From the Laboratories of Pulmonary
Pathobiology, § Structural Biology, and
¶ Experimental Pathology, NIEHS, National Institutes of
Health, Research Triangle Park, North Carolina 27709 and the
Department of Medicine, Vanderbilt University,
Nashville, Tennessee 37232
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ABSTRACT |
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A cDNA encoding a new cytochrome P450 was
isolated from a mouse liver library. Sequence analysis reveals that
this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that
is 69-74% identical to CYP2J subfamily P450s and is designated
CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450
oxidoreductase in Sf9 cells using a baculovirus system.
Microsomal fractions of CYP2J5/NADPH-cytochrome P450
oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 11- and
15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of
product/nmol of cytochrome P450/min at 37 °C); thus CYP2J5 is
enzymologically distinct. Northern analysis reveals that CYP2J5
transcripts are most abundant in mouse kidney and present at lower
levels in liver. Immunoblotting using a polyclonal antibody against a
CYP2J5-specific peptide detects a protein with the same electrophoretic
mobility as recombinant CYP2J5 most abundantly in mouse kidney
microsomes. CYP2J5 is regulated during development in a tissue-specific
fashion. In the kidney, CYP2J5 is present before birth and reaches
maximal levels at 2-4 weeks of age. In the liver, CYP2J5 is absent
prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical
staining of kidney sections with anti-human CYP2J2 IgG reveals that
CYP2J protein(s) are present primarily in the proximal tubules and
collecting ducts, sites where the epoxyeicosatrienoic acids are known
to modulate fluid/electrolyte transport and mediate hormonal action.
In situ hybridization confirms abundant CYP2J5 mRNA
within tubules of the renal cortex and outer medulla.
Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney
thus providing direct evidence for the in vivo metabolism
of arachidonic acid by the mouse renal epoxygenase(s). Based on these
data, we conclude that CYP2J5 is an enzymologically distinct,
developmentally regulated, protein that is localized to specific
nephron segments and contributes to the oxidation of endogenous renal
arachidonic acid pools. In light of the well documented effects of
epoxyeicosatrienoic acids in modulating renal tubular transport
processes, we postulate that CYP2J5 products play important functional
roles in the kidney.
Cytochrome P450s have been the subject of intense investigation by
toxicologists and pharmacologists over the past 4 decades because they
catalyze the oxidative, peroxidative, and reductive metabolism of a
wide variety of drugs, industrial chemicals, environmental pollutants,
and carcinogens (1, 2). Some of these enzymes also catalyze the
NADPH-dependent oxidation of arachidonic acid to
cis-epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and
14,15-EET),1 mid-chain
cis-trans-conjugated dienols (5-, 8-, 9-, 11-, 12-, and
15-HETE), and/or P450-derived eicosanoids have been extensively studied in the kidney
and have been shown to substantially contribute to integrated renal
function (6, 7). Thus, the EETs and/or their hydration products (the
DHETs) inhibit Na+ transport and mediate angiotensin
II-induced rises in cytosolic Ca2+ in the proximal tubule
(15, 16), stimulate prostaglandin synthesis, and inhibit
Na+ reabsorption, K+ secretion, and
vasopressin-stimulated water reabsorption in the collecting duct
(17-19), activate Na+/H+ exchange, amplify
vasopressin-induced increases in cytosolic Ca2+, and
stimulate cellular proliferation in renal glomerular mesangial cells
(20, 21), modulate renal Na+/K+-ATPase (22),
affect intrarenal vascular tone, and renal vascular smooth muscle cell
K+ channel activity (23-25), and inhibit renal cortical
renin release (26). Recent studies demonstrating that (a)
the rat renal epoxygenase(s) are under regulatory control by dietary
salt, (b) clotrimazole inhibition of the rat renal
epoxygenase(s) leads to the development of salt-dependent
hypertension, (c) the salt-sensitive phenotype in the Dahl
rat model of genetic hypertension is associated with decreased renal
microsomal arachidonic acid epoxygenase activity and an inability to
up-regulate this activity in response to salt loading, (d)
the developmental phase of hypertension in spontaneously hypertensive
rats is associated with alterations in kidney P450 arachidonic acid
metabolism, and (e) the urinary excretion of epoxygenase
metabolites is increased during pregnancy-induced hypertension in
humans have supported the hypothesis that P450-derived arachidonic acid
metabolites may be involved in the pathophysiology of hypertension (6,
7, 27-32). Together, these findings suggest that the renal P450
arachidonic acid monooxygenases may be important in controlling blood
pressure and body fluid volume/composition and may be part of an
adaptive response of the kidney to excess dietary salt intake (6,
7).
Despite considerable investigations into the physiological aspects of
the renal epoxygenase pathway, little is known with regard to the
biochemical and molecular properties of the renal P450 enzyme(s) active
in the biosynthesis of the EETs. A member of the CYP2C subfamily was
purified from rabbit kidney and shown to catalyze arachidonic acid
epoxidation and Materials--
[ Screening of the cDNA Library--
An oligo(dT)-primed
Lambda ZAP B6/CBAF1J male mouse liver cDNA library (Stratagene, La
Jolla, CA) was screened with the 1.8-kb CYP2J3 cDNA probe under
conditions identical to those described previously (14). Approximately
100 duplicate positive clones were identified, of which 21, selected at
random, were plaque-purified and rescued into pBluescript SK(+)
(Stratagene). Plasmid DNAs were isolated using a Qiagen Plasmid
Purification Kit (Qiagen Inc., Chatsworth, CA), and insert sizes were
determined by agarose gel electrophoresis after EcoRI
digestion. The pBluescript cDNA inserts were partially sequenced by
the dideoxy chain termination method using Sequenase version 2.0 (U. S. Biochemical Corp.) and T3/T7 oligonucleotide primers.
Nucleotide sequences were analyzed by searching GenBankTM
and EMBL data bases utilizing GCG software (Genetics Computer Group,
Inc., Madison, WI). Sixteen of the duplicate positive clones contained
sequences that were identical and shared homology with several human
and rodent CYP2 family P450s (1). One of these clones (clone
JM-6)2 was completely
sequenced utilizing a total of 16 oligonucleotide primers (21-25
nucleotides, each) that spanned the entire length of the sense and
antisense cDNA strands. Oligonucleotides were synthesized and
purified as described (14).
