From the Division of Biopharmaceutics, Leiden Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, Leiden, Zuid-Holland 2300 RA, The Netherlands
Received for publication, February 4, 2003 , and in revised form, April 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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The identification of SR-BI and novel members of the ABC transporter family, including ABCA1, ABCG1, ABCG5, and ABCG8, has allowed the molecular characterization of the individual transporters responsible for the intracellular trafficking and excretion of cholesterol (derivatives). In the liver, it has been shown that SR-BI is responsible primarily for the selective uptake of cholesterol esters from HDL (8), whereas ABCG5/G8 and ABCG1, and ABCA1, are proposed mediators of efflux to the bile and to HDL, respectively (6, 9). However, the liver is a complex tissue and contains, in addition to the parenchymal cells, which are localized around the bile canaliculi, endothelial cells, and tissue macrophages (Kupffer cells). To assess the individual function of the ABC transporters and their regulation by nuclear hormone receptors it is therefore essential to establish their cellular localization in the liver.
Here we report that key mediators in liver cholesterol homeostasis, in
particular PPAR, PPAR
, and ABCG1, are expressed differentially
in specific cell types of the rat liver. Our data stress that it is necessary
to focus on the regulation of genes involved in cholesterol homeostasis in the
different cell types of the liver to get molecular insight in their mechanism
of regulation and the consequences for liver cholesterol transport.
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EXPERIMENTAL PROCEDURES |
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Analysis of Gene Expression by Real Time Quantitative PCRTotal RNA was isolated from rat liver parenchymal, endothelial, and Kupffer cells using TriZol reagent (Invitrogen) according to the manufacturer's instructions. Purified RNA was DNase treated (DNase I, 10 units/µg of total RNA) and reverse transcribed (RevertAid M-MuLV reverse transcriptase) according to the protocols supplied by the manufacturer.
Quantitative gene expression analysis was performed on an ABI PRISM 7700 machine (Applied Biosystems, Foster City, CA) using SYBR Green technology. PCR primers (Table I) were designed using Primer Express 1.7 software with the manufacturer's default settings (Applied Biosystems) and validated for identical efficiencies (slope = 3.3 for a plot of the threshold cycle number (Ct) versus log ng cDNA). In 96-wells optical plates, 12.5 µl of SYBR Green master mix was added to 12.5 µl of cDNA (corresponding to 50 ng of total RNA input) and 300 nM forward and reverse primers in water. Plates were heated for 2 min at 50 °C and 10 min at 95 °C. Subsequently 40 PCR cycles consisting of 15 s at 95 °C and 60 s at 60 °C were applied. At the end of the run, samples were heated to 95 °C with a ramp time of 20 min to construct dissociation curves to check that single PCR products were obtained. The absence of genomic DNA contamination in the RNA preparations was confirmed by using total RNA samples that had not been subjected to reverse transcription. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as the standard housekeeping gene. Ratios of target gene and HPRT expression levels (relative gene expression numbers) were calculated by subtracting the Ct of the target gene from the Ct of HPRT and raising 2 to the power of this difference. Ct values are defined as the number of PCR cycles at which the fluorescent signal during the PCR reaches a fixed threshold. Target gene mRNA expressions are thus expressed relative to HPRT expression.
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Data AnalysisThe significance of differences in relative gene expression numbers among the different liver cell types, from three different cell isolations, measured by real time quantitative PCR was calculated using a two-tailed Student's t test on the differences in Ct (Ct(HPRT)Ct(target gene)). Probability values less than 0.05 were considered significant.
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RESULTS |
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In the liver, cholesterol is used for bile acid synthesis. Therefore, the mRNA expression patterns of two key enzymes in bile acid synthesis, CYP7A1 and CYP27, in the different hepatic cell types were examined. A relatively high level of CYP7A1 expression was observed in parenchymal cells, which was more than 200-fold (p < 0.001) higher than the expression levels found in endothelial and Kupffer cells (Fig. 2a). Accordingly, CYP27 expression (Fig. 2b) was observed in parenchymal cells and also in endothelial cells.
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Because a second route of cholesterol disposal from the liver is through direct excretion of cholesterol into the bile via the half-transporters ABCG5 and ABCG8, we investigated whether the expression of ABCG5/8 is also higher in parenchymal cells than in non-parenchymal cells. Fig. 3 clearly indicates that ABCG5 (a) and ABCG8 (b) expression was indeed 510-fold higher in parenchymal cells compared with endothelial and Kupffer cells.
