Tumor Necrosis Factor--Independent Downregulation of Hepatic Cholesterol 7-Hydroxylase Gene in Mice Treated with Lead Nitrate
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《毒物学科学杂志》
Laboratory of Animal Gene Function, Department of Physiology and Gene Regulation, Institute of Insect and Animal Sciences, National Institute of Agrobiological Sciences, Kannondai 2–1–2, Tsukuba 305-8602, Japan
Department of Molecular Biology and Immunology, Institute of Insect and Animal Sciences, National Institute of Agrobiological Sciences, Kannondai 2–1–2, Tsukuba 305-8602, Japan
Department of Molecular Toxicology and COE Program for the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52–1 Yada, Shizuoka 422-8526, Japan
ABSTRACT
We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor- (TNF-), downregulated gene expression of cholesterol 7-hydroxylase. Herein, to clarify the role of TNF- in LN-induced downregulation of cholesterol 7-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7-hydroxylase (Cyp7a1) in TNF--knockout (KO) and TNF--wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6–12 h after treatment with LN (100 μmol/kg body weight, iv). Levels of hepatic TNF- protein in either WT or KO mice were below the detection limit, although expression levels of the TNF- gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1 protein, which is known to be a suppressor of the cholesterol 7-hydroxylase gene in hamsters, were significantly increased 3–6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor , retinoid X receptor , fetoprotein transcription factor, and hepatocyte nuclear factor 4) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-–dependent pathway and might occur mainly through an IL-1–dependent pathway.
Key Words: lead nitrate; Cyp7a1; TNF-; IL-1; liver; mouse.
INTRODUCTION
Lead salt is a ubiquitous environmental pollutant and has been known to cause impairment of male reproduction (Gennart et al., 1992; Pinon-Lataillade et al., 1995; Ronis et al., 1996; Winder 1993), neurological problems (Finkelstein et al., 1998; Goyer 1993; Pirkle et al., 1998; Silbergeld et al., 1992), and decrease in the level of hepatic cytochrome P-450 (CYP) enzymes (Degawa, et al., 1993; Hammond and Dietrich, 1990). Furthermore, lead nitrate (LN) shows ability to induce developments of hypercholesterolemia (Dessi et al., 1984) and liver hyperplasia (Columbano et al., 1983) after undergoing induction of tumor necrosis factor- (TNF-) in the rat liver (Ledda-Columbano et al., 1994; Shinozuka et al., 1994).
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-1 (IL-1) 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.
MATERIALS AND METHODS
Treatment of mice with lead nitrate (LN).
TNF--knockout (KO) mice were constructed from WT C57BL/6J mice as described previously (Taniguchi et al., 1997) and were bred in our laboratory. Wild-type C57BL/6J mice were purchased from CLEA Inc. (Tokyo, Japan). All mice used in the present experiments were kept in plastic cages in an air-conditioned room with a 12-h light/dark cycle, and given a basal diet, MF (Oriental Yeast, Co., Tokyo, Japan) and water ad libitum. All mice were used at 8–10 weeks of age. Lead nitrate was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Female KO and WT mice were administered a single dose of LN (100 μmol/kg body weight, iv) dissolved in distilled water at the concentration of 20 mM as described previously (Degawa et al., 1993). Control mice were treated with vehicle alone (5 ml/kg body weight). All mice used were sacrificed by decapitation at 10:00–11:00 a. m. The mice were killed at 3, 6, 12, 24, and 48 h after LN treatment, and their livers were removed quickly and frozen in liquid nitrogen for storage at –80°C until use.
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)12–18 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.
The level of each cDNA was assessed by the relative standard curve method as described in PE Applied Biosystems User Bulletin #2 (1997). Standard curves for determining the expression levels of Cyp7a1, SHP, and HNF4 were generated using an RT-reaction mixture with total RNA from control (LN-untreated) WT mouse livers, and those for other genes (TNF-, LXR, RXR, FTF, and G3PDH) were generated with total RNA from the WT mouse livers at 6 h after LN treatment. In addition, the source of RNA used for making each standard curve was the experimental group of mice having the highest level of the corresponding mRNAs.
Levels of TNF- and IL-1 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-1. Briefly, a portion (50 μl/well) of each S-105 was transferred to anti-mouse TNF- antibody-coated and anti-mouse IL-1 antibody-coated 96-well plates contained in a Quantikine mouse TNF- kit and a Quantikine mouse IL-1 kit (R&D Systems Inc., Minneapolis, MN), respectively. Amounts of TNF- and IL-1 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-1 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.
