* Laboratory of Animal Gene Function, Department of Physiology and Gene Regulation, Institute of Insect and Animal Sciences, National Institute of Agrobiological Sciences, Kannondai 212, Tsukuba 305-8602, Japan; Department of Molecular Biology and Immunology, Institute of Insect and Animal Sciences, National Institute of Agrobiological Sciences, Kannondai 212, Tsukuba 305-8602, Japan;
Department of Molecular Toxicology and COE Program for the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 521 Yada, Shizuoka 422-8526, Japan
1 To whom correspondence should be addressed at Laboratory of Animal Gene Function, Department of Physiology and Gene Regulation, Institute of Insect and Animal Sciences, National Institute of Agrobiological Sciences, Kannondai 212, Tsukuba 305-8602, Japan. Fax: +81-298388662. E-mail: misaki{at}affrc.go.jp.
Received May 15, 2005; accepted July 22, 2005
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ABSTRACT |
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Key Words: lead nitrate; Cyp7a1; TNF-; IL-1ß; liver; mouse.
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INTRODUCTION |
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More recently, Kojima et al. (2002 and 2004
) have suggested that LN-induced development of hypercholesterolemia in rats occurs not only through an increase in expression level of hepatic cholesterogenic enzymes including 3-hydroxy-3-methylglutaryl-CoA reductase, a rate-limiting enzyme in a cholesterol biosynthesis pathway (Goldstein and Brown, 1990
), but also through a decrease in expression level of hepatic cholesterol 7
-hydroxylase (CYP7A1 in rats), a rate-limiting enzyme in a bile acid biosynthesis pathway (Russell and Setchell, 1992
), and further demonstrated that LN shows the ability to induce not only hepatic TNF-
but also hepatic interleukin-1ß (IL-1ß) in rats.
The constitutive expression level of cholesterol 7-hydroxylase is controlled under feed-forward regulation by cholesterol and feed-back regulation by bile acids in rats and mice (Chiang, 1998
; Russell, 1999
). A heterodimer of oxysterol-activated liver X receptor
(LXR
) and retinoid X receptor (RXR) mediates transcriptional upregulation of the cholesterol 7
-hydroxylase gene (Lehmann et al., 1997
), while bile acid-activated farnesoid X receptor (FXR) suppresses the expression of the gene by inducing small heterodimer partner (SHP), which inactivates fetoprotein transcription factor (FTF) responsible for gene activation of cholesterol 7
-hydroxylase (Goodwin et al., 2000
; Lu et al., 2000
; Nitta et al., 1999
). In addition, hepatocyte nuclear factor 4
(HNF4
) is also a positive transcription factor for the hepatic cholesterol 7
-hydroxylase gene (Hayhurst et al., 2001
; Stroup and Chaing, 2000
). Gene expressions of these transcription factors, LXR
(Beigneux et al., 2000
), RXR
(Beigneux et al., 2000
), FXR (Kim et al., 2003
), FTF (Kim et al., 2003
), SHP (Kim et al., 2003
), and HNF4
(Fabiani et al., 2001
), as well as that of cholesterol 7
-hydroxylase (Feingold et al., 1996
), have been reported to be affected by cytokines including TNF-
. These previous findings suggest that LN-induced downregulation of the hepatic CYP7A1 gene occurs through induction of TNF-
. However, no direct evidence is obtained; TNF-
protein is not detected in LN-treated rats, although a significant increase in the level of TNF-
mRNA by LN has been demonstrated (Kojima et al., 2004
; Ledda-Columbano et al., 1994
).
In the present study, to clarify whether LN-induced downregulation of the cholesterol 7-hydroxylase gene occurs through induction of TNF-
, effects of LN on gene expressions of cholesterol 7
-hydroxylase (Cyp7a1 in mice) and its transcription factors were comparatively examined in TNF-
-knockout (KO) and TNF-
-wild-type (WT) mice. The results are presented and discussed here.
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MATERIALS AND METHODS |
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Real-time reverse transcription (RT)-polymerase chain reaction (PCR).
Total RNA preparations were obtained from a part of the liver of individual rats using Trizol reagent (Life Technologies Inc., Rockville, MD) and were used to determine the expression levels of each indicated gene. Briefly, a portion (4 µg) of the total RNA was converted to cDNA in a 20 µl RT-reaction mixture using the Super Script First-Strand Synthesis System for RT-PCR (Life Technologies Inc.) with oligo d(T)1218 as described in the manufacturer's instructions. Real-time RT-PCR was performed with an ABI PRISM 7700 Sequence Detection System with SYBR green master mix (PE Applied Systems, Tokyo, Japan) in 25 µl total reaction mixtures containing 0.5 µl of the RT-reaction mixture and 100 nM of each primer (forward and reverse) for the all genes examined. Primer sets used in this study are shown in Table 1. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was used as an internal standard. The amplification protocol consisted of AmpliTaq Gold pre-activation for 10 min at 95°C, 50 cycles of denaturation for 15 s at 95°C, annealing for 15 s at 55°C, and extension for 1 min at 72°C.
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Levels of TNF- and IL-1ß proteins in liver.
Livers from individual mice in each experimental group were homogenized with 2 volumes (w/v) of 1.15% KCl. Each liver homogenate was centrifuged at 9,000 x g for 20 min at 4°C, and the supernatant was further centrifuged at 105,000 x g for 1 h at 4°C. The resulting supernatant (S-105) was used to determine the amounts of TNF- and IL-1ß. Briefly, a portion (50 µl/well) of each S-105 was transferred to anti-mouse TNF-
antibody-coated and anti-mouse IL-1ß antibody-coated 96-well plates contained in a Quantikine mouse TNF-
kit and a Quantikine mouse IL-1ß kit (R&D Systems Inc., Minneapolis, MN), respectively. Amounts of TNF-
and IL-1ß proteins were then determined according to the manufacturer's instructions. Protein levels of each S-105 were measured by the method of Lowry et al. (1951)
, and amounts of TNF-
and IL-1ß were represented as picograms per milligram of protein.
Statistical analyses.
Statistical significance was determined using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test.
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RESULTS |
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DISCUSSION |
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Interestingly, marked downregulation (less than 5% of the control) of the Cyp7a1 gene by LN treatment occurred earlier in KO mice (3 h later) than in WT mice (6 h later). Although an exact mechanism for the difference in the time-dependency remains unclear, as a possible explanation, difference between KO mice and WT mice in the production pattern of IL-1ß, which shows ability to inhibit gene expression of CYP7A1 in hamsters (Feingold et al., 1996), might be considered, because the level of IL-1ß protein produced at 3 h after LN treatment was about twofold higher in KO mice than in WT mice. Recently, Isoda et al. (2005)
reported that level of the Cyp7a1 gene expression was lower in null mice of the IL-1 receptor antagonist (IL-1Ra) than in the wild-type mice, whereas the expression level of the IL-1ß gene was higher in null mice than in the wild-type mice. The previous report and the present findings strongly suggest that IL-1ß plays an important role in LN-induced downregulation of the Cyp7a1 gene.
To clarify the mechanism underlying downregulation of the Cyp7a1 gene, we further examined the changes in gene expression levels of positive transcription factors responsible for Cyp7a1 gene expression, including LXR, RXR
, HNF4
, and FTF (Chiang, 1998
; Hayhurst et al., 2001
; Lehmann et al., 1997
; Nitta et al., 1999
; Russell, 1999
; Stroup and Chiang, 2000
), after LN treatment in WT and KO mice. No significant change in gene expression of LXR
and its heterodimer partner RXR
after LN treatment was observed in either WT or KO mice, although we have previously found that LN shows a definite capacity for suppressing the hepatic LXR
gene in rats (Kojima et al., 2004
). Accordingly, there might be species differences between mice and rats in the LN-induced downregulation of the LXR
gene. The gene expression level of hepatic HNF4
significantly decreased at 624 h in WT mice, but not in KO mice, suggesting that LN-induced downregulation of the hepatic HNF4
gene occurs through a TNF-
dependent pathway. In addition, gene expression of hepatic FTF was significantly increased 612 h after LN treatment in both WT and KO mice. The present findings indicate that the LN-induced altered gene expression of positive transcription factors, including LXR
, RXR
, HNF4
, and FTF, responsible for the Cyp7a1 gene would not contribute to an LN-induced downregulation of the hepatic Cyp7a1 gene in mice.
Transcription factor SHP is thought to be a negative regulator for the Cyp7a1 gene (Goodwin et al., 2000; Lu et al., 2000
). Therefore, effects of LN treatment on the expression level of the SHP gene in both WT mice and KO mice were examined, and the results showed that the expression level of the SHP gene was reduced in WT mice but not in KO mice. These findings indicate that SHP would not act as a main factor in the LN-induced downregulation of the Cyp7a1 gene in mice. In addition, constitutive expression levels of the SHP gene in KO mice were half of those in WT mice, suggesting that constitutive expression of the SHP gene might be regulated, at least in part, through a TNF-
associated pathway.
In conclusion, we have demonstrated the presence of a TNF-independent pathway for LN-induced downregulation of hepatic Cyp7a1 gene with KO mice, and we further suggest that IL-1ß rather than TNF-
plays an important role in the LN-induced downregulation of the Cyp7a1 gene in mice.
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ACKNOWLEDGMENTS |
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