (Craig and Denhardt 1991). In the peri-implanta-
tion pig and mouse uterus, estrogen is known to induce OPN
mRNA expression in the endometrial luminal epithelium
(White et al. 2005; White et al. 2006). Indeed, estrogen and
progesterone appear to regulate OPN expression in the human
uterus. Similar induction of OPN mRNA has been observed in
our model, with higher doses of estrogen. However, lower
doses of estrogen decreased OPN expression. This finding is
in concordance with the studies by Turner et al. (1990), where
estrogen (diethylstilbestrol) was reported to down-regulate
OPN levels in the bone tissue of OVX rats. It can be argued that
in addition to E2, other hormones such as progesterone may
play a role in OPN induction. In fact, there are studies in the
literature that have shown that the hormone progesterone also
controls OPN expression (Craig and Denhardt 1991; Johnson
et al.2000; White et al. 2006).The OPN genehas been reported
to contain a progesterone-response element in its 50-flanking
region that is likely induced by progesterone in vivo in mice
(Craig and Denhardt 1991). Also, OPN is up-regulated in
human cytotrophoblasts by progesterone (Omigbodun et al.
1997), indicating that in addition to estrogen, the expression
of OPN in our model could also be influenced by progesterone,
and this possibility is worthy of future investigation.
FIGURE 5.—In situ hybridization analysis of OPN mRNA in the liver of (A) a female rat fed EtOH-containing Lieber DeCarli diet followed by a
single LPS injection. Corresponding bright and dark field images are shown. (B) A section hybridized with a radiolabeled sense cRNA probe
served as the negative control. BE, biliary epithelium.
498BANERJEE ET AL.TOXICOLOGIC PATHOLOGY
In conclusion, estrogen was found to have a protective effect
on ethanol-mediated liver injury, although the protection
appeared to diminish with increasing doses of E2 in the rat
model of alcoholic liver injury. The effects of E2 in this model
appear to be mediated by OPN. Based on the deleterious effect
of OPN on hepatic inflammation during liver injury in the rat
FIGURE 6.—In situ hybridization analysis of OPN mRNA in the liver of estradiol-supplemented, ovariectomized rats fed EtOH-containing Lieber
DeCarli diet followed by a single LPS injection. Corresponding bright and dark field images are shown. Representative liver section of (A)
EtOHþLPS, O, 0.18 mg E2; (B) EtOHþLPS, O, 0.36 mg E2; (C) EtOHþLPS, O, 0.72 mg E2; and (D) EtOHþLPS, O, 1.7 mg E2. BE, biliary
epithelium; EtOH, ethanol; LPS, lipopolysaccharide; O, ovariectomized; E2, estrogen.
Vol. 37, No. 4, 2009 ESTROGEN AND OSTEOPONTIN RELATIONSHIP IN ALCOHOLIC STEATOHEPATITIS499
ALD model employed, it is possible that OPN levels can be
altered by E2 and that can potentially exaggerate or diminish
liver injury. Although further studies are needed to confirm this
observation, potential novel therapeutic strategies, such as E2
to influence OPN induction, can be developed to treat human
alcoholic liver disease patients.
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