RESULTS
Change in Expression Levels of Hepatic TNF- and IL-1 after LN Treatment
We first examined the levels of hepatic mRNA and protein of TNF- in WT and KO mice after LN treatment. In WT mice, expression levels of hepatic TNF- gene were enhanced to about tenfold over the control level at 3 h and 20-fold over the control level at 6 h after LN treatment, and returned to control levels at 24 h (Fig. 1). In contrast, in KO mice, no expression of the gene was confirmed at any time points examined (data not shown). In addition, the level of TNF- protein in both the KO and the WT mice was below the detection limit (0.25 pg/50 μl/well) at any time points examined (data not shown).
Levels of hepatic IL-1 protein were increased 3–6 h after LN treatment in both WT and KO mice (Fig. 2): in WT mice, levels were threefold over the control level (21.9 pg/mg protein) at 3 and 6 h; in KO mice, levels were sixfold and threefold over the control level (18.9 pg/mg protein) at 3 h and 6 h, respectively. Thereafter, levels of IL-1 protein in the WT and KO mice recovered to the corresponding control levels at 12 h.
Change in Expression Levels of Hepatic Cyp7a1 Gene after LN Treatment
No differences between KO and WT mice in constitutive expression levels of hepatic Cyp7a1 gene were observed (data not shown). After LN treatment, the expression level of hepatic Cyp7a1 gene in WT mice decreased to 50% of the control levels at 3 h and to less than 5% of the control levels at 6–12 h. The expression level 24–48 h later recovered to up to 50% of the control (Fig. 3). Similarly, LN-induced downregulation of the gene was observed in KO mice. Interestingly, the marked decrease (5% of the control level) in expression level of the Cyp7a1 gene occurred in KO mice (3 h later) earlier than in WT mice (6 h later).
Altered Gene Expression of LXR, RXR, HNF4, FTF, and SHP
LXR, RXR, HNF4, and FTF are known to act as positive regulators for the Cyp7a1 gene (Chiang, 1998; Hayhurst et al., 2001; Lehmann et al., 1997; Nitta et al., 1999; Russell, 1999; Stroup and Chiang, 2000). Therefore, we examined the changes in hepatic gene expression of these regulators after LN treatment. No significant changes in gene expression of LXR and RXR after LN treatment were observed in either KO or WT mice (Fig. 4). Expression levels of the HNF4 gene were significantly decreased at 6–24 h after LN treatment in WT mice, but not in KO mice. Expression levels of the FTF gene in both KO and WT mice increased to 1.5–2-fold over the corresponding control levels at 6–12 h after LN treatment. In addition, no significant differences between KO and WT mice in the constitutive expression levels of the LXR, RXR, HNF4, and FTF genes were observed (data not shown).
After LN treatment, altered gene expression of SHP, a negative regulator of the Cyp7a1 gene (Goodwin et al., 2000; Lu et al., 2000), was also examined. Constitutive expression levels of hepatic SHP gene in KO mice were half those in WT mice (Fig. 5). Expression levels of the SHP gene in TNF- WT mice decreased to 50% of the control levels 3 h after LN treatment, and the decreased levels were maintained up to 48 h (Fig. 5). In KO mice, no such decrease was observed.
DISCUSSION
We have recently demonstrated that gene expression of hepatic cholesterol 7-hydroxylase (CYP7A1) in rats was downregulated by LN treatment (Kojima et al., 2004) and that LN treatment resulted in increases in levels of the mRNA and/or protein of cytokines such as TNF- and IL-1 in the liver (Kojima et al., 2004; Ledda-Columbano et al., 1994; Shinozuka et al., 1994). In the present study, to clarify a role of TNF- in LN-induced downregulation of the cholesterol 7-hydroxylase gene (Cyp7a1 in mice), we comparatively examined the effects of LN on expression of the hepatic Cyp7a1 gene in TNF--knockout and wild-type mice. A significant decrease in expression of the hepatic Cyp7a1 gene was observed 3–48 h after LN treatment in both WT mice and KO mice, demonstrating that LN-induced downregulation of the hepatic Cyp7a1 gene occurs, even through a TNF-–independent pathway. In addition, the level of TNF- protein was under the detection limit even in WT mice. No detection of its protein has been reported in LN-treated rat liver (Kojima et al., 2004; Ledda-Columbano et al., 1994).
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-1, which shows ability to inhibit gene expression of CYP7A1 in hamsters (Feingold et al., 1996), might be considered, because the level of IL-1 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-1 gene was higher in null mice than in the wild-type mice. The previous report and the present findings strongly suggest that IL-1 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 6–24 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 6–12 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-1 rather than TNF- plays an important role in the LN-induced downregulation of the Cyp7a1 gene in mice.
