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J. Biol. Chem., Vol. 278, Issue 34, 31504-31511, August 22, 2003
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From the
EA 3508, Université Paris 7, Denis Diderot, Case 7104, 2 Place
Jussieu, 75251 Paris Cedex, France,
INSERM U490,
Faculté de Médecine Necker, 75743 Paris Cedex 15, France,
¶Central Nervous System Department, Aventis,
94403 Vitry sur Seine, France, ||Laboratoire de
Biochimie Médecine B, Hopital Necker-Enfants Malades, France, and
**Department of Pathology, University of North
Carolina, Chapel Hill, North Carolina 27599
Received for publication, December 20, 2002 , and in revised form, June 2, 2003.
| ABSTRACT |
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-synthase (CBS) deficiency causes severe
hyperhomocysteinemia and other signs of homocystinuria syndrome, in particular
a premature atherosclerosis with multiple thrombosis. However, the molecular
mechanisms by which homocysteine could interfere with normal cell function are
poorly understood in a whole organ like the liver, which is central to the
catabolism of homocysteine. We used a combination of differential display and
cDNA arrays to analyze differential gene expression in association with
elevated hepatic homocysteine levels in CBS-deficient mice, a murine model of
hyperhomocysteinemia. Expression of several genes was found to be reproducibly
abnormal in the livers of heterozygous and homozygous CBS-deficient mice. We
report altered expression of genes encoding ribosomal protein S3a and
methylthioadenosine phosphorylase, suggesting such cellular growth and
proliferation perturbations may occur in homozygous CBS-deficient mice liver.
Many up- or down-regulated genes encoded cytochromes P450, evidence of
perturbations of the redox potential in heterozygous and homozygous
CBS-deficient mice liver. The expression of various genes involved in severe
oxidative processes was also abnormal in homozygous CBS-deficient mice liver.
Among them, the expression of heme oxygenase 1 gene was increased, concomitant
with overexpression of heme oxygenase 1 at the protein level. Commensurate
with the difference in hepatic mRNA paraoxonase 1 abundance, the mean hepatic
activity of paraoxonase 1, an enzyme that protects low density lipoprotein
from oxidation, was 3-fold lower in homozygous CBS-deficient mice.
Heterozygous CBS-deficient mice, when fed a hyperhomocysteinemic diet, have
also reduced PON1 activity, which demonstrates the effect of
hyperhomocysteinemia in the paraoxonase 1 activity. | INTRODUCTION |
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-synthase (CBS), and
then cystathionine is used to yield cysteine. Cysteine is a major precursor of
glutathione, the principal redox metabolite in the cell.
CBS deficiency, an inborn metabolic defect that displays an autosomal recessive inheritance, is the most common identifiable cause of hyperhomocysteinemia (HH) (2). HH is associated with an increased incidence of vascular thrombosis and development of premature atherosclerosis (3). Severe monogenic HH (also referred to as homocystinuria) is commonly caused by mutations in the CBS gene that prevent the use of Hcy in the biosynthesis of cysteine and lead to abnormally high plasma total Hcy (tHcy) concentrations in patients with severe HH (100250 µM). Normal plasma tHcy concentrations range from 5 to 15 µM. Mild HH, usually defined as plasma levels of tHcy between 15 and 30 µM, is associated with increased risk of atherosclerosis (4).
The relationship between high Hcy concentrations in the blood and thrombosis has been a subject of considerable research. Endothelial dysfunction is a key process in atherosclerosis and has been reported in patients with HH (5) and in experimental HH, which is induced by methionine loading in normal subjects (6). Administration of Hcy causes vascular injury and thrombosis in animals (7). It is thought to predispose to atherosclerosis by injuring the vascular endothelium; after exposure to Hcy, endothelial dysfunction occurs at several levels, suggesting that Hcy contributes to enhanced vascular inflammation and hemostatic changes linked to the development of atherosclerosis and a prothrombotic state.