Heterologous Expression of Recombinant CYP2J5--
Co-expression
of the protein encoded by the cloned 1.886-kb JM-6 cDNA insert
(CYP2J5) with CYPOR in Sf9 insect cells was accomplished with
the pAcUW51-CYPOR shuttle vector (kindly provided by Dr. Cosette
Serabjit-Singh, Glaxo Wellcome, Research Triangle Park, NC) and the
BaculoGold Baculovirus Expression System (PharMingen, San Diego, CA)
using methods similar to those previously described (14, 39). The
CYP2J5 cDNA (nucleotides 42-1886) including the entire coding
region was ligated into a slightly modified pAcUW51-CYPOR vector, and
the orientation and identity of the resulting expression vector
(pAcUW51-CYPOR-CYP2J5) were confirmed by sequence analysis. In this
construct, the expression of CYPOR was controlled by the p10 promoter,
whereas the expression of CYP2J5 was independently controlled by the
polyhedrin promoter. Cultured Sf9 cells were co-transfected with
the pAcUw51-CYPOR-CYP2J5 vector and linear wild-type BaculoGold viral
DNA, and recombinant viruses were purified as described (14). Cultured
Sf9 cells grown in spinner flasks at a density of 1.5-2 × 106 cells/ml were then infected with high titer
CYP2J5-CYPOR recombinant baculovirus stock in the presence of
Incubations of Recombinant CYP2J5 with Arachidonic Acid, Product
Characterization--
Reaction mixtures containing 0.05 M
Tris-Cl buffer (pH 7.5), 0.15 M KCl, 0.01 M
MgCl2, 8 mM sodium isocitrate, 0.5 IU/ml
isocitrate dehydrogenase, 0.1-0.2 mg of CYP2J5-CYPOR-transfected
Sf9 cell microsomal protein/ml, and
[1-14C]arachidonic acid (25-55 µCi/µmol; 100 µM, final concentration) were constantly stirred at
37 °C. After temperature equilibration, NADPH (1 mM,
final concentration) was added to initiate the reaction. At different
time points, aliquots were withdrawn, and the reaction products were
extracted and analyzed by reverse-phase HPLC as described (41).
Products were identified by comparing their reverse- and normal-phase
HPLC properties with those of authentic standards and by GC/MS (42,
43). For kinetic experiments, arachidonic acid concentrations were
varied from 5 to 100 µM, and reactions were only allowed
to proceed for 2-3 min to ensure that the quantitative assessment of
the rates of product formation reflected initial rates. For chiral
analysis, the EETs were collected from the HPLC eluent, derivatized,
and resolved into the corresponding antipodes by chiral-phase HPLC as
described previously (44, 45). Control studies were performed by
incubating uninfected Sf9 cell microsomes and
baculovirus-infected Sf9 cell microsomes expressing recombinant
CYPOR but containing no spectrally evident P450 with arachidonic acid
under identical conditions.
Detection of Endogenous EETs in Mouse Kidney--
Methods used
to quantify endogenous EETs present in mouse kidney were similar to
those used to quantify EETs in rat liver and heart (14, 46). Briefly,
kidneys were frozen in liquid nitrogen and homogenized in
phosphate-buffered saline containing triphenylphosphine. The homogenate
was extracted as described (14), and the combined organic phases were
evaporated in tubes containing mixtures of
[1-14C]8,9-EET, [1-14C]11,12-EET, and
[1-14C]14,15-EET internal standards (56-57 µCi/µmol,
80 ng each). Saponification, silica column purification, HPLC
resolution, GC/MS analysis, and quantification were done as described
elsewhere (14, 45, 46).
Northern Blot Hybridization and RNA PCR Analysis--
Normal
mouse tissues were obtained from adult male C57BL/6J mice fed NIH
31 rodent chow (Agway, St. Mary, OH) ad libitum and sacrificed by lethal CO2 inhalation. RNA was prepared by
the guanidinium thiocyanate/cesium chloride density gradient
centrifugation method as described previously (47). Total RNA (25 µg)
prepared from mouse testes, small intestine, muscle, lung, liver,
kidney, heart, colon, and brain was denatured and electrophoresed in
1.2% agarose gels containing 2.2 M formaldehyde. After
capillary pressure transfer to Hybond-N+ membranes
(Amersham Pharmacia Biotech), the blots were hybridized with either the
cloned 1.886-kb JM-6 cDNA insert or with the following JM-6
sequence-specific oligonucleotide probe
(5'-TCAAAAACATAATCATATTTGGGATCCTTGCACAC-3', complementary to
nucleotides 23-57 of the JM-6 cDNA). With the cDNA probe,
hybridizations were performed at 42 °C in 50% formamide containing
0.7 M NaCl, 70 mM sodium citrate, 5×
Denhardt's solution, 0.5% (w/v) SDS, and 0.1 mg of heatdenatured
salmon sperm DNA/ml. With the oligonucleotide probe, hybridizations
were performed at 50 °C in 10% formamide containing 0.9 M NaCl, 90 mM sodium citrate, 2× Denhardt's
solution, 0.5% (w/v) SDS, and 0.1 mg of heat-denatured salmon sperm
DNA/ml. The double-stranded cDNA probe was labeled with
[ Protein Immunoblotting and Immunohistochemistry--
Microsomal
fractions were prepared from adult male C57BL/6J mouse tissues by
differential centrifugation at 4 °C as described previously (12).
Polyclonal antibodies against the purified, recombinant human CYP2J2
proteins (anti-CYP2J2 IgG) were raised in New Zealand White rabbits and
affinity purified as described previously (38). A peptide corresponding
to amino acids 103-117 of the deduced CYP2J5 sequence was designed
based upon sequence alignments with known CYP2 family P450s. The
peptide was synthesized and HPLC-purified by the Protein Chemistry
Facility at the University of North Carolina (Chapel Hill, NC). The
peptide was coupled to keyhole limpet hemocyanin via a
carboxyl-terminal cysteine to enhance antigenicity. Polyclonal
antibodies against this CYP2J5-specific peptide (anti-CYP2J5pep IgG)
were raised in New Zealand White rabbits using previously described
methods (48). Purified recombinant human CYP2J2 was prepared as
described previously (38). Microsomes prepared from Sf9 insect
cells infected with recombinant CYP2J6 baculovirus3 were used as a
source of mouse CYP2J6. Purified preparations of mouse CYP2A4 and
CYP2A5 were a gift from Dr. Masahiko Negishi (NIEHS, National
Institutes of Health). Purified recombinant CYP2C29, CYP2C37, CYP2C38,
CYP2C39, and CYP2C40 were a gift from Dr. Joyce Goldstein (NIEHS). For
immunoblotting, microsomal fractions or purified P450s were
electrophoresed in SDS-10% (w/v) polyacrylamide gels (80 × 80 × 1 mm), and the resolved proteins were transferred electrophoretically onto nitrocellulose membranes. Membranes were immunoblotted using either rabbit anti-CYP2J2 IgG or rabbit
anti-CYP2J5pep IgG, goat anti-rabbit IgG conjugated to horseradish
peroxidase (Bio-Rad), and the ECL Western blotting Detection System
(Amersham Pharmacia Biotech) as described (38). Protein determinations were performed according to the method of Bradford (49). Preimmune serum, collected from the rabbits prior to immunization, did not cross-react with CYP2J5 or with microsomal fractions prepared from
mouse tissues.
For immunohistochemistry, adult mouse, rat, and human kidney tissues
were fixed in 10% neutral buffered formalin, processed routinely, and
embedded in paraffin. Normal human kidney was obtained through the
Cooperative Human Tissue Network (National Disease Research
Interchange, Philadelphia, PA). Normal rat and mouse kidneys were
obtained from adult male Fischer 344 rats and C57BL/6J mice that had
been fed ad libitum and killed by lethal CO2
inhalation. Localization of CYP2J protein expression was investigated
on serial kidney sections (5-6 µm) with the anti-CYP2J2 IgG (1:100
dilution) using methods previously described for heart and intestine
(14, 41). Preimmune rabbit IgG was used as the negative control in place of the primary antibody.