ACKNOWLEDGMENTS
This work was supported in part by a Science Research Grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (M.K.) and by a Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (M.D.).
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Department of Molecular Biology and Immunology, Institute of Insect and Animal Sciences, National Institute of Agrobiological Sciences, Kannondai 2–1–2, Tsukuba 305-8602, Japan
Department of Molecular Toxicology and COE Program for the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52–1 Yada, Shizuoka 422-8526, Japan
ABSTRACT
We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor- (TNF-), downregulated gene expression of cholesterol 7-hydroxylase. Herein, to clarify the role of TNF- in LN-induced downregulation of cholesterol 7-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7-hydroxylase (Cyp7a1) in TNF--knockout (KO) and TNF--wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6–12 h after treatment with LN (100 μmol/kg body weight, iv). Levels of hepatic TNF- protein in either WT or KO mice were below the detection limit, although expression levels of the TNF- gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1 protein, which is known to be a suppressor of the cholesterol 7-hydroxylase gene in hamsters, were significantly increased 3–6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor , retinoid X receptor , fetoprotein transcription factor, and hepatocyte nuclear factor 4) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-–dependent pathway and might occur mainly through an IL-1–dependent pathway.
Key Words: lead nitrate; Cyp7a1; TNF-; IL-1; liver; mouse.
INTRODUCTION
Lead salt is a ubiquitous environmental pollutant and has been known to cause impairment of male reproduction (Gennart et al., 1992; Pinon-Lataillade et al., 1995; Ronis et al., 1996; Winder 1993), neurological problems (Finkelstein et al., 1998; Goyer 1993; Pirkle et al., 1998; Silbergeld et al., 1992), and decrease in the level of hepatic cytochrome P-450 (CYP) enzymes (Degawa, et al., 1993; Hammond and Dietrich, 1990). Furthermore, lead nitrate (LN) shows ability to induce developments of hypercholesterolemia (Dessi et al., 1984) and liver hyperplasia (Columbano et al., 1983) after undergoing induction of tumor necrosis factor- (TNF-) in the rat liver (Ledda-Columbano et al., 1994; Shinozuka et al., 1994).
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-1 (IL-1) 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.
MATERIALS AND METHODS
Treatment of mice with lead nitrate (LN).
TNF--knockout (KO) mice were constructed from WT C57BL/6J mice as described previously (Taniguchi et al., 1997) and were bred in our laboratory. Wild-type C57BL/6J mice were purchased from CLEA Inc. (Tokyo, Japan). All mice used in the present experiments were kept in plastic cages in an air-conditioned room with a 12-h light/dark cycle, and given a basal diet, MF (Oriental Yeast, Co., Tokyo, Japan) and water ad libitum. All mice were used at 8–10 weeks of age. Lead nitrate was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Female KO and WT mice were administered a single dose of LN (100 μmol/kg body weight, iv) dissolved in distilled water at the concentration of 20 mM as described previously (Degawa et al., 1993). Control mice were treated with vehicle alone (5 ml/kg body weight). All mice used were sacrificed by decapitation at 10:00–11:00 a. m. The mice were killed at 3, 6, 12, 24, and 48 h after LN treatment, and their livers were removed quickly and frozen in liquid nitrogen for storage at –80°C until use.
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)12–18 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.
The level of each cDNA was assessed by the relative standard curve method as described in PE Applied Biosystems User Bulletin #2 (1997). Standard curves for determining the expression levels of Cyp7a1, SHP, and HNF4 were generated using an RT-reaction mixture with total RNA from control (LN-untreated) WT mouse livers, and those for other genes (TNF-, LXR, RXR, FTF, and G3PDH) were generated with total RNA from the WT mouse livers at 6 h after LN treatment. In addition, the source of RNA used for making each standard curve was the experimental group of mice having the highest level of the corresponding mRNAs.
Levels of TNF- and IL-1 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-1. Briefly, a portion (50 μl/well) of each S-105 was transferred to anti-mouse TNF- antibody-coated and anti-mouse IL-1 antibody-coated 96-well plates contained in a Quantikine mouse TNF- kit and a Quantikine mouse IL-1 kit (R&D Systems Inc., Minneapolis, MN), respectively. Amounts of TNF- and IL-1 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-1 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.