An understanding of these processes at the molecular level is required to elucidate the mechanism of cell injury by Hcy. Expression of a range of genes has been shown to be modulated in cultured human umbilical vein endothelial cells, vascular smooth muscle cells, human megakaryocytic cell line (DAMI), hepatic stellate cells, human hepatoma cell line (HepG2), and primary neuronal cells exposed to exogenous excess Hcy (816). In vitro, high levels of Hcy affect the expression of the endoplasmic reticulum stress-response genes, the acute translational responses genes, mitochondrial genes, genes encoding antioxidant enzymes and transcription factors, and genes implicated in cell growth, cellular differentiation and proliferation, and regulation of extracellular matrix homeostasis. Unfortunately, most in vitro studies use physiologically irrelevant concentrations or forms or both of Hcy, concentrations much higher than those found in patients with HH. Therefore, studies need to be conducted using a pathophysiologically relevant model of in vivo HH.
A well characterized genetic model of HH in mice was obtained by targeted deletion of the CBS gene by homologous recombination (17) and has been used to show the influence of HH on vascular function (1822). Homozygous CBS-deficient mice (CBS / mice) develop very high plasma tHcy concentrations, and heterozygous CBS-deficient mice (CBS +/ mice) have mildly elevated plasma concentrations of tHcy (1723). Both CBS +/ and CBS / mice exhibit some pathological features of HH such as endothelial dysfunction (1822), which can be negated by overexpression of glutathione peroxidase (21), and hepatic steatosis (17, 24).
Although the mechanisms that lead to high plasma Hcy have been well established, the molecular basis of Hcy-mediated alteration of cellular function in whole organs is not completely understood. To elucidate the molecular mechanisms responsible for the interference in normal tissue function, we searched for genes whose expression is abnormal in CBS-deficient mice. Impaired liver function has been associated with elevated plasma levels of tHcy (2527), evidence of the central role of the liver in the metabolism of methionine and consequently in the catabolism of Hcy (28). A recent study shows that the liver is indeed a key organ in Hcy metabolism and contributes to much of the plasma Hcy (29). We therefore focused our research on the liver of CBS-deficient mice and monitored gene expression by a combination of differential display reverse transcriptase (RT)-PCR and cDNA expression array analysis. We found that 13 genes were up- or down-regulated in liver from 3- and 4-month-old CBS +/ and/or CBS / mice. Several of these genes affected in CBS / mouse liver suggest altered cellular growth and severe oxidative processes.
| EXPERIMENTAL PROCEDURES |
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Plasma and Hepatic tHcy AssaysAt the time of sacrifice,
blood samples were collected in tubes containing a
volume of 3.8% sodium
citrate and placed on ice immediately, and plasma was isolated by
centrifugation at 2500 x g for 15 min at 4 °C. Liver was
harvested, snap-frozen, and stored at 80 °C until use. tHcy was
assayed in plasma and crude liver extracts with a fluorescence polarization
immunoassay using an Abbott IMX analyzer (Abbott Laboratories, Mississauga,
Ontario, Canada) (30) and
normalized to total protein concentration for liver extracts.
Western Blot AnalysisTotal protein liver extracts (20
µg) were subjected to SDS electrophoresis on 12% gels under reducing
conditions by the method of Laemmli
(31). Proteins were
transferred electrophoretically to nitrocellulose membranes (Bio-Rad). The
membranes were blocked with Tris buffer (1.5 mM Tris base, pH 8, 5
mM NaCl, 0.1% Tween 20) containing 5% skimmed milk, and the
immobilized proteins were immunostained with a polyclonal antibody directed
against rat CBS (1:10000 dilution)
(32) or with anti-Cu,Zn
superoxide dismutase 1 (SOD1) (1:1000 dilution) (Calbiochem) or with anti-HO-1
(1:200 dilution) (Sigma-Aldrich), or with anti-
-tubulin (1:2000
dilution) (Sigma-Aldrich). After incubation with the primary and
peroxidase-conjugated secondary antibodies, blots were developed with the
enhanced chemiluminescence reagent (Amersham Biosciences).