In Situ Hybridization--
Radiolabeled antisense and sense RNA
probes to mouse CYP2J5 were transcribed from the linearized CYP2J5
(JM-6)/pBluescript construct using the T7 and T3 RNA polymerases and
[ Developmental Regulation of CYP2J5--
Kidneys and livers from
male and female C57BL/6J mice ages 18 days fetal to 16 weeks postnatal
(adult) were obtained from Hilltop Labs (Scottdale, PA). Animals were
weaned at 3 weeks of age and then fed NIH 31 rodent chow (Agway, St.
Mary, OH) ad libitum and sacrificed by lethal
CO2 inhalation. Tissues were frozen in liquid nitrogen
immediately following collection and shipped on dry ice. Microsomes
were prepared from pooled frozen tissues obtained from 3 to 6 animals
at each age, and protein immunoblotting was performed as detailed above.
Synthetic Procedures--
The [1-14C]EET internal
standards were synthesized from [1-14C]arachidonic acid
(56-57 µCi/µmol) by nonselective epoxidation as described
previously (51). EETs and HETE standards were prepared by total
chemical synthesis according to published procedures (52-54). DHETs
and [1-14C]DHETs were prepared by chemical hydration of
individual EETs as described (55). All synthetic compounds were
purified by reverse-phase HPLC (42). Methylations were performed using
an ethereal solution of diazomethane (56). Pentafluorobenzyl esters were formed by reaction with pentafluorobenzyl bromide as described (46).
Cloning of Mouse CYP2J5 cDNA--
Screening of the mouse liver
cDNA library with the rat CYP2J3 cDNA probe yielded 16 duplicate positive clones that contained identical sequences. One of
these clones (clone JM-6) was selected for further study. Complete
nucleic acid sequence analysis of clone JM-6 revealed that the cDNA
was 1886 nucleotides long, contained an open reading frame between
nucleotides 42 and 1544 flanked by initiation (ATG) and termination
(TGA) codons, had a short 5'-untranslated region, and had a
342-nucleotide 3'-untranslated region with a polyadenylation tail (Fig.
1). The cDNA encoded a 501-amino acid
polypeptide that had a derived molecular mass of 57,784 Da. The deduced
amino acid sequence for JM-6 contained a putative heme binding peptide
(FSMGKRACLGEQLA)
with the underlined conserved residues and the invariant cysteine at
position 447. The polypeptide encoded by JM-6 also contained other
structural features associated with CYP2 family P450s including an
amino-terminal hydrophobic peptide and a proline cluster between
residues 40 and 51 (Fig. 1). A comparison of the deduced JM-6 amino
acid sequence with that of other P450s indicated that it was
(a) <32% identical to members of the CYP1, CYP3, CYP4,
CYP5, CYP6, and CYP7 families, (b) 38-44% identical with
several CYP2 family P450s, and (c) 69-74% identical to
human, rabbit, and rat CYP2J P450s (1, 14, 38, 57, 58). Based on the
amino acid sequence homology with other CYP2Js, the new mouse
hemoprotein was designated CYP2J5 by the Committee on Standardized
Cytochrome P450 Nomenclature (1).
Heterologous Expression and Enzymatic Characterization of
Recombinant CYP2J5--
The protein encoded by clone JM-6 (CYP2J5) was
expressed with/without CYPOR in Sf9 insect cells using the
baculovirus expression system according to previously described methods
(14, 38, 39). The level of expression of recombinant CYP2J5 was 10-15 nmol of P450/liter of infected Sf9 cells using the
pAcUW51-CYPOR-CYP2J5 vector. CYP2J5 expression was significantly higher
(~100 nmol of P450/liter of infected Sf9 cells) in the absence
of CYPOR coexpression using the pBlueBacIV vector. Coomassie Brilliant
Blue-stained SDS-polyacrylamide gels of lysates prepared from CYP2J5
baculovirus-infected Sf9 cells revealed a prominent band with an
estimated molecular mass of 57-58 kDa that was not present in
uninfected insect cell lysates (data not shown).
Microsomal fractions prepared from Sf9 insect cells
co-expressing CYP2J5 and CYPOR metabolized arachidonic acid to EETs and midchain HETEs as the principal reaction products (catalytic turnover, 4.5 nmol of products/nmol of P450/min at 100 µM
arachidonic acid) (Fig. 2A).
We identified these metabolites by comparing their reverse- and
normal-phase HPLC properties with those of authentic standards and by
GC/MS analysis. None of the metabolites were formed when NADPH was
omitted from the reaction. Furthermore, uninfected Sf9 insect
cell microsomes (data not shown) and baculovirus-infected Sf9
cell microsomes expressing recombinant CYPOR but containing no
spectrally evident P450 (Fig. 2B) did not metabolize
arachidonic acid to EETs or midchain HETEs. This indicated that
nonspecific oxidation from sources other than P450 was minimal and
demonstrated that the eicosanoid products were generated in a
P450-dependent fashion. Thus, based on the chromatogram in
Fig. 2, we conclude that CYP2J5 is both an arachidonic acid
epoxygenase and an arachidonic acid midchain
hydroxylase.
Regiochemical analysis of the EETs, which accounted for approximately
half of the reaction products, revealed a preference for epoxidation at
the 14,15-olefin (64% of total EETs) as evidenced by the recovery of
14,15-EET and its hydration product, 14,15-DHET (Fig. 2A,
Table I). Epoxidation at the 11,12- and
8,9-olefins occurred less often (24 and 11% of total EETs,
respectively), whereas epoxidation at the 5,6-olefin occurred only
rarely (1% of the total EETs) (Table I). Stereochemical analysis of
CYP2J5-derived EETs revealed a preference for
(14S,15R)-, (11R,12S)-, and
(8R,9S)-EETs (optical purity, 60, 69, and 58%,
respectively) (Table I). Regiochemical analysis of the midchain
hydroxylase products revealed a preference for 15- and 11-HETEs (26 and
15% of total HETEs, respectively). Hydroxylation at the 12-, 9-, 8-, and 5-positions occurred less often. Thus, CYP2J5 is enzymologically
distinct from other CYP2J P450s in that (a) arachidonic acid
epoxidation occurs with greater regioselectivity, and (b) it
is the only CYP2J isoform that produces appreciable quantities of
midchain HETEs.
CYP2J5 containing microsomes metabolized arachidonic acid in a
time-dependent manner with linear reaction kinetics up to
10-min incubation times at P450 concentrations up to 500 pmol/ml.
14,15-EET, the major product, was formed at a rate of 1.3 nmol of
EET/nmol of P450/min. 11,12- and 8,9-EETs were formed at lower rates
(0.5 and 0.2 nmol of EET/nmol of P450/min, respectively), whereas
epoxidation at the 5,6-olefin occurred very slowly (<0.02 nmol
EET/nmol P450/min). 15- and 11-HETEs, the major hydroxylase products,
were formed at rates of 0.7 and 0.4 nmol of HETE/nmol of P450/min,
respectively. Other midchain HETEs were formed at lower rates. A more
detailed kinetic analysis revealed that the metabolism of arachidonic
acid seemed to exhibit simple Michaelis-Menten kinetics over the range of substrate concentrations used (5-100 µM) (Fig.