RESULTS
Change in Expression Levels of Hepatic TNF- and IL-1 after LN Treatment
We first examined the levels of hepatic mRNA and protein of TNF- in WT and KO mice after LN treatment. In WT mice, expression levels of hepatic TNF- gene were enhanced to about tenfold over the control level at 3 h and 20-fold over the control level at 6 h after LN treatment, and returned to control levels at 24 h (Fig. 1). In contrast, in KO mice, no expression of the gene was confirmed at any time points examined (data not shown). In addition, the level of TNF- protein in both the KO and the WT mice was below the detection limit (0.25 pg/50 μl/well) at any time points examined (data not shown).
Levels of hepatic IL-1 protein were increased 3–6 h after LN treatment in both WT and KO mice (Fig. 2): in WT mice, levels were threefold over the control level (21.9 pg/mg protein) at 3 and 6 h; in KO mice, levels were sixfold and threefold over the control level (18.9 pg/mg protein) at 3 h and 6 h, respectively. Thereafter, levels of IL-1 protein in the WT and KO mice recovered to the corresponding control levels at 12 h.
Change in Expression Levels of Hepatic Cyp7a1 Gene after LN Treatment
No differences between KO and WT mice in constitutive expression levels of hepatic Cyp7a1 gene were observed (data not shown). After LN treatment, the expression level of hepatic Cyp7a1 gene in WT mice decreased to 50% of the control levels at 3 h and to less than 5% of the control levels at 6–12 h. The expression level 24–48 h later recovered to up to 50% of the control (Fig. 3). Similarly, LN-induced downregulation of the gene was observed in KO mice. Interestingly, the marked decrease (5% of the control level) in expression level of the Cyp7a1 gene occurred in KO mice (3 h later) earlier than in WT mice (6 h later).
Altered Gene Expression of LXR, RXR, HNF4, FTF, and SHP
LXR, RXR, HNF4, and FTF are known to act as positive regulators for the Cyp7a1 gene (Chiang, 1998; Hayhurst et al., 2001; Lehmann et al., 1997; Nitta et al., 1999; Russell, 1999; Stroup and Chiang, 2000). Therefore, we examined the changes in hepatic gene expression of these regulators after LN treatment. No significant changes in gene expression of LXR and RXR after LN treatment were observed in either KO or WT mice (Fig. 4). Expression levels of the HNF4 gene were significantly decreased at 6–24 h after LN treatment in WT mice, but not in KO mice. Expression levels of the FTF gene in both KO and WT mice increased to 1.5–2-fold over the corresponding control levels at 6–12 h after LN treatment. In addition, no significant differences between KO and WT mice in the constitutive expression levels of the LXR, RXR, HNF4, and FTF genes were observed (data not shown).
After LN treatment, altered gene expression of SHP, a negative regulator of the Cyp7a1 gene (Goodwin et al., 2000; Lu et al., 2000), was also examined. Constitutive expression levels of hepatic SHP gene in KO mice were half those in WT mice (Fig. 5). Expression levels of the SHP gene in TNF- WT mice decreased to 50% of the control levels 3 h after LN treatment, and the decreased levels were maintained up to 48 h (Fig. 5). In KO mice, no such decrease was observed.
DISCUSSION
We have recently demonstrated that gene expression of hepatic cholesterol 7-hydroxylase (CYP7A1) in rats was downregulated by LN treatment (Kojima et al., 2004) and that LN treatment resulted in increases in levels of the mRNA and/or protein of cytokines such as TNF- and IL-1 in the liver (Kojima et al., 2004; Ledda-Columbano et al., 1994; Shinozuka et al., 1994). In the present study, to clarify a role of TNF- in LN-induced downregulation of the cholesterol 7-hydroxylase gene (Cyp7a1 in mice), we comparatively examined the effects of LN on expression of the hepatic Cyp7a1 gene in TNF--knockout and wild-type mice. A significant decrease in expression of the hepatic Cyp7a1 gene was observed 3–48 h after LN treatment in both WT mice and KO mice, demonstrating that LN-induced downregulation of the hepatic Cyp7a1 gene occurs, even through a TNF-–independent pathway. In addition, the level of TNF- protein was under the detection limit even in WT mice. No detection of its protein has been reported in LN-treated rat liver (Kojima et al., 2004; Ledda-Columbano et al., 1994).
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-1, which shows ability to inhibit gene expression of CYP7A1 in hamsters (Feingold et al., 1996), might be considered, because the level of IL-1 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-1 gene was higher in null mice than in the wild-type mice. The previous report and the present findings strongly suggest that IL-1 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 6–24 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 6–12 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-1 rather than TNF- plays an important role in the LN-induced downregulation of the Cyp7a1 gene in mice.
ACKNOWLEDGMENTS
This work was supported in part by a Science Research Grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (M.K.) and by a Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (M.D.).
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