Total RNA IsolationTotal RNA was prepared from two mixtures of three different livers from 3- or 4-month-old mice by the guanidinium thiocyanate procedure (33). The quantity and purity of the RNA was assessed by measuring absorbance at 260 and 280 nm.
Analysis of Differential Gene Expression Using Differential
Display Total RNA was analyzed by RT-PCR-based differential
display as described by Liang and Pardee
(34). DNase-treated RNA (400
ng) was reverse-transcribed using anchored primers D1
(5'-T11GC-3') or D2
(5'-T11GG-3') or D4
(5'-T11CG-3') and superscript II reverse transcriptase
(Invitrogen). One-fortieth of the reverse transcriptase mixture was used for
the subsequent amplification by PCR with 2.5 µM concentrations
of the same D1 and D2 and D4 as 3' primers and 0.5 µM
concentrations of an arbitrary 5' primer: U2
(5'-TTGATCCGAG-3'), 2.5 mM MgCl2, 10
µM dNTPs, 1 µCi of [
-33P]dATP (PerkinElmer
Life Sciences), and 1 unit of Taq polymerase (Invitrogen) in 20
mM Tris-HCl, pH 8.0, 50 mM KCl. The mixture was
subjected to the following cycles: 1x (5 min at 94 °C, 5 min at 40
°C, 5 min at 72 °C), 39x (15 s at 95 °C, 2 min at 40 °C,
1 min at 72 °C), 1x (5 min at 72 °C). The amplified cDNAs were
separated on a 6% DNA sequencing gel. Total RNA from two extractions of
mixtures of three different tissues was used, and duplicate RT-PCRs were
performed and repeated at least twice to ensure reproducibility.
Cloning and Sequencing of cDNA FragmentsDNA bands of interest were cut from the sequencing gels and eluted, then reamplified by PCR with the same primer pairs. The amplified fragments were inserted directly into the pCR2.1 TA vector by using the TA cloning kit (Invitrogen) and sequenced using the T7 sequencing kit (Amersham Biosciences). The sequences were compared with the GenBankTM data base using the BLAST program.
Analysis of Differential Gene Expression Using a Mouse cDNA
ArrayRadiolabeled cDNA probes were generated by reverse
transcription of total RNA (2.5 µg) with Moloney murine leukemia virus
reverse transcriptase and 35 µCi of [
-32P]dATP
(PerkinElmer Life Sciences). The radiolabeled cDNA probes were purified from
unincorporated nucleotides by column chromatography using the NucleoSpin
extraction kit (Clontech) and hybridized overnight at 68 °C to an atlas
mouse stress cDNA expression array consisting of 140 known mouse genes as
described by the manufacturer (Clontech catalog number 7749-10). The membranes
were washed four times for 30 min in 2x saline-sodium citrate (SSC;
1x SSC = 0.15 M NaCl and 0.015 M sodium citrate),
1% SDS followed by one time for 30 min in 0.1x SSC, 0.5% SDS at 68
°C and exposed to x-ray film (Kodak Biomax MS). The x-ray films were
scanned, and the images were analyzed using the AtlasImageTM Software
(Clontech).
Northern Blot AnalysisSamples of denatured RNA (15 µg)
from each group were electrophoresed for 3 h on a 2.2 M
formaldehyde, 1% agarose gel. After a capillary transfer onto a nylon membrane
(Appligene Oncor), the membrane was cross-linked using a UV transilluminator
(Stratalinker, Appligene Oncor). The isolated cDNA fragments of RT-PCR
analysis were used as probes for verification of differential expression by
Northern blotting. cDNA-specific probes of genes included on the cDNA array
were obtained by reverse transcription of DNA-free RNA (500 ng) from liver
tissues with specific primers complementary to the studied cDNA and
superscript II reverse transcriptase (Invitrogen) followed by PCR
amplification using nested primers before labeling. Specific probes were
generated by labeling the cDNA fragments with [
-32P]dCTP
(PerkinElmer Life Sciences) using the Multiprime DNA labeling system (Amersham
Biosciences). After an overnight incubation in the probe solution (ExpressHyb
solution, Clontech) at 68 °C, the membrane was washed 3 times for 30 min
in 0.1x SSC, 0.1% SDS at 68 °C. An autoradiogram was generated by
exposing the washed membrane to film (Fuji) for overnight at 80 °C.