2C). The Lineweaver-Burke double-reciprocal plot shown in
Fig. 2D was used to derive an apparent Km
of 83 µM and Vmax of 10.9 nmol of
product/nmol of P450/min for CYP2J5.
Tissue Distribution of CYP2J5 mRNA and Protein--
To
ascertain the relative organ abundance of CYP2J5 transcripts, total RNA
extracted from various mouse tissues was blot hybridized under high
stringency conditions with the full-length CYP2J5 cDNA probe. As
shown in Fig. 3A, the CYP2J5
cDNA hybridized with kidney RNA to produce three transcripts as
follows: (a) an abundant 1.8-2.0-kb transcript consistent
with the size of the CYP2J5 mRNA; (b) a larger 3.2-kb
transcript of similar intensity; and (c) a 4.2-kb transcript
of significantly lower intensity. The identities of the 3.2- and 4.2-kb
transcripts remain unknown, but these larger transcripts may represent
other mouse P450s that share nucleic acid sequence homology with CYP2J5
or alternate splice variants of CYP2J5. The 1.9- and 3.2-kb CYP2J5
transcripts were also present at lower levels in mouse liver but were
absent from other mouse tissues including testes, small intestine,
skeletal muscle, lung, heart, colon, and brain (Fig. 3A).
The differences in mRNA abundance were not due to differences in
the amount of RNA applied to each lane as assessed by ethidium bromide
staining of the gel prior to transfer (Fig. 3A) and the
membrane following transfer (data not shown). Identical results were
obtained using a CYP2J5 sequence-specific oligonucleotide probe (data
not shown). To confirm independently the narrow tissue distribution of
CYP2J5 mRNAs, we used a sensitive and specific RNA PCR method to
amplify a 284-base pair DNA fragment (predicted size) from
reverse-transcribed mouse kidney and liver RNA (Fig. 3B). We
were unable to amplify a fragment from testis, small intestine,
skeletal muscle, lung, heart, colon, or brain RNA indicating that
CYP2J5 mRNA expression was below the limit of detection of the RNA
PCR assay in these tissues (Fig. 3B). RNA-PCR analysis using
sequence-specific primers for
Immunoblotting of microsomal fractions prepared from Sf9 insect
cells infected with recombinant CYP2J5 baculovirus stock using either
the anti-CYP2J2 IgG (Fig. 4A)
or the anti-CYP2J5pep IgG (Fig. 4B) showed a primary band at
approximately 57-58 kDa indicating that both of these antibodies
cross-reacted with mouse CYP2J5. Control studies demonstrated that the
anti-CYP2J5pep IgG did not cross-react with uninfected Sf9
insect cell microsomes or with the following recombinant mouse P450s:
CYP2J6, CYP2A4, CYP2A5, CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40
(Fig. 4B). The anti-CYP2J2 IgG, which has been previously
shown to immunoreact with CYP2J2 and CYP2J3 (14, 38), also
immunoreacted with recombinant CYP2J6 but did not react with uninfected
Sf9 insect cell microsomes (Fig. 4A). In order to
examine the tissue distribution of CYP2J5 protein, we performed
immunoblotting of microsomal fractions prepared from various mouse
tissues. As shown in Fig. 4C, anti-CYP2J5pep IgG detected a
protein with the same electrophoretic mobility as recombinant CYP2J5 in
mouse kidney microsomes and, at lower levels, in mouse liver
microsomes. This band was not present in microsomal fractions prepared
from other mouse tissues including stomach, small intestine, skeletal
muscle, lung, heart, colon, and brain (Fig. 4C). There was
little inter-animal variation in the tissue expression of CYP2J5
protein (data not shown). The CYP2J5 peptide antibody also
immunoreacted, albeit less intensely, with two higher molecular mass
bands (~66-67 and ~75-76 kDa) (Fig. 4C). Although the
identity of these additional immunoreactive bands that were present in
virtually all tissues tested remains unknown, it is unlikely that they
represent P450s based upon their molecular mass. To confirm the tissue
distribution of CYP2J5 protein, we immunoblotted mouse microsomes with
the anti-CYP2J2 IgG. As in the case of the anti-CYP2J5pep IgG, we
detected a protein with the same electrophoretic mobility as
recombinant CYP2J5 predominantly in mouse kidney and, at lower levels,
in mouse liver microsomes (data not shown). Based on these data, we
concluded that CYP2J5 mRNA and protein are abundant in mouse
kidney, present at lower levels in liver, and absent from other mouse
tissues. Importantly, CYP2J5 is the only mouse P450 arachidonic acid
epoxygenase known to be expressed predominantly in the kidney.
Immunolocalization of CYP2J Protein Expression in the
Kidney--
To determine the distribution of CYP2J protein(s) within
the kidney, we stained formalin-fixed paraffin-embedded kidney sections with the anti-CYP2J2 IgG. Similar staining patterns were observed in
mouse, rat, and human kidney. As shown in Fig.
5, CYP2J protein expression was most
abundant in the renal cortex, the outer stripe of the outer medulla,
and the renal papilla. CYP2J immunopositivity was generally intense
throughout the proximal tubules; however, staining in the straight part
of the proximal tubules (corresponding to the P2/P3 segments) was
generally more intense than in the convoluted part (corresponding to
the P1 segment) (Fig. 5, B, C, D, and H). Within
the collecting ducts, immunostaining was most intense in the papillary
collecting ducts and was present at lower levels in the inner and outer
medullary collecting ducts (Fig. 5, B, E, and F).
Strong positive immunostaining was present in the transitional
epithelium lining the renal pelvis (Fig. 5F). Immunoreactivity was also present, albeit in significantly lower amounts, in distal tubules (including the thick ascending limb and
distal convoluted tubule), and was generally absent from vascular structures, glomeruli, the thin limbs of Henle's loop, and intervening stromal tissue (Fig. 5, C, D, F, G, and H).
Preimmune IgG produced negative staining throughout the kidney (Fig.
5A). The immunolocalization of CYP2J protein(s) to regions
of the nephron where energy-dependent absorption and
secretion take place has potentially important functional implications
given the well known effects of EETs and HETEs in modulating fluid and
electrolyte transport in the renal proximal tubules and collecting
ducts (6, 7, 15-19, 22). To our knowledge, this report is the first to
localize expression of a P450 epoxygenase to these portions of the
nephron.
Localization of CYP2J5 mRNA by in Situ Hybridization--
To
ascertain the distribution of CYP2J5 mRNAs within the kidney, we
performed in situ hybridization of paraformaldehyde-fixed, paraffin-embedded mouse kidney sections with the
35S-labeled CYP2J5 antisense RNA probe. As shown in Fig.
6, CYP2J5 mRNAs were primarily
localized to the renal cortex and the outer stripe of the outer
medulla. Intense labeling was observed in both the convoluted and the
straight portions of the proximal tubules (Fig. 6, B-D). No
significant labeling was present in glomeruli, renal blood vessels, the
thin limb of Henle's loop, or the thick ascending limb.