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In the liver, ABCA1 was recently suggested to be involved in the efflux of
cholesterol for production of HDL
(6). Although no conclusive
evidence has been shown, in the liver ABCG1, like ABCG5/G8, is proposed to
play a role in biliary efflux
(11). In macrophages, ABCA1
and ABCG1 expression is induced in response to cholesterol loading, and both
proteins are potentially involved in cholesterol efflux to apoA-I
(12). Because both ABCA1 and
ABCG1 are implicated in the same physiological functions, we determined
whether both genes also have a comparable expression distribution profile over
the different cell types of the liver. Contrary to the assumption, ABCA1 was
expressed mainly in parenchymal and Kupffer cells
(Fig. 4a), whereas
ABCG1 was expressed 76-fold (p < 0.001) and 27-fold (p
< 0.01) higher in Kupffer and endothelial cells than in parenchymal cells,
respectively (Fig.
4b). In contrast to ABCA1, ABCG1 is thus mainly expressed
in non-parenchymal cells, which suggests a limited role of ABCG1 in the
excretion of cholesterol directly into the bile under the standard feeding
conditions. In addition, ABCG1 expression was analyzed in the different
hepatic cells isolated from rats on a high cholesterol diet. Interestingly,
hepatic parenchymal cell ABCG1 expression increased 4-fold (p
< 0.05) in response to a high cholesterol diet, whereas no significant
effect on endothelial and Kupffer cell ABCG1 expression was observed
(Fig. 5). Although ABCG1 levels
were significantly increased in parenchymal cells in response to a high
cholesterol diet, ABCG1 expression levels were still, respectively, 10- and
12-fold higher in endothelial and Kupffer cells compared with parenchymal
cells.
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Analysis of LXR expression in the different cell types was performed to
investigate a potential relation with expression patterns of the ABC
transporters. LXR had a distribution pattern comparable to that found
for ABCA1, with a relatively high expression in parenchymal (p <
0.01) and Kupffer cells compared with endothelial cells
(Fig. 6a). A
significantly higher expression of LXR
was found in endothelial liver
cells compared with parenchymal (p < 0.05) and Kupffer cells
(p < 0.05), respectively (Fig.
6b), which suggests that LXR
may be a more
important mediator in endothelial cells.
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It has been shown that PPAR activators are able to regulate LXR
expression, and thereby indirectly influence ABCA1 mRNA levels
(13), and that ligands for
PPAR
directly regulate the expression of ABCA1 via an unknown mechanism
(14). Therefore, we also
investigated the PPAR gene expression in the different cell types.
Fig. 7a clearly indicates that
PPAR
expression is found mostly in parenchymal cells, with an 82- and
23-fold higher expression (p < 0.001 in both cases) in these cells
than in endothelial cells and Kupffer cells, respectively. The PPAR
distribution pattern is comparable with that found for PPAR
(Fig. 7b), suggesting
a major function of these genes in parenchymal cells. PPAR
mRNA levels
were almost equal in the different cells, although endothelial cell
PPAR
expression was somewhat higher as compared with parenchymal and
Kupffer cells (Fig.
7c). These data indicate that within the various liver
cell types PPAR
will have a more general function.
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DISCUSSION |
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In the liver, SR-BI plays a crucial role in the selective uptake of cholesterol esters from HDL (2). Additionally, studies on ABC transporters suggested that hepatic ABCA1 is involved in HDL production (6), whereas ABCG5/G8 and ABCG1 were indicated to mediate biliary efflux of cholesterol from the liver (7, 9). Repa et al. (17) showed that treatment of mice with synthetic ligands of LXR markedly increased liver ABCG5/G8 expression. In vitro observations by Malerod et al. (18) also indicate that LXR is able to regulate hepatic SR-BI expression through a direct interaction with a newly discovered LXR/retinoid X receptor response element in the SR-BI promoter. It is however still unclear how hepatic ABCA1 and ABCG1 expression is regulated and what the precise consequence of their regulation is on hepatic cholesterol levels and transport.
The liver consists of several different cell types, including parenchymal, endothelial, and Kupffer cells. It is therefore important to study the expression levels of SR-BI and the ABC transporters in the individual hepatic cell types to get a more detailed view of their specific functions and regulatory mechanisms in the liver.
Earlier studies performed by Pieters et al. (19) showed that uptake of HDL cholesterol esters into liver parenchymal cells is efficiently coupled to a rapid synthesis of bile acids. Accordingly, Fluiter et al. (20) observed that the receptor responsible for the selective uptake of cholesterol esters into the liver, SR-BI, has a relatively high expression in parenchymal cells compared with endothelial and Kupffer cells. These combined observations stressed an important role for parenchymal cells in the last step of the reverse cholesterol transport process.