We ensured that loading was equal and the RNA from the liver samples had not
been degraded by hybridization with a probe specific for mouse SOD1 gene.
Changes in steady-state mRNA levels were quantified by densitometric scanning
using Image Analysis Software and normalized to SOD1 to correct for
differences in gel loading.
Paraoxonase Enzyme AssaysPON activity toward phenyl acetate
(Sigma-Aldrich) was quantified spectrophotometrically using 50 mM
Tris-HCl, pH 8, containing 1 mM CaCl2 and 5
mM phenyl acetate. The reaction was monitored for 10 min at 35
°C by measuring the appearance of phenol at 270 nm with the use of
continuously and automated recording spectrophotometer (PowerWaveX microplate
spectrophotometer of Biotek instruments with KC4 software). All rates were
determined in duplicate and corrected for non-enzymatic hydrolysis
(35). The enzyme activity is
expressed as
A270 per min and per mg of cellular
proteins of liver.
| RESULTS |
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Hepatic CBS protein production and hepatic tHcy concentrations were measured. We carried out Western blotting experiments using a polyclonal antibody against rat CBS (32) to probe protein extracts from the livers of CBS +/+, CBS +/, and CBS / mice. Two bands, 68 and 50 kDa, were detected (Fig. 1). The band at 50 kDa was found to be nonspecific; its intensity did not differ significantly between the three extracts and, thus, could serve as an internal control. The intensity of the specific band of 68 kDa in crude liver extracts from CBS +/ mice was half that in CBS +/+ mice; the band was absent from extracts from CBS / mice (Fig. 1). The same results were obtained with protein samples isolated from the brains of CBS +/+, CBS +/, and CBS / mice (37).
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Commensurate with this difference in hepatic CBS protein abundance, the mean hepatic concentration of tHcy in CBS / mice was approximately 20-fold higher than that in CBS +/+ mice (tHcy concentrations were 6.44 ± 3.86 versus 0.34 ± 0.14 nmol/mg of cellular protein; p < 0.001 by Student's t test n = 5 for each). In contrast, hepatic tHcy concentrations in CBS +/ mice fed a standard rodent chow enriched in choline chloride that is sufficient in folate and vitamin B6 were not significantly different from those in CBS +/+ mice (tHcy levels were 0.43 ± 0.15 versus 0.34 ± 0.14 nmol/mg of cellular protein; p < 0.35 by Student's t test n = 5 for each).
Plasma tHcy concentration was also higher in CBS / mice than
in CBS +/+ mice (205 ± 86 versus 3.9 ± 0.9
µM; p < 0.0001 by Student's t test
n = 4 for each). CBS +/ mice had intermediate plasma tHcy
levels
2-fold that in CBS +/+ mice (tHcy levels were 9.1 ± 2.4
versus 3.9 ± 0.9 µM; p < 0.0001 by
Student's t test n = 8 for each). Plasma levels of tHcy in
mice are generally lower than those in humans; nevertheless, the plasma tHcy
concentrations resulting from partial CBS deficiency in mice have been shown
to be sufficient to cause endothelial dysfunction
(18,
21,
22). Therefore, CBS +/
mice and CBS / mice may be suitable for pathophysiological study
of mild and severe HH and can be used to identify the genes involved.
Identification by Differential Display of Transcripts Differentially
Expressed in Liver from CBS +/ and CBS
/ MiceWe sought to identify genes that
are differentially expressed in the liver of CBS-deficient mice. We compared
liver RNA fingerprint patterns from CBS +/+ mice with those from CBS +/
mice and CBS / mice of 3- or 4-month-old mice. After screening
using 20 primer combinations, a total of 15 cDNA fragments differentially
expressed in the livers of CBS /, CBS +/, and CBS +/+
were identified on sequencing gels. They were isolated, cloned, and sequenced.
Fig. 2A shows the
typical banding patterns for the PCR products obtained with three different
primer combinations. Band D contained two different cDNAs
(Table I). To confirm the
results of differential display RT-PCR of mRNA, Northern blot analysis was
performed using probes obtained from the isolated differential display
fragments (Fig. 2B).
The genes that were confirmed by Northern blot analysis as having differential
expression are listed in Table
I. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) is widely used
as an internal control in mRNA expression studies. Unexpectedly, G3PDH
expression was lower in livers from CBS / mice than in controls
(Fig. 2B). The gene
encoding SOD1, an antioxidant enzyme, was expressed at equal levels in all
three groups of mice (Fig.
2B). We have also shown no difference in hepatic SOD1
protein production (Fig. 5) and
in SOD1 activity (data not shown) between CBS +/+, CBS +/, and CBS
/ mice. The mouse SOD1 probe was therefore used as the loading
control. The relative expression levels of these genes were also quantified by
densitometric scanning of Northern blots
(Table I). Three of the cDNAs
corresponding to genes down-regulated in livers of CBS / mice
had sequences similar to those of known mouse genes
(Table I): mitochondrial
cytochrome c oxidase subunit I gene (GenBankTM accession number
NP 008110); the gene for cytochrome P450 (CYP) 3A25 (GenBankTM accession
number AF 204959), a testosterone 6
-hydroxylase; and the gene for
ribosomal protein S3a (RPS3a) (GenBankTM accession number NM_016959
[GenBank]
), a
ribosomal protein that is essential for cell growth and cellular proliferation
(38,
39). One of the cDNAs
revealing up-regulation in CBS / mice was similar to the
methylthioadenosine phosphorylase gene (GenBankTM accession number AB
056100), encoding an enzyme implicated in the salvage pathways of purines and
methionine (40), and another
had sequences EST (expressed sequence tag) identical to those in the cDNA
clone 620429009 (GenBankTM accession number AV 324910), confirming the
expression of this novel transcript of unknown function. These results
demonstrate notably altered mitochondrial gene expression in livers of 3- and
4-month-old CBS / mice. Mitochondrial alterations in the liver
of CBS / mice are known to generate reactive oxygen species.
Therefore, we searched for specific changes in the expression of
stress-response genes.
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Identification of Differentially Expressed Stress Gene ProductsAtlas mouse stress cDNA expression membranes (Clontech) were used to identify genes modulated in response to hepatic stress in CBS-deficient mice. The membrane used contains 140 cDNA species involved in stress. The complete list of genes is available at www.Clontech.com. Total RNA obtained from two mixtures of three different livers of 3- or 4-month-old CBS +/+ and CBS / mice were used to generate 32P-labeled cDNA probes. The probes were hybridized to the membranes. Identical arrays were differentially screened in triplicate to eliminate false positives, and only hybridization signals of at least 3-fold background levels were considered. Fig. 3 is an example of one hybridization. To allow comparisons, hybridization signals were normalized to the signals obtained from housekeeping gene controls on the same array and to the relative decrease in expression of the G3PDH gene in CBS / liver (Fig. 3, arrow 1, and Fig. 2B).