Interestingly, distal portions of the nephron (including the distal
convoluted tubules and collecting ducts) did not significantly label
with the CYP2J5 RNA probe (Fig. 6, B and C),
despite positive staining of these regions with the anti-CYP2J2 IgG
which cross-reacts with CYP2J5 and other CYP2J proteins (Figs. 4 and
5). Hybridization of kidney sections with the sense RNA probe yielded
no appreciable staining (Fig. 6A). Therefore, in
situ hybridization confirms that, like CYP2J protein(s), CYP2J5
mRNAs are abundant within the proximal tubules; however, the CYP2J
immunostaining observed within distal nephron segments may be due to
the presence of other CYP2J proteins in this region or, alternatively,
may reflect inherent differences in CYP2J5 mRNA and protein
stability.
Regulation of CYP2J5 Expression during
Development--
Immunoblotting of microsomal fractions prepared from
kidneys of mice ages 18 days fetal to 16 weeks postnatal (adult) with the anti-CYP2J5pep IgG showed that CYP2J5 protein was present before
birth, increased gradually during the 1st week of life, reached maximal
levels at 2-4 weeks of age, decreased at 8-10 weeks of age, and
achieved adult levels by 16 weeks of age (Fig. 7). In contrast, immunoblotting of
microsomal fractions prepared from mouse livers with the anti-CYP2J5pep
IgG showed that CYP2J5 protein was undetectable prenatally and, during
the early postnatal period, first appeared at 1 week of age, remained
relatively constant from 1 to 10 weeks of age, and achieved adult
levels by 16 weeks of age (Fig. 7). Similar results were obtained using
the anti-CYP2J2 IgG that cross-reacts with CYP2J5 (data not shown).
Based upon these data, we conclude that the pattern of expression of
CYP2J5 during development is different in mouse kidney and liver.
Importantly, the age-related changes in CYP2J5 protein expression in
mouse kidney correspond to known age-related changes in rodent kidney microsomal arachidonic acid epoxygenase activity (30, 59). Furthermore,
maximal CYP2J5 levels occur at a critical time during postnatal kidney
development when the pressure-natriuresis relationship is established
and when abnormalities in renal function are demonstrable in animals
that develop hypertension (60, 61).
Measurement of Endogenous EETs in Mouse Kidney--
By using a
combination of HPLC and GC/MS techniques, we detected substantial
amounts of EETs in mouse kidney tissue. Mouse kidney contained ~250
ng of EET/g of kidney. 14,15-, 11,12-, and 8,9-EET were each present in
roughly equal amounts (31, 31, and 38% of the total, respectively).
The labile 5,6-EET suffers extensive decomposition during the
extraction and purification process used and therefore cannot be
quantified. Chiral analysis of endogenous mouse kidney EETs showed a
slight preference for (14R,15S)- and (11R,12S)-EET (optical purity, 59 and 57%,
respectively), whereas 8,9-EET was recovered from mouse kidney as a
racemic mixture. The documentation of EETs as endogenous constituents
of mouse kidney provides evidence supporting the in vivo
metabolism of arachidonic acid by the mouse kidney epoxygenase(s).
P450-derived eicosanoids have been shown to make important
contributions to integrated kidney function either by directly affecting tubular ionic transport processes, vascular tone, and cellular proliferation within the kidney or by mediating the action of
several hormones including renin, angiotensin II, and arginine vasopressin (6, 7, 15-26). Furthermore, considerable evidence has
accumulated regarding the potential role of these arachidonic acid
metabolites in the pathogenesis of hypertension in rodents and humans
(6, 7, 27-32). Despite extensive work on the relevance of P450-derived
lipid mediators to kidney physiology and pathophysiology, remarkably
little is known about the biochemical, molecular, and regulatory
properties of the renal P450 enzymes that synthesize these compounds.
Herein, we report the cDNA cloning and heterologous expression of a
new mouse P450 (CYP2J5) that is abundant in the kidney and active in
the biosynthesis of EETs and midchain HETEs. We provide immunologic
data to demonstrate that expression of CYP2J5 is regulated during
postnatal development, and we present immunohistochemical and in
situ hybridization data to show that the CYP2J5 mRNA and CYP2J
protein(s) are localized to regions of the nephron where the EETs are
known to be biologically active. Finally, we provide GC/MS data to
document that the EETs are present endogenously in mouse kidney tissue.
The recombinant CYP2J5 protein catalyzed the
NADPH-dependent metabolism of arachidonic acid to 14,15-, 11,12-, and 8,9-EETs, and 11- and 15-HETE; hence, CYP2J5 is
both an arachidonic acid epoxygenase and midchain
hydroxylase. The CYP2J5 product profile is distinct from that
previously reported for other CYP2J enzymes (14, 38, 57, 58). The
CYP2J5 product profile is also different from non-CYP2J P450s that are
known to metabolize arachidonic acid including members of the CYP1A,
CYP2B, CYP2C, CYP2E, and CYP4A subfamilies (5, 12, 33-36, 62-64).
Therefore, we conclude that CYP2J5 possesses unique enzymological properties.
The highest concentrations of P450 enzymes in mammalian tissues are
found in the liver. As a result, most studies have focused on the
molecular and biochemical characterization of hepatic heme-thiolate proteins and their regulatory properties. Despite the fact that the
P450 content of kidney microsomes is substantial (20-25% of that in
liver) (65), little is known about the renal hemoproteins. Members of
the CYP2C and CYP4A subfamilies are constitutively expressed in the
rat, rabbit, and human kidney (32-36, 64, 66). In addition, members of
the CYP1A, CYP2B, and CYP4A subfamilies are inducible in the rat and
rabbit kidney by aromatic hydrocarbons, phenobarbital, and clofibrate,
respectively (67, 68). Recent work from our laboratory shows that
CYP2J2 is constitutively expressed in human kidney; however, the
primary sites of expression of this human hemoprotein are the heart and
intestinal tract (38, 41). Work presented in this article confirms our
earlier observations that CYP2J enzymes are members of the renal P450
monooxygenase system and shows that a member of the mouse CYP2J
subfamily is expressed primarily in the kidney. To our
knowledge, this is the first report of a P450 arachidonic acid
epoxygenase that is expressed predominantly in mouse kidney.
The CYP4A enzymes, which are active in the The cellular distribution of CYP2J enzymes within the kidney may have
important functional implications. For example, the localization of
CYP2J5 to the proximal tubules suggests that CYP2J5 products may
mediate angiotensin II actions, since this portion of the nephron has
the highest density of angiotensin II receptors. In this regard,
Douglas and co-workers (15, 16) have shown that EETs mediate the
angiotensin II-induced rise in cytosolic Ca2+ and mimic the
inhibitory effect of angiotensin II on Na+ fluxes from the
lumen to the basolateral compartment in the rabbit proximal tubule.