In the current study, using real time quantitative PCR, we investigated the mRNA expression of genes involved in hepatic cholesterol transport and metabolism in liver parenchymal, endothelial, and Kupffer cells. Real time PCR is a highly sensitive method to quantify mRNA expression levels in vitro and in vivo. mRNA levels have been shown to correlate strongly with protein expression levels, indicating that a substantial portion of changes in protein levels is a consequence of altered mRNA levels rather than post-transcriptional modifications (21).
Importantly, Wellington et al.
(22) showed a high concordance
of ABCA1 mRNA and protein levels in the liver. Additionally, we observed in
the present study the highest SR-BI mRNA expression in the parenchymal cells
compared with endothelial and Kupffer cells, which is in accordance with the
high Western blot protein expression data for SR-BI as reported by Fluiter
et al. (20). We thus
suggest that our quantitative mRNA data for the various cell types are
indicative for the activity of the particular genes of interest and their
metabolic function. For SR-BI, the mRNA and protein expression data in
parenchymal cells are consistent with our data that the parenchymal
cholesterol ester uptake is reduced dramatically in SR-BI deficient mice as
compared with non-transgenic
littermates.2 CYP7A1
and CYP27 are the key enzymes in classical and alternative bile acid synthesis
pathways (23). The relatively
high expression of these two bile acid synthesis enzymes observed in
parenchymal cells is in agreement with the data provided by Pieters et
al. (19), as it was shown
that uptake of HDL cholesterol esters by the parenchymal cells is coupled
efficiently to bile acid synthesis. Interestingly, an equally high expression
of CYP27 compared with parenchymal cells was observed in liver endothelial
cells. In agreement, Reiss et al.
(24) have detected the same
high levels of CYP27 activity in cultured vascular endothelial cells. The
observed difference between the CYP7A1 and CYP27 expression patterns suggests
differential functions for these bile acid synthesizing enzymes.
Interestingly, Babiker et al.
(25) suggested that
CYP27-mediated elimination of cholesterol from macrophages and endothelial
cells may be an alternative or complement to HDL-mediated reverse cholesterol
transport under low HDL conditions. They observed a high secretion of
3-hydroxycholestenoic acid, an intermediate of the CYP27-mediated
alternative bile acid formation pathway, from endothelial cells and
macrophages to albumin containing medium.
Efflux of hepatic cholesterol to the serum compartment by ABCA1 for the
production of native HDL is a second important route in maintaining
cholesterol homeostasis (6). An
equally high relative expression of ABCA1 was observed in parenchymal and
Kupffer cells, whereas a 4-fold lower expression of ABCA1 was seen in
liver endothelial cells. In macrophages, ABCA1 is a critical regulator of the
specific ATP-dependent cholesterol efflux to apoA-I, leading to an inhibition
of foam cell formation (26).
Kupffer cells are liver macrophages, which play an important role in the
uptake of (modified) lipoproteins
(19,
27). The high uptake and an
accordingly high efflux of cholesterol from Kupffer cells might be the
metabolic mechanism for the relatively high ABCA1 expression level observed in
these cells. Haghpassand et al.
(28) and Van Eck et
al. (29) have shown that
monocyte/macrophage ABCA1 only minimally contributes to the overall plasma HDL
levels. The observed high expression levels of ABCA1 in parenchymal cells,
combined with the observation that ABCA1 functions on the basolateral surface
of hepatocytes (30), suggest
that the liver does contribute to HDL production by the efflux of
cholesterol from parenchymal cells via ABCA1.
A third catabolic route for hepatic cholesterol is the direct excretion
into the bile, which accounts for 40% of the total liver catabolism.
Recently, two members of the ABC transporters, ABCG5 and ABCG8, have been
shown to participate coordinately in the hepatic sterol secretion into bile
(31). Mutations in either
ABCG5 or ABCG8 are sufficient to cause sitosterolemia, a disorder that is
characterized by elevated plasma levels of sterols
(32). Because parenchymal
cells are responsible for bile acid formation, a relatively high expression of
the biliary transporters such as ABCG5/G8 in these cells compared with
endothelial and Kupffer cells is consistent with their suggested function. The
expression pattern of ABCG5 resembled ABCG8, which is in agreement with the
statements that these transporters operate as heterodimers to regulate biliary
cholesterol efflux (9,
33).
Interestingly, a novel member of the ABC transporter family, ABCG1, has
also been proposed to have a function in the intracellular trafficking and
biliary efflux of cholesterol in the liver
(11). Contrary to the
expectations, ABCG1 expression was observed mainly in non-parenchymal cells of
the rat liver. A 76-fold and 27-fold higher ABCG1 expression was observed in
Kupffer and endothelial cells than in parenchymal cell under standard feeding
conditions. Although Kupffer and endothelial cells only contribute 2.5 and
3.3% to the total liver protein, they do contain 51 and 24% of total liver
ABCG1 expression, respectively. Such a high specific ABCG1 expression in
Kupffer cells was not expected, although ABCG1 has also been proposed to play
a role in the cholesterol efflux from peripheral macrophages
(7). Importantly, after putting
rats on a high cholesterol diet for 2 weeks, ABCG1 expression increased 4-fold
in parenchymal cells, whereas no significant change in ABCG1 expression in
endothelial and Kupffer cells was observed. The absence of a similar induction
of ABCG1 in endothelial and Kupffer cells in response to diet feeding may well
be caused by an already maximal activity of ABCG1 in these cells even on a
chow diet. Also, the differences in expression and intracellular localization
of direct activators (e.g. LXR) and repressors (e.g.
ZNF202) of ABCG1 might contribute to the difference in its transcriptional
regulation between different cell types as earlier mentioned by Schmitz and
Langmann (11). Although
endothelial and Kupffer cell ABCG1 expressions were still 10- and 12-fold
higher than that in parenchymal cells, the relative contribution of ABCG1 in
the parenchymal cells to total liver increased from 25 to 60%. This suggests
that under high cholesterol conditions ABCG1 might indeed contribute to the
transport of cholesterol in the parenchymal cells.
Recent pharmacological interest is focused upon the regulation of SR-BI and the ABC transporters by newly discovered nuclear hormone receptors, the LXRs and PPARs, respectively (34, 35). Therefore, we also studied their cellular localization in the different cell types of the liver.
Two different types of the LXR have been discovered so far, LXR and
LXR
. A relatively high expression of LXR
was observed in
parenchymal cells. In the liver, LXR
plays an essential role in the
regulation of CYP7A1 and thus the formation of bile acids
(36). CYP7A1 was found almost
exclusively in the parenchymal cells, which coincides with the high expression
of LXR
in these cells. Contrarily, equally high LXR
expression
levels were observed in Kupffer cells, where CYP7A1 expression was almost
absent. In macrophages, however, LXR
plays a crucial role in the
regulation of lipid efflux via ABCA1
(37). Kupffer cells also
contain high expression levels of ABCA1, which is consistent with a role for
LXR
in the regulation of ABCA1 in these cells. Among the different cell
types of the liver, LXR
was ubiquitously expressed, with a somewhat
higher expression in endothelial cells versus parenchymal and Kupffer
cells. The expression distribution of LXR
thus does not resemble that
found for LXR
, which suggests that in the liver, LXR
may have a
function different from that of LXR
.
In the liver, PPAR is suggested to play a role in the formation of
bile acids because it is able to bind a PPAR response element in the sterol
12
-hydroxylase promoter, leading to increased levels of cholic acid
(38). This might explain the
extremely high expression of PPAR
found in parenchymal cells compared
with endothelial and Kupffer cells. The high expression of PPAR
observed in parenchymal cells suggests that PPAR
, like PPAR
,
also has a major function in these cells. Contrarily, PPAR
is expressed
ubiquitously among the different cell types of the liver, suggesting a more
general function for PPAR
in all cell types of the liver.
In conclusion, we have provided data that several ABC transporters and nuclear hormone receptors involved in liver cholesterol homeostasis are expressed differentially in the specific cell types of the liver. To study their intracellular transport function inside the liver it appears to be essential to take into account their cellular localization, as especially evident for ABCG1. This appears specifically true for studies on the regulation of the transporters by nuclear receptors because metabolic changes are coupled directly to the specific (intra)cellular expression level of the cholesterol transporters.
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FOOTNOTES |
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Supported by Grant 2001 D041 from the Netherlands Heart Foundation.
To whom correspondence should be addressed. Tel.: 31-71-527-6238; Fax:
31-71-527-6032; E-mail:
Hoekstra{at}LACDR.Leidenuniv.nl.
1 The abbreviations used are: HDL, high density lipoprotein; ABC, ATP-binding
cassette; Ct, threshold cycle number; CYP7A1, cholesterol
7-hydroxylase; CYP27, cholesterol 27-hydroxylase; HPRT,
hypoxanthine-guanine phosphoribosyltransferase; LXR, liver X receptor; PPAR,
peroxisome proliferator-activated receptor; SR-BI, scavenger receptor class
BI.
2 J. K. Kruijt and Th. J. C. van Berkel, unpublished data.
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REFERENCES |
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