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Computer analysis of hybridization intensities of the three hybridizations
indicated that seven known mouse genes were differentially expressed. All of
the cDNA spots indicated with an arrow in
Fig. 3 were selected for
further examination. The differential expression was evaluated by Northern
blot analysis; this confirmed four down-regulated and three up-regulated CBS
/ mice mRNA in two independent experiments. In each case, the
relative expression levels of these genes observed on Northern blots were
consistent with the differential gene expression identified by array
hybridization (Table II). We
also analyzed by Northern blotting the modulations in liver RNA of CBS
+/ mice. Seven comparative Northern blots were obtained with the CBS
/, CBS +/, and CBS +/+ livers probed with the
differentially expressed cDNAs listed in
Table II
(Fig. 4). The genes with higher
expression in CBS / liver included HO-1, encoding
stress-response protein that is induced by a variety of agents causing
oxidative stress (41), CYP7B1
(which was also overexpressed in liver from male +/ mice), encoding an
oxysterol 7
-hydroxylase, which plays a crucial role in lipid and bile
acid metabolism (42), and
CYP3A11, encoding a testosterone 6
-hydroxylase
(Fig. 4). Genes found to be
down-regulated in CBS / liver included CYP2B9/10 (which were
also underexpressed in liver from female CBS +/ mice), encoding
testosterone 16
-hydroxylases, CYP2C29, encoding an aldehyde oxygenase,
the genes for sulfotransferases 1,2 (STA1/2) (which were also underexpressed
in liver from female CBS +/ mice), two members of
alcohol/hydroxysteroid sulfotransferase gene family that catalyze the
sulfation of hydroxysteroids, and PON1, encoding a high density lipoprotein
(HDL)-associated enzyme that can protect low density lipoprotein (LDL) against
oxidation by aortic wall cells
(43). CYP2B9 and CYP2B10 mRNAs
are very similar in size and nucleotide sequence. Similarly, STA1 and STA2 are
very similar. Consequently, in both cases, probes recognize both members of
the pair, which cannot be easily discriminated. Therefore, the mRNA detected
on the Northern blot is referred to as CYP2B9/10 and STA1/2, respectively
(Fig. 4).
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Analysis of HO-1 Protein Expression and PON1 Activity Expression of HO-1 was examined in CBS +/+, CBS +/, and CBS / liver mice by Western blotting to determine whether HO-1 is also overexpressed at the protein level. In agreement with increased levels of HO-1 mRNA, HO-1 protein expression was induced in liver of CBS / mice (Fig. 5).
Activity of PON1 was also examined in liver extracts of CBS +/+, CBS
+/, and CBS / mice. The mean hepatic activity of PON1 in
CBS +/ mice was not different from those in CBS +/+ mice (data not
shown). Commensurate with the difference in hepatic mRNA PON1 abundance, the
mean hepatic activity of PON1 in CBS / mice was
3-fold
lower than that in CBS +/+ mice (PON1 activities were 8.8 ± 2.6
versus 24.4 ± 4.2
A270/min/mg of
cellular protein; p < 0.03 by Student's t test n
= 4 for each).
To determine whether the difference in PON1 activity is due to the loss of
CBS expression or due to the resultant HH, activity of PON1 was also examined
in liver extracts of CBS +/+ and CBS +/ fed a hyperhomocysteinemic
diet. At the time of weaning, CBS +/+ mice and CBS +/ mice were
provided with drinking water that was supplemented with 0.5%
L-methionine (defined as high methionine diet)
(20), and mice were studied at
3 months of age. The mean hepatic activity of PON1 in CBS +/+ mice fed a
hyperhomocysteinemic diet was not different from those in CBS +/ mice
fed a standard rodent diet (data not shown), and the mean plasma concentration
of tHcy was 2-fold that in CBS +/+ mice fed a standard rodent diet (tHcy
levels were 9.3 ± 1 versus 3.9 ± 0.9 µM;
p < 0.0001 by Student's t test n = 4 for each).
However, CBS +/ mice fed a hyperhomocysteinemic diet have a significant
decrease in mean hepatic activity of PON1 (PON1 activities were 13.2 ±
1.7 versus 24.4 ± 4.2
A270/min/mg
of cellular protein; p < 0.005 by Student's t test
n = 4 for each), and the mean plasma concentration of tHcy was 2-fold
that in CBS +/+ mice fed a hyperhomocysteinemic diet and in CBS +/ mice
fed a standard rodent diet (tHcy levels were 15 ± 0.6 versus
9.2 ± 1.8 µM; p < 0.0001 by Student's
t test n = 4 for each). These results show that the decrease
in PON1 activity is inversely correlated with moderate to severe HH in
mice.
| DISCUSSION |
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We used differential screening of mRNAs prepared from the liver of CBS /, CBS +/, and CBS +/+ mice to provide insight into perturbations associated with CBS deficiency and, thus, with high hepatic tHcy concentrations. We also analyzed by Northern blotting the modulations in the liver RNA of CBS +/+ and CBS +/ mice with or without the supplemented diet with choline, and we have obtained the same results (data not shown). Some of the modifications of expression were only observed in the livers of CBS / mice, and others were only observed in the livers of CBS / mice and CBS +/ mice. Note that hepatic tHcy levels in CBS +/ mice do not differ significantly from those in CBS +/+ mice. We did not observe modifications of genes in the liver of CBS +/ mice that were not modified in CBS / mice.
We report altered expression of genes encoding RPS3a and methylthioadenosine phosphorylase, suggesting such cellular growth and proliferation perturbations may occur in homozygous CBS-deficient mice liver. The gene encoding RPS3a was down-regulated, and it has been shown that apoptosis is induced in certain cell lines by lowering constitutively high levels of RPS3a expression using antisense RPS3a gene sequences (44). The gene encoding methylthioadenosine phosphorylase was up-regulated. Methylthioadenosine, the substrate of methylthioadenosine phosphorylase, is a potent inhibitor of S-adenosylmethionine-dependent transmethylation reactions (40). CBS / mice have elevated plasma levels of tHcy and a concomitant increase in S-adenosyl-L-Hcy, a potent inhibitor of methyltransferases, notably in liver (45). Thus, other metabolic perturbations caused by CBS deficiency might have possible effects upon gene expression patterns in liver of CBS / mice.
We demonstrated the presence of oxidative stress in liver of CBS-deficient mice. The expression of many CYP was affected in CBS / and CBS +/ liver cells, indicating perturbation of the redox potential. One mitochondrial mRNA transcript (mitochondrial cytochrome c oxidase subunit I) was down-regulated in CBS / liver. This suggests that all mitochondrial transcripts are down-regulated because all mitochondrial transcription depends on a single promoter (46). This effect could also be attributed to oxidative damage, which is particularly important in the mitochondrial compartment. High concentrations of Hcy on human megakaryocytic cell line cells decrease mitochondrial gene expression in the presence of physiological concentrations of Cu2+ (14). The down-regulation of mitochondrial RNAs may be a general mechanism by which cells of liver of CBS / mice protect themselves against oxidative stress.
Changes in the intracellular redox state associated with elevated tHcy levels in liver of CBS / mice can alter the expression of intracellular defense genes induced in response to stressful conditions. The activity of several antioxidant enzymes including glutathione peroxidase was decreased in liver of CBS / mice (47). Glutathione, which plays a key role in the cellular defense against oxidative stress, is less abundant in liver of CBS / than control mice (47). Approximately half of the intracellular glutathione pool in human liver cells is derived from Hcy via the transsulfuration pathway (48). The Hcy-dependent transsulfuration pathway is important for maintaining the intracellular glutathione pool, and abnormality in this pathway compromises the redox buffering capacity of cells. Therefore, the susceptibility of hepatic tissues to oxidative stress may be enhanced by CBS deficiency. We have shown that expression of HO-1 is powerfully induced in liver of CBS / mice at the transcriptional and protein expression levels. Agents that are oxidants or can generate active intermediates lead to induction of HO-1 in cultured human skin fibroblasts (49). Induction of HO-1 mRNA may be a result of oxidative stress in the liver of CBS / mice, and overexpression of HO-1 protein could contribute to a cytoprotective mechanism to prevent further oxidative injury in the liver.
Werstuck et al.
(24) demonstrate that
HH-induced endoplasmic reticulum stress in hepatocytes results in an enhanced
lipid biosynthesis by activation of the sterol regulatory element-binding
protein, thus leading to hepatic steatosis. CBS +/ mice fed a
hyperhomocysteinemic diet have increased hepatic cholesterol and triglyceride
levels through increased hepatic expression of genes involved in cholesterol
and triglyceride synthesis, uptake, and storage
(24). We have also seen by
histological analysis of liver sections from CBS / mice that
hepatocytes are engorged with lipid vesicles (data not shown), but we have not
found expression modulations of genes involved in hepatic metabolism of
cholesterol. Even if we have not screened all the hepatic transcripts of CBS
/ mice, we have used a mouse stress cDNA expression membrane
that contains the gene encoding peroxisome proliferator-activated receptor
, and overexpression of peroxisome proliferator-activated receptor
has been associated to the development of hepatic steatosis.
Nevertheless, we have not found gene expression modulation of peroxisome
proliferator-activated receptor
in the liver of CBS /
mice.
There is growing evidence that elevated plasma levels of tHcy are associated with an increased risk of atherosclerosis. Several studies show that Hcy induces endothelial dysfunction and injury in CBS +/ (18, 19, 21, 22) and CBS / (50) mice. Moreover, endothelial dysfunction is believed to be an important mechanism initiating the pathogenesis of atherosclerosis. We have found altered expression of gene encoding PON1 concomitant with reduced PON1 activity in the liver of CBS / mice but also in the liver of CBS +/ mice fed a hyperhomocysteinemic diet, which demonstrates the importance of HH in the PON1 activity. Most serum PON1 is synthesized in the liver, and PON1 is a protein component of HDL. It destroys biologically active lipids in mildly oxidized LDL, thereby preventing oxidized LDL build-up when antioxidant protection is not sufficient (43, 51). This protection is very important because oxidized LDL is highly cytotoxic to the endothelium, promotes conversion of macrophages to foam cells, and stimulates monocyte recruitment and immunological responses (52). PON1-deficient mice, when crossed to apolipoprotein E (apoE, a protein that mediates removal of plasma lipoproteins via the LDL receptors and other receptors)-deficient mice and fed on a high fat high cholesterol diet, also exhibited increased expression of genes related to oxidative stress like HO-1 and were more susceptible to atherosclerosis than wild type from the same litter (53). Moreover, HO-1 expression in cultured artery wall cells is up-regulated by mildly oxidized LDL (54). Therefore, increased and decreased levels of HO-1 and PON1, respectively, in the liver of CBS / mice provide indirect evidence that severe HH may lead to higher LDL oxidation in mice. A recent study shows that PON1 contributes to the protective role of HDL against the oxidative damage and against toxicity exerted by Hcy involved in the development of atherosclerosis (55). Moreover, mice deficient in CBS and apoE develop accelerated aortic atherosclerosis (56). Increased lesion formation was observed in 1-year-old CBS //apoE / mice on regular diet and in a diet-induced milder HH in 5-month-old CBS +//apoE / mice (56) and suggests that isolated HH is atherogenic. We have not observed atherosclerosis lesions in CBS / mice at 3 or 4 months of age (data not shown). Nevertheless, mice used in our study are younger than mice used by Wang et al. (56).
In summary, the reduced PON1 activity indicates that HH in mice could lead to higher LDL oxidation, which may influence the development and progression of atherosclerosis lesions. Our work also increases our understanding of the gene pathways involved in the pathophysiological consequences of HH.
| FOOTNOTES |
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Supported by a Fellowship of the Fondation Jérome Lejeune. ![]()

To whom correspondence should be addressed. Tel.: 33-1-44-27-77-45; Fax:
33-1-44-27-83-38; E-mail:
janel{at}paris7.jussieu.fr.
1 The abbreviations used are: Hcy, homocysteine; tHcy, total Hcy; apoE,
apolipoprotein E; CBS, cystathionine
-synthase; CBS +/+ mice, wild type
mice; CBS +/ mice, heterozygous CBS-deficient mice; CBS /
mice, homozygous CBS-deficient mice; CYP, cytochrome P450; G3PDH,
glyceraldehyde-3-phosphate dehydrogenase; HDL, high density lipoprotein; LDL,
low density lipoprotein; HepG2, human hepatoma cell line; HH,
hyperhomocysteinemia; HO-1, heme oxygenase 1; PON1, paraoxonase 1; RPS3a,
ribosomal protein S3a; RT, reverse transcriptase; STA, sulfotransferases;
SOD1, Cu/Zn superoxide dismutase 1. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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