The CYP monooxygenase, CYP2B12, is the first
identified skin-specific cytochrome P450 enzyme. It is characterized by
high, constitutive expression in an extrahepatic tissue, the sebaceous glands of cutaneous tissues. It is expressed exclusively in a subset of
differentiated keratinocytes called sebocytes, as demonstrated by
Northern blot analysis, in situ hybridