Wadsworth Center, New York State Department of Health and School of
Public Health, State University of New York at Albany, Albany, New
York
Rat CYP2A3 and mouse CYP2A5 are predominantly expressed in the
olfactory mucosa. CYP2A3 is also expressed in the lung at a low level,
whereas CYP2A5 is expressed in several additional tissues. To better
understand the transcriptional regulation of the CYP2A genes, transgenic mice were generated with a full-length
CYP2A3 gene fragment containing 3.4 kilobases of the
5'-flanking region. CYP2A3 mRNA was detected in the brain and olfactory
bulb in four transgenic mouse lines, in the olfactory mucosa in three
lines, and in kidney, liver, lung, and small intestine in two lines. Thus, the expression of the CYP2A3 transgene mimicked
the tissue distribution pattern of mouse CYP2A5 rather than that of rat
CYP2A3. Furthermore, the levels of CYP2A3 mRNA were very low in all
lines examined, suggesting that more distal regulatory regions may be involved in the abundant expression of the CYP2A genes
in the olfactory mucosa.
 |
Introduction |
Rat
CYP2A3 and its orthologs, mouse CYP2A5, human CYP2A6, and human
CYP2A13, are cytochrome P450 enzymes highly active in the metabolism of
many xenobiotics, including several known chemical carcinogens
(Fernandez-Salguero and Gonzalez, 1995
; Honkakoski and Negishi, 1997;
Su et al., 2000). Despite high similarities in their amino acid
sequence and substrate specificity, however, these enzymes differ in
tissue distribution. CYP2A3 and CYP2A5 are abundantly and
preferentially expressed in the olfactory mucosa (Su et al., 1996).
CYP2A3 is also expressed in the lung at a low level and at trace levels
in the breast and esophagus but not in the liver or other tissues
(Kimura et al., 1989; Su et al., 1996; Hellmold et al., 1998;
Gopalakrishnan et al., 1999), whereas CYP2A5 is expressed in many
additional tissues, including the brain (unpublished results), liver,
kidney, and small intestine (Su et al., 1998). In humans, CYP2A6 is
predominantly expressed in the liver, whereas CYP2A13 is selectively
expressed in the nasal mucosa and other parts of the respiratory tract
(Su et al., 2000).
The difference in tissue distribution of rat CYP2A3 and mouse CYP2A5
could be due to differences in their promoter sequences or to the
availability and nature of relevant transcription factors. An alignment
of the available sequences of the 5'-flanking regions of the rat
CYP2A3 and mouse Cyp2a5 genes revealed an 85%
identity. In the present study, transgenic mice were generated with a
full-length CYP2A3 gene, with 3.4 kilobases
(kb2) of the 5'-flanking region and 1.5 kb of the
3'-flanking region (Ueno and Gonzalez, 1990). The tissue distribution
and the levels of transgene expression were determined in multiple
transgenic lines. The results suggest that more distal regulatory
regions may be important for the abundant expression of the
CYP2A genes in the olfactory mucosa and that the species
differences in CYP2A distribution in other tissues may not be due to
differences in the promoter sequences. These findings provide a
foundation for future studies on the mechanisms of tissue-selective
gene expression in the CYP2A subfamily.
| |
Materials and Methods |
Generation of CYP2A3 Transgenic Mice.
A 13.0-kb CYP2A3 gene fragment containing 3.4 kb of the
5'-flanking region, all exons and introns, and 1.5 kb of the
3'-flanking region was obtained by a PacI and
SalI double digestion of a CYP2A3 genomic clone
(Ueno and Gonzalez, 1990). Transgenic mice were produced at the
Transgenic/Knockout Mice Core Facility of the Wadsworth Center (Albany,
NY) using standard procedures (Hogan et al., 1994). The transgene
construct was purified, and approximately 2 pl of the DNA fragment (2 µg/ml) was microinjected into the pronuclei of fertilized eggs from
the C57BL/6 strain or the B6/CBA strain. The eggs were then cultured to
the two-cell stage, reimplanted into the oviducts of pseudopregnant
B6/CBA F1 mice, and allowed to develop to term. The transgenic mice
were crossed with wild-type C57BL/6 mice to generate F1 and F2
heterozygous offspring, which were used for analysis of transgene expression.
Southern Blot Analysis.
Genomic DNA was isolated from mouse tail biopsy (Hogan et al., 1994).
Aliquots of 10 µg of DNA were digested with 100 units each of
BamHI and PstI or with 100 units of
SacI at 37°C overnight. The samples were subsequently
fractionated by electrophoresis on 0.8% agarose gels, and transferred
to nylon membranes. Hybridization and washing were performed as
described previously (Su et al., 1996), using a 312-base pair (bp)
CYP2A3 DNA probe derived from the proximal promoter region of the
CYP2A3 gene (
254 to +58; Zhang and Ding, 1998). The
intensity of the detected bands was quantified with a densitometer
(Personal Densitometer SI; Molecular Dynamics, Sunnyvale, CA) for
determination of transgene copy number.
Qualitative RNA-PCR Analysis.
Total RNAs were isolated from various tissues of 2-month-old mice or
the lung of an adult male Wistar rat (Charles River Laboratories, Inc.,
Wilmington, MA) using TRIzol reagent (Invitrogen, Carlsbad, CA).
First-strand cDNA was synthesized by reverse transcription (RT) from 2 µg of total RNA with use of Moloney murine leukemia virus reverse
transcriptase (Promega, Madison, WI) and an
oligo-d(T)16 primer (Applied Biosystems, Foster
City, CA). For analysis of CYP2A3 transgene expression, PCR was carried
out with a set of CYP2A3-specific primers (forward primer: 2A3UE34,
5'-tcgaaagacgaacggtgccc-3'; reverse primer: 2A3LE67,
5'-tcctcgtggaccttagcctcaatatcc-3'). The expected product was 510 bp in
length. PCR mixtures contained 1× reaction buffer (Promega), 1 mM
MgCl2, 0.4 mM dNTPs, 0.4 µM each primer, 2.5 µl of a RT mixture, and 1.25 units of Taq DNA polymerase
in a total volume of 25 µl. PCR was performed using a two-step
protocol (30 s at 94°C and 45 s at 68°C) for 35 cycles.
For detection of the full-length CYP2A3 cDNA coding region, PCR was
initially carried out with a set of CYP2A3-specific primers (forward
primer: 2A35UTRF, 5'-tgtgtctgtcatcccatcgcc-3'; reverse primer:
2A33UTRR, 5'-cacgcaatgctggctcaacg-3'). PCR mixtures contained 25 µl
of Hotstart Master Mix (QIAGEN, Hilden, Germany), 0.4 µM each primer,
and 3 µl of an RT mixture in a total volume of 50 µl. PCR was
performed at 95°C for 15 min to activate the Taq DNA polymerase, followed by 35 cycles of 94°C for 30 s, 55°C for
30 s, and 72°C for 90 s. Nested PCR was then performed
under the same condition as for the first-round PCR but with 1 µl of
the reaction mixture from the initial PCR as the template and a set of
CYP2A primers that do not distinguish CYP2A3 from CYP2A5 (forward primer: 2A3E1F, 5'-ctcaggactccttctggtgg-3'; reverse primer: 2A3E9R, 5'-cacaagtctaggagacacatcg-3'). The nested-PCR products were subjected to restriction mapping with ClaI to confirm their identities.
For analysis of the 3'-untranslated region (UTR) of the transcripts of
the CYP2A3 transgene, PCR was carried out with a forward primer (5'-ctacactatgagtttcttgtc-3') complementary to exon 9 of CYP2A3
mRNA and an anchored oligo-dT reverse primer
(5'-tttttttttttttttgcca-3'). PCR mixtures contained 1× reaction
buffer, 2 mM MgCl2, 0.4 mM dNTPs, 0.4 µM each
primer, 2.5 µl of a RT mixture, and 1.25 units of Taq DNA
polymerase in a total volume of 25 µl. PCR was performed for 35 cycles of 94°C for 30 s, 47°C for 30 s, and 72°C for
30 s. The 3'-UTRs of both CYP2A3 and CYP2A5 mRNAs would be
amplified with this set of primers. The expected sizes of the PCR
products derived from CYP2A5 and CYP2A3 mRNAs were 232 and 223 bp,
respectively. The two products were distinguished by digestion of a
15-µl reaction mixture with 10 units of Tsp509 I at 37°C
for 1.5 h. Tsp509 I cuts the PCR product derived from
CYP2A5 but not that from CYP2A3. PCR products were analyzed on agarose
gels and visualized by staining with ethidium bromide. The uncut CYP2A3
band was gel-purified and used as a template in a nested PCR with the
same primer set, generating enough material for definitive
identification of the PCR products by DNA sequencing. The CYP2A3 band
from the second round of Tsp509 I digestion was isolated for
DNA sequencing.
Real-Time Quantitative RNA-PCR Analysis.
Quantitation of CYP2A3 mRNA was carried out using a LightCycler
(Roche Applied Science, Indianapolis, IN). RT was performed as
described above. The CYP2A3-specific primers used were the same as
described earlier. PCR mixtures contained 2 µl of FastStart DNA
Master SYBR Green I (Roche Applied Science), 2 mM
MgCl2, 0.4 µM each primer, and 0.5 µl of a RT
mixture in a total volume of 20 µl. PCR reactions were initiated with
a denaturation/Taq activation step at 95°C for 6 min. For
the detection of CYP2A3 mRNA, the reactions were performed for 40 cycles of 94°C for 2 s and 68°C for 35 s. For the
detection of 
-actin mRNA, the reactions were performed for 35 cycles
of 94°C for 2 s, 60°C for 5 s, and 72°C for 20 s.
For the detection of CYP2A5 mRNA, the reactions were performed with a
pair of CYP2A4/5 primers (forward, 2A5LE3:
5'-gggctttctcatcgatttatttcgg-3'; reverse, 2A5LE67:
5'-tcctcatggaccttggcctcaatatct-3') at 94°C for 2 s, 58°C for
5 s, and 72°C for 35 s for 40 cycles. Detection of the
fluorescence in each reaction was carried out at 3°C below the
melting temperature (Tm) of the corresponding PCR product, which
eliminates the interference from potential PCR primer dimers. The
specificity of the PCR products was confirmed by melting-curve analysis. Quantification and melting temperature were analyzed using
the LightCycler data analysis software (Roche Applied Science).
| |
Results |
Characterization of the CYP2A3 Transgene.
A total of eight transgenic lines were generated, as summarized in
Table 1. Presence of the
CYP2A3 transgene was initially detected by PCR and then
confirmed by Southern blot analysis after digestion of genomic DNA with
BamHI and PstI (not shown). The integrity of the
transgene was verified by digesting genomic DNA samples with
SacI, which yields a 10-kb fragment from the 13-kb transgene. The CYP2A3 probe detected the 10-kb band in genomic DNA
samples prepared from six transgenic lines, but it detected a shorter
band (about 7 kb) in line 1786 and 1854 (data not shown). Thus, the
transgene was truncated in the latter two lines (which were not studied
further), but it seemed to be intact in the other six lines. The copy
numbers of the CYP2A3 transgene were determined using
Southern blot analysis, with rat genomic DNA as a standard for
quantification; CYP2A3 is a single-copy gene in the rat
genome (Kimura et al., 1989). The copy numbers of the transgene varied from 1 to 12 among different transgenic lines (Table 1). As later observed, however, the transgene copy numbers were not correlated with
the levels of transgene expression (data not shown).
Expression of the CYP2A3 Transgene.
CYP2A3 transgene expression was examined using both
qualitative and quantitative RNA-PCR approaches. Assays for detecting cytochrome P450 protein, such as immunoblot and enzyme activity assays,
were not used because enzyme-specific antibodies, substrates, or
inhibitors are currently not available. RNA blot analysis was also
inadequate because of the low levels of transgene expression and the
expected sequence homology between the transgene mRNA and the mouse
CYP2A transcripts. The specificity of the PCR primers was first
confirmed using CYP2A3 and CYP2A5 cDNA as positive and negative control
templates, respectively (Fig. 1). Various
tissues, including brain, heart, kidney, liver, lung, olfactory bulb,
olfactory mucosa, small intestine, and spleen, were collected from each transgenic line. The expression of the CYP2A3 transgene was
not detected in any of the tissues in two of the six transgenic lines studied (Table 1), presumably due to the integration of the transgene into a transcriptionally inactive area of the mouse genome. The results
from the other four lines are listed in Table
2. The expression of the
CYP2A3 transgene varied among different transgenic lines,
most likely affected by the transgene integration sites. Interestingly,
CYP2A3 mRNA was detected, in more than one line, in a number of tissues
where CYP2A5 was coexpressed, including brain, heart, kidney, liver,
lung, olfactory bulb, olfactory mucosa, and small intestine (see Fig.
1). In contrast, CYP2A3 mRNA was not detected in heart or spleen, where
the constitutive levels of CYP2A5 mRNA were barely detectable, at least
100 times lower than the level in the lung (data not shown).
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Fig. 1.
Qualitative RNA-PCR analysis of CYP2A3 mRNA
expression in transgenic mice.
RNA-PCR was performed with the use of a set of CYP2A3-specific primers,
as described under Materials and Methods. The samples
analyzed include total RNAs from olfactory mucosa of an adult male
Wistar rat (as a positive control; lane 1), olfactory mucosa of an
adult male C57BL/6 wild-type mouse (as a negative control; lane 2), and
brain (lane 3), heart (lane 4), kidney (lane 5), liver (lane 6), lung
(lane 7), olfactory mucosa (lane 8), olfactory bulb (lane 9), small
intestine (lane 10), and spleen (lane 11) of a 2-month-old male mouse
of transgenic line 1482. The position of the expected PCR product from
CYP2A3 mRNA is indicated by an arrow; it was clearly distinguished from
the weak, diffused, nonspecific product that was visible in most
samples (probably a primer dimer). The positive band in lane 7 is
rather weak and may not reproduce well.
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TABLE 2
Qualitative analysis of the tissue distribution of CYP2A3 mRNA in
transgenic mice
Detection of CYP2A3 transgene mRNA by qualitative RNA-PCR was performed
as described under Materials and Methods. For each tissue,
four mice (two male and two female) were analyzed, except for line 2714 for which only two females were examined.
|
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The PCR method used for qualitative detection of CYP2A3 mRNA was
designed to maximize specificity and sensitivity, but it only amplifies
cDNA sequences between exons 3 and 7. To determine whether the
transgene transcript was processed correctly, we also performed
experiments to evaluate whether all exons were present and whether the
3'-UTR was the same as that found in CYP2A3 mRNA from rats. In the
first set of experiments, the CYP2A3 cDNA was detected by a nested PCR
designed to amplify the entire coding region, from exons 1 to 9, of the
transgene mRNA (data not shown). The identity of the PCR product was
confirmed by DNA sequencing. More importantly, PCR products with a
smaller size than that of the full-length coding region were not
detected in any of the tissues examined, indicating that the transgene
transcript was correctly spliced.
In the second set of experiments, the sequence of the 3'-UTR of the
CYP2A3 transgene mRNA was determined with use of a forward PCR primer
from exon 9 and an anchored oligo-dT reverse primer. As shown in Fig.
2, the PCR product corresponding to the
3'-UTR of CYP2A5, but not to the 3'-UTR of CYP2A3, was cut by
Tsp509 I, as predicted from their sequences. With RNA
samples prepared from the transgenic mice, not all of the PCR products
were cut by Tsp509 I, but sequence analysis of the residual,
uncut PCR product confirmed that it was derived from CYP2A3 mRNA. Thus, the authentic polyadenylation signal of the CYP2A3 transgene
was used. However, these results do not rule out the potential
occurrence of alternatively polyadenylated CYP2A3 transcripts with a
much longer 3'-UTR, which would not be detected under the PCR
conditions used. The results in Fig. 2 also confirmed that the
expression level of the transgene was quite low; the PCR product
corresponding to CYP2A3 was much less abundant than that generated from
the endogenous CYP2A5 mRNA in various tissues of the transgenic mice.
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Fig. 2.
Analysis of the 3'-UTR of the CYP2A3 mRNA in
transgenic mice.
Procedures for RNA-PCR and digestion of the PCR products with
Tsp509 I are described under Materials and
Methods. Samples analyzed include RNAs from the lung of an
adult male Wistar rat (as a positive control, lane 2), lung of a male
C57BL/6 wild-type mouse (as a negative control, lane 3), kidney of a
male mouse from line 1482 (lane 4), kidney of a female mouse from line
1482 (lane 5), liver of a female mouse from line 2699 (lane 6), and
liver of a male mouse from line 2699 (lane 7). All mice were about 2 months old. DNA markers (100 bp) are included for size determination
(lane 1). The positions of the Tsp509 I-digested CYP2A3
and CYP2A5 bands are indicated.
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Quantitative Analysis of Levels of CYP2A3 mRNA in Transgenic Mice.
Real-time PCR was performed to quantify the levels of CYP2A3 mRNA in
various tissues of different transgenic lines. The CYP2A3 cDNA
(101 to 107 copies) was
used as a standard for quantification. A linear standard curve was
generated from a starting copy number of 100 or higher (Fig.
3, A and B). No product was generated
from CYP2A5 cDNA (Fig. 3, A), thus demonstrating the specificity of the
PCR primers. Melting-curve analysis showed that the CYP2A3 primer pair
amplified a single predominant product with a Tm at 86°C, which was
the same as predicted for the product of CYP2A3 cDNA (Fig. 3, C and D).
This product was not detected in reactions with the CYP2A5 cDNA (not
shown) or in the H2O (no-template) controls. The
length of the PCR product was further confirmed by agarose gel
electrophoresis (data not shown).
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Fig. 3.
CYP2A3 cDNA standard curve and melting-curve
analyses.
Serial dilutions of the CYP2A3 cDNA standards from 107 to
101 were analyzed, and the fluorescence was measured at
3°C below the Tm of the specific product. A, demonstrates the
sensitivity and specificity of the PCR reaction. The negative controls
contained CYP2A5 or H2O instead of the CYP2A3 template. B,
a linear standard curve, which was generated from the data in A using
the LightCycler data analysis software, was used to quantify the levels
of CYP2A3 mRNA in the transgenic mice. C and D, the specificities of
the PCR products were further analyzed by melting-curve analysis; the
examples shown are PCR products generated from the brain RNA of a mouse
from transgenic line 2701 (Tg brain RNA) and those from the
105 dilution of the CYP2A3 cDNA standard.
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As shown in Table 3, although CYP2A3
transgene mRNA was detected at relatively high levels in the olfactory
mucosa in lines 2699 and 2714, a preferential expression in this tissue
was not observed, in contrast to the known high levels of nasal
expression of rat CYP2A3 and mouse CYP2A5 (Su et al., 1996). In control
experiments not presented, the mRNA levels of a house-keeping gene,
-actin, were also determined in each RNA sample, using a
LightCycler. The results were consistent with the total RNA
concentrations measured spectrally, confirming that equal amounts of
total RNA were used in the RT reactions.
| |
Discussion |
The levels of CYP2A3 transgene expression were very low in this
study, despite the fact that the gene (including 3.4 kb of 5'-flanking
region, 1.5 kb of 3'-flanking region, and all introns), instead of a
cDNA, was included in the transgene construct. Furthermore, although
both CYP2A3 and CYP2A5 are expressed predominantly in the olfactory
mucosa in rats and mice, respectively (Su et al., 1996, 2000), this
strong tissue preference was not reproduced by the CYP2A3
transgene in any of the transgenic lines analyzed. These observations
imply that additional enhancers may be located further upstream or
downstream of the gene fragment included in the transgene.
Alternatively, although less likely, the putative olfactory
mucosa-selective transcriptional activators may not be highly conserved
to the extent that the mouse proteins would not activate the cognate
regulatory element in the CYP2A3 transgene as efficiently as
with the mouse Cyp2a5 gene.
An earlier study identified a nasal predominant transcriptional
activating (NPTA) element in the 5'-flanking region of the rat
CYP2A3 gene (Zhang and Ding, 1998). The NPTA element was
found to be essential for transcriptional activity of the
CYP2A3 promoter in in vitro transcription assays, and it
specifically interacted with proteins from the olfactory mucosal
nuclear extracts but not with the proteins from the liver, lung,
kidney, or brain. Moreover, the NPTA element is conserved in mouse
Cyp2a5 and human CYP2A6 genes. Other evidence
supporting the hypothesis that rat CYP2A3 and mouse
Cyp2a5 genes may use the same cis-elements for transcriptional activation include the identical location of a TATA box
in their proximal promoter region and the identical location of their
transcription initiation sites, both at 24-bp upstream from the ATG
translation start codon (Lindberg et al., 1989; Ueno and Gonzalez,
1990). However, further studies are needed to better define the role of
the NPTA element in the regulation of the CYP2A genes,
particularly in light of the implication that more distal regulatory
regions may also be required for their transcriptional activation in
the olfactory mucosa.
The species difference in tissue distribution of orthologous
xenobiotic-metabolizing enzymes is one of the important factors to
consider when risk assessment of chemical toxicity is made by
extrapolating from metabolism and toxicity data obtained in animal
experiments. It also provides a useful model for studying the basic
mechanisms of regulation of tissue-specific gene expression. Mechanistically, the lack of CYP2A3 expression in the liver and kidney
in rats and other tissues in which CYP2A5 is expressed in mice may be
due to the lack of necessary transcriptional activators or the presence
of tissue-selective transcriptional repressors. Alternatively, the more
ubiquitous expression of CYP2A5 in mice may be due to the presence of
unique, mouse-specific regulatory elements in the Cyp2a5
promoter, which allows transcriptional activation of the
Cyp2a5 gene in multiple tissues. However, the latter
mechanism is unlikely if a mouse-like tissue distribution is achieved
with the CYP2A3 gene in transgenic mice, as was found in
this study, despite the low levels of transgene expression and the
apparent impact of integration site on the transgene expression in
different lines. Thus, the results of the present study suggest that
the expression of CYP2A3 in rats and that of CYP2A5 in mice are
probably controlled by similar cis-elements. Consequently, a
more restrictive tissue distribution or different properties of the
relevant transcription factors may at least partly account for the lack
of CYP2A3 expression in rat liver and kidney.
Within the olfactory mucosa, the CYP2A proteins are expressed in
Bowman's glands and sustentacular cells but not in olfactory receptor
neurons (Zupko et al., 1991; Chen et al., 1992; Walters et al., 1993).
The cellular expression profile of the CYP2A3 transgene in
the olfactory mucosa was not examined in this study because of
difficulties in distinguishing CYP2A3 protein or mRNA from the
endogenous CYP2A5. Of interest, a 3.6-kb 5'-flanking sequence of the
mouse olfactory mucosa-specific Cyp2g1 gene was found to contain regulatory elements sufficient for olfactory mucosa-specific expression of a LacZ reporter gene in a recent study (Zhuo
et al., 2001). However, the cellular expression patterns of the
transgene failed to reproduce the pattern of the endogenous gene, which implicated the presence of additional regulatory sequences that are
necessary for the correct cell type-selectivity within the olfactory
mucosa. Additional transgenic studies with much longer upstream
sequences are warranted. In addition, it will be interesting to
determine whether the CYP2A and CYP2G genes are
regulated under a common locus control region.
We thank Dr. Frank Gonzalez of the National Cancer Institute (National
Institutes of Health, Bethesda, MD) for providing the CYP2A3 genomic clone and Drs. Laurence Kaminsky and
Adriana Verschoor for reading the manuscript. We also gratefully
acknowledge the use of the Transgenic and Molecular Genetics Cores of
the Wadsworth Center.
This research was supported in part by National Institute of
Environmental Health Sciences Grant ES-07462 (National Institutes of
Health, Bethesda, MD).
Abbreviations used are:
kb, kilobase;
bp, base
pair;
PCR, polymerase chain reaction;
RT, reverse transcription;
UTR, untranslated region;
Tm, melting temperature;
NPTA, nasal predominant
transcriptional activating.
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Abstract
Introduction
-gene as major steroid 15-