Localization of CYP2J protein(s) to the collecting ducts suggests that
CYP2J products may be involved in arginine vasopressin action and in
ionic transport within this nephron segment. In this regard, P450
epoxygenase metabolites have been shown to inhibit the hydroosmotic
effect of arginine vasopressin, decrease net Na+
reabsorption and K+ secretion, and increase cytosolic
Ca2+ concentrations in the rabbit cortical collecting
tubules (17-19). Importantly, the observed biological effects of EETs
within the proximal tubule and cortical collecting ducts occur at
concentrations of 10 Several lines of evidence support the hypothesis that P450-derived
arachidonic acid metabolites may be involved in the pathogenesis of
hypertension as follows: (a) these eicosanoids have potent effects on vascular tone and fluid/electrolyte transport in the kidney
(6, 7, 15-26); (b) the rat renal epoxygenase(s) are under
regulatory control by dietary salt, and their inhibition leads to the
development of salt-dependent hypertension (27, 28);
(c) the salt-sensitive phenotype in the Dahl rat model of
genetic hypertension is associated with altered renal metabolism of
arachidonic acid (28, 32, 59, 72); (d) alterations in kidney
P450 arachidonic acid metabolism exist during the developmental phase
of hypertension in spontaneously hypertensive rats (30, 31, 69,
73-75); (e) treatment of spontaneously hypertensive rats
with agents that deplete renal P450 normalizes blood pressure (76, 77);
and (f) the urinary excretion of epoxygenase metabolites is
increased during pregnancy-induced hypertension in humans (29). Recently, the CYP4A genotype has been found to cosegregate with the
hypertensive phenotype in Dahl salt-sensitive rats (72). These
findings, together with our recent observation that the Cyp2j cluster maps to a region of mouse Chromosome 4 that is
close to the mouse Cyp4a cluster (78), suggest that further
investigation is warranted to determine if Cyp2j5 is also a
viable candidate gene for hypertension.
Many factors are known to alter the levels of expression of hepatic
P450s including genetic polymorphism, enzyme induction by xenobiotics
and physiological manipulations, and developmental factors. Much less
is known about the factors that regulate expression of kidney P450s.
Several groups have documented age-related changes in the renal
expression of rat CYP4A subfamily P450s (69, 75, 79). The expression of
CYP4A1, CYP4A3, and CYP4A8 was maximal between 3 and 5 weeks of age,
whereas CYP4A2 increased steadily after 5 weeks of age and was maximal
in adult animals (75). None of the CYP4A isoforms were expressed at
appreciable levels before 2 weeks of age. In contrast, CYP2J5 was
present before birth and reached maximal levels at 2-4 weeks of age.
Interestingly, the age-related pattern of CYP2J5 expression was
different in kidney and liver, thus suggesting that tissue-specific
mechanisms may govern the expression of this hemoprotein during
development. Transcriptional activation of liver CYP2E1 during early
postnatal development is coincident with specific demethylations in the 5'-end of the CYP2E1 gene which are thought to influence
chromatin accessibility to transcription factors (80). Whether similar mechanisms also underlie the regulation of Cyp2j5 gene
expression during development are unknown. The developmental pattern of
CYP2J5 protein expression in mouse kidney parallels known age-related changes in rodent kidney microsomal arachidonic acid epoxygenase activity (30, 59). Importantly, maximal CYP2J5 protein levels occur
during a critical time of postnatal kidney development when the
pressure-natriuresis relationship is established and when abnormalities
in renal function exist among animals who subsequently develop
hypertension (60, 61). Although these temporal relationships are
intriguing, they do not prove that the regulation of Cyp2j5 gene expression during this important developmental period impacts on
renal function, maintenance of the pressure-natriuresis relationship, or blood pressure.
It is difficult to determine the quantitative contribution of CYP2J5 to
renal arachidonic acid metabolism given that specific CYP2J5 inhibitors
are not available, the CYP2J antibodies that have been developed are
not immunoinhibitory, and CYP2J5 knock-out mice have not yet been
generated. However, several lines of evidence support the hypothesis
that CYP2J5 plays a major role in renal EET biosynthesis. First, CYP2J5
is present at high levels in mouse kidney. Other mouse P450
epoxygenases, including members of the CYP2C subfamily, appear to be
less abundant in the kidney.4
Second, CYP2J5 is localized to regions of the nephron where EETs are
known to be produced. Third, age-related changes in CYP2J5 protein
expression in mouse kidney correspond to known age-related changes in
kidney microsomal arachidonic acid epoxygenase activity. Fourth, CYP2J5
is active in the metabolism of arachidonic acid and has kinetic
parameters that are favorable when compared with other renal P450s that
metabolize this substrate. For example, CYP2E1 and CYP4A11 have
apparent Km values of 228 and 62 µM,
respectively (81, 82). Finally, CYP2J5 is more active in the metabolism
of arachidonic acid than any other endogenous or xenobiotic substrate
we have tested including retinoic acid, testosterone, diclofenac,
bufuralol, and various aromatic and heterocyclic
amines.5
In summary, we report the cDNA cloning and heterologous expression
of CYP2J5, a new mouse P450 that is abundant in the kidney and active
in the biosynthesis of EETs and midchain HETEs. We show that this
enzyme is regulated during postnatal development and that regulation is
tissue-specific. We also demonstrate, using immunohistochemical
techniques and in situ hybridization, that CYP2J5 mRNA
and CYP2J protein(s) are primarily localized to regions of the nephron
where EETs have direct effects on fluid/electrolyte transport and are
known to mediate the actions of hormones such as angiotensin II. We
conclude that CYP2J5 is an enzymologically distinct, developmentally
regulated, heme-thiolate protein that is highly localized to specific
nephron segments and contributes to the oxidation of endogenous renal
arachidonic acid pools. In light of the well documented effects of
epoxyeicosatrienoic acids in modulating renal tubular transport
processes, we postulate that CYP2J5 products play important functional
roles within the kidney.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-terminal alcohols of arachidonic acid (16-, 17-, 18, 19-, and 20-HETE) (3-5). A particular interest in the epoxygenase
reaction has developed because the EETs are endogenous constituents of
numerous tissues and because they possess a myriad of potent biological
activities (3-7). For example, the EETs have been shown to control
peptide hormone secretion in the pancreas, pituitary, and hypothalamus
(8, 9), regulate vascular tone in the intestine and brain (10, 11),
affect ion transport and airway smooth muscle tone in the lung (12,
13), and improve functional recovery following global ischemia in the
heart (14).
/
-1 hydroxylation (33). Immunochemical studies
suggested that CYP2C isoforms were responsible for some of the
arachidonic acid epoxygenase activity present in rat and rabbit kidney
microsomal fractions (27, 33). Work by Karara et al. (34)
and Imaoka et al. (35) demonstrated that CYP2C23 was highly
expressed in rat kidney and active in the regio- and
stereoselective epoxidation of arachidonic acid (34, 35). CYP2C8,
cloned from a human kidney cDNA library, was also an active
arachidonic acid epoxygenase; however, this P450 was expressed at
relatively low levels in human kidney (36, 37). More recently, our
laboratory identified a human P450 of the CYP2J subfamily that was
highly expressed in a number of extrahepatic tissues including the
kidney and active in the metabolism of arachidonic acid to EETs (38).
However, studies on the regulation, importance, and functional role of
this new human P450 epoxygenase in the kidney have not been possible
because of the limited availability of fresh, histologically normal and
abnormal human kidney tissues. Herein, we report the cDNA cloning
and heterologous expression of CYP2J5, a new mouse P450 that is
abundant in the kidney and active in the epoxidation and midchain
hydroxylation of arachidonic acid. We show that this heme-thiolate
protein is regulated during development and in a tissue-specific
fashion. We further demonstrate, using immunohistochemical techniques
and in situ hybridization, that CYP2J5 mRNA and protein
are localized to sites within the nephron where the EETs are known to
affect fluid/electrolyte transport and mediate hormonal action.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP and
[1-14C]arachidonic acid were purchased from NEN Life
Science Products. Restriction enzymes were purchased from New England
Biolabs (Beverly, MA). PCR reagents including AmpliTaq® DNA
polymerase were purchased from Perkin-Elmer. Triphenylphosphine,
-bromo-2,3,4,5,6-pentafluorotoluene,
N,N-diisopropylethylamine, N,N-dimethylformamide,
and diazald were purchased from Aldrich. All other chemicals and
reagents were purchased from Sigma unless otherwise specified.
-aminolevulinic acid and iron citrate (100 µM each).
Cells co-expressing recombinant CYP2J5 and CYPOR were harvested 72 h after infection, washed twice with phosphate-buffered saline, and
were used to prepare microsomal fractions by differential
centrifugation at 4 °C as described previously (12). P450 content
was determined spectrally according to the method of Omura and Sato
(40) using a Shimadzu UV-3000 dual-wavelength/double-beam
spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD).
CYP2J5 protein was also expressed without CYPOR in Sf9 cells
using the pBlueBacIV expression vector (Invitrogen, San Diego, CA).
Sf9 cells expressing recombinant CYPOR but not P450 were
prepared as described (39).
-32P]dATP by nick translation using
Escherichia coli polymerase I. The oligonucleotide probe was
end-labeled using T4 polynucleotide kinase and
[
-32P]ATP. Northern blot results were confirmed
by PCR amplification of reversetranscribed mouse RNAs using the
GeneAmp® RNA PCR Kit (Perkin-Elmer). The following JM-6
sequence-specific oligonucleotides were used: forward primer,
5'-ATCAGAGAAGCGAAAAGAATGTAG-3', corresponding to nucleotides 1549-1572
of the JM-6 cDNA; reverse primer, 5'-CCATTTCCTCTGATTCTGACTCAT-3',
complementary to nucleotides 1810-1833 of the JM-6 cDNA. Reverse
transcription was performed with 1 µg of total RNA in a buffer
containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl,
5 mM MgCl2, 2.5 µM random
hexamers, 1 mM each of dGTP, dATP, dTTP, and dCTP, and 50 units of Moloney murine leukemia virus reverse transcriptase incubated
at 42 °C for 1 h. The PCR amplifications were performed in the
presence of 2 mM MgCl2, 0.15 µM
forward and reverse primers, and 2.5 units of AmpliTaq®
DNA polymerase. Following an initial incubation for 105 s at
95 °C, samples were subjected to 35 cycles of 15 s at 95 °C
and 30 s at 62 °C. The PCR products were electrophoresed on
1.2% agarose gels containing ethidium bromide. The following
-actin
sequence-specific primers were used to control for the quality and
amount of RNA: forward primer, 5'-GAGCTATGAGCTGCCTGACG-3', corresponding to nucleotides 776-795 of the mouse
-actin cDNA; reverse primer, 5'-AGCACTTGCGGTGCACGATG-3', complementary to
nucleotides 1166-1185 of the mouse
-actin cDNA.
-35S]UTP. The 35S-labeled probes were
then subjected to alkaline hydrolysis. In situ hybridization
was performed on 4% paraformaldehyde-fixed, paraffin-embedded mouse
kidney sections as described previously (50). Briefly, 6-7-µm
sections were deparaffinized, refixed in paraformaldehyde, treated with
proteinase K (20 µg/ml), washed with phosphate-buffered saline,
refixed in paraformaldehyde, treated with triethanolamine plus acetic
anhydride (0.25% v/v), and then dehydrated with 100% ethanol. Sense
or antisense RNA probes were hybridized to the sections at 55 °C for
approximately 18 h. After hybridization, the sections were washed
once in 5× SSC plus 10 mM
-mercaptoethanol for 30 min
and once more in 50% formamide, 2× SSC, and 100 mM
-mercaptoethanol for 60 min at 65 °C. After two additional washes
in 10 mM Tris-Cl, 5 mM EDTA, 500 mM
NaCl (TEN) at 37 °C, sections were treated with RNase A (10 µg/ml) at 37 °C for 30 min, followed by another wash in TEN at 37 °C. Sections were then washed twice in 2× SSC and twice more in 0.1× SSC
at 65 °C. Slides were dehydrated with graded ethanol containing 300 mM ammonium acetate, dipped in photoemulsion (Ilford K5,
Knutsford, Cheshire, UK) diluted 1:1 with 2% glycerol, and exposed for
4-5 days at 4 °C. After developing in Kodak D-19,
slides were counterstained with hematoxylin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequences for CYP2J5. The
putative heme-binding peptide is underlined, and the
conserved residues are in bold. An asterisk
marks the termination codon. The location of the peptide used to
prepare the anti-CYP2J5pep IgG is enclosed within a
box.
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Fig. 2.
Metabolism of arachidonic acid by recombinant
CYP2J5. A, reverse-phase HPLC chromatogram of the
organic soluble metabolites generated during an incubation of
Sf9 insect cell microsomes containing recombinant CYP2J5 and
CYPOR with [1-14C]arachidonic acid. Chromatogram is
representative of 15 separate experiments. B, reverse-phase
HPLC chromatogram of the organic soluble metabolites generated during
incubation of Sf9 insect cell microsomes containing CYPOR but
not containing P450 with [1-14C]arachidonic acid. Results
are representative of 5 separate experiments. C, plot of
reaction velocity versus substrate concentration. The
arachidonic acid concentration was varied from 5 to 100 µM, and reactions were allowed to proceed for 2-3 min to
ensure that the quantitative assessment of the rates of product
formation reflected initial rates. D, Lineweaver-Burk
transformation of the data shown in C. The apparent
Km and Vmax values were
derived by fitting the results to the Michaelis-Menten equation.
Results are representative of 7 separate experiments.
Regio- and stereochemical composition of EETs produced by recombinant
CYP2J5
-actin confirmed that the observations
were not due to differences in the quality or amount of RNA used (Fig.
3B). Thus, based upon the Northern analysis and RNA PCR, we
conclude that CYP2J5 mRNA is primarily present in mouse kidney,
expressed at lower levels in mouse liver, and absent from other mouse
tissues.
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Fig. 3.
Tissue distribution of CYP2J5 mRNAs by
Northern analysis and RNA PCR. A, total RNA (25 µg)
isolated from various mouse tissues was denatured and electrophoresed
in a 1.2% agarose gel containing 2.2 M formaldehyde. After
capillary-pressure transfer to Hybond-N+ membranes, the
blot was hybridized with the cloned 1.886-kb CYP2J5 cDNA labeled
with [ -32P]dATP by nick translation. Top,
autoradiograph of blot after 24-h exposure time. Bottom,
ethidium bromide-stained gel before transfer. B, total RNA
(1 µg) was reverse-transcribed using random hexamers and Moloney
murine leukemia virus reverse transcriptase as described under
"Experimental Procedures." PCR amplifications were then performed
using either CYP2J5 or mouse
-actin sequence-specific
oligonucleotide primers and AmpliTaq® DNA polymerase as
described. The PCR products were electrophoresed on 1.2% agarose gels
containing ethidium bromide. Top, ethidium bromide-stained
gel of DNAs amplified using CYP2J5-specific primers. Bottom,
ethidium bromide-stained gel of DNAs amplified using
-actin-specific
primers.
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Fig. 4.
Tissue-specific expression of CYP2J5 by
protein immunoblotting. A, microsomal fractions
prepared from uninfected Sf9 insect cells or insect cells
infected with recombinant CYP2J5 or CYP2J6 baculovirus stock (0.5 pmol
of P450/lane) were electrophoresed on SDS-10% polyacrylamide gels and
the resolved proteins transferred to nitrocellulose membranes.
Membranes were immunoblotted with the anti-human CYP2J2 IgG and goat
anti-rabbit IgG conjugated to horseradish peroxidase as described under
"Experimental Procedures." The immunoreactive proteins were then
visualized using the ECL detection system and autoradiography.
B, purified recombinant CYP2J2, CYP2A4, CYP2A5, CYP2C29,
CYP2C37, CYP2C38, CYP2C29, or CYP2C40, microsomal fractions prepared
from Sf9 cells infected with recombinant CYP2J5 or CYP2J6
baculovirus stock (0.5 pmol P450/lane), or microsomes prepared from
uninfected Sf9 cells were electrophoresed, transferred to
nitrocellulose, and immunoblotted with the anti-CYP2J5pep IgG as
described. C, microsomal fractions prepared from mouse
stomach, small intestine, muscle, lung, liver, kidney, heart, colon,
brain (25 µg of microsomal protein/lane), or Sf9 cells
infected with recombinant CYP2J5 or CYP2J6 baculovirus stock (0.25 pmol
P450/lane) were electrophoresed, transferred to nitrocellulose, and
immunoblotted with the anti-CYP2J5pep IgG as described.
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Fig. 5.
Immunohistochemical localization of CYP2J in
mouse, rat, and human kidney. A, mouse kidney section
stained with preimmune IgG showing absence of positive immunostaining
(negative control). B, adjacent mouse kidney section stained
with anti-human CYP2J2 IgG showing positive immunostaining (brown
color) in proximal convoluted tubules in the cortex (C)
and the straight segments of the proximal tubules in the outer stripe
(O) of the outer medulla. Tubules in the inner stripe
(I) of the outer medulla are largely negative for CYP2J
immunoreactivity. C, higher magnification of central portion
of B. D, higher magnification of upper
left portion of C showing strong positive staining in
proximal convoluted tubules, minimal staining in distal tubules
(asterisks), and negative staining in an arcuate artery
(A). E, rat kidney section stained with
anti-human CYP2J2 IgG showing positive brown immunostaining of the
collecting ducts in the renal papilla. F, renal papilla
(P) from another rat showing positive immunostaining of
collecting ducts and pelvic transitional epithelium
(arrowheads). G, higher magnification of the
large renal artery shown in E demonstrating an absence of
CYP2J immunostaining in both vascular smooth muscle and endothelium.
H, human kidney section showing positive immunostaining in
proximal convoluted tubules in renal cortex with minimal staining of
distal tubules (asterisks) and negative staining in an
arcuate artery (A). Magnifications × 33 (A,
B, and E), 82 (C and F), and 164 (D, G, and H).
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Fig. 6.
Distribution of CYP2J5 mRNA in mouse
kidney by in situ hybridization. A,
mouse kidney section stained with an 35S-labeled sense
CYP2J5 RNA probe showing absence of labeling (negative control).
B, adjacent mouse kidney section stained with an
35S-labeled antisense CYP2J5 RNA probe showing intense
labeling in the renal cortex and the outer stripe of the outer medulla.
C, higher magnification of B showing labeling in
both the convoluted and the straight portions of the proximal tubules.
D, higher magnification of C showing intense
staining throughout the proximal tubules but no labeling in glomeruli
or distal tubules. Magnifications × 15 (A and
B), 33 (C), and 82 (D).
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Fig. 7.
Expression of CYP2J5 during development.
Microsomal fractions prepared from kidneys and livers of mice ages 18 days fetal to 16 weeks postnatal (adult) were electrophoresed on
SDS-10% polyacrylamide gels, transferred to nitrocellulose, and
immunoblotted with the anti-CYP2J5pep IgG as described under
"Experimental Procedures." Each microsomal sample represents kidney
and liver tissues pooled from 3 to 10 animals. The results shown are
representative of three separate experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-terminal hydroxylation
of arachidonic acid to 20-HETE, are expressed at highest levels in
renal proximal tubules, although CYP4A expression has also been
described in renal microvessels (64, 68, 69). The renal distribution of
the CYP4A isoforms correlates well with the distribution of renal
arachidonic acid
-hydroxylase activity, which is also highly
concentrated in the proximal tubules (69, 70). This suggests that the
CYP4A enzymes are the main P450s that biosynthesize 20-HETE within the
kidney. In contrast to the localization of 20-HETE formation,
epoxygenase activity is distributed throughout the entire nephron with
the highest levels of EETs present in proximal tubules and collecting
ducts (70). The localization of CYP2J5 mRNA and protein to proximal
tubules, together with the demonstration that recombinant CYP2J5 is an
arachidonic acid epoxygenase, suggests a role for this enzyme in the
biosynthesis of EETs within this portion of the nephron. Interestingly,
we also observed CYP2J immunostaining in distal nephron segments, most
prominently within the collecting ducts. We are unaware of previous
reports documenting expression of a P450 within this region of the
nephron. Prostaglandin synthesis is known to occur in collecting ducts,
and the renal biological actions of these cyclooxygenase-derived
eicosanoids are well described (71). The EETs have been shown to
stimulate prostaglandin synthesis in primary cultures of rabbit
cortical collecting ducts and to release vasodilator prostaglandins in
the isolated-perfused rabbit kidney (19, 25). Thus, CYP2J products
could potentially modulate prostaglandin action within distal nephron
segments of the kidney.
11 to 10
6
M. These levels are well within the physiologically
relevant concentration range based upon estimates of kidney EET
concentrations reported herein and described by others (23,
27-29).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Joyce Goldstein for providing purified preparations of CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 and to Dr. Masahiko Negishi for providing CYP2A4 and CYP2A5. We also thank Drs. Richard Philpot and Robert Langenbach for their helpful comments during preparation of this manuscript.
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FOOTNOTES |
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* These studies were supported in part by National Institutes of Health P01-DK38226 (to M. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U62294.
** To whom correspondence should be addressed: Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1169; Fax: 919-541-4133; E-mail: zeldin{at}niehs.nih.gov.
2 The new sequence that is reported in this paper was submitted to the Committee on Standardized Cytochrome P450 Nomenclature and has been designated CYP2J5.
3 J. Ma and D. C. Zeldin, unpublished observations.
4 D. C. Zeldin and J. A. Goldstein, unpublished observations.
5 C. Crespi, F. Kadlubar, and D. C. Zeldin, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: EET, cis-epoxyeicosatrienoic acid; DHET, vic-dihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HPLC, high performance liquid chromatography; P450, cytochrome P450; CYPOR, NADPH-cytochrome P450 oxidoreductase; kb, kilobase; PCR, polymerase chain reaction; GC/MS, gas chromatography/mass spectrometry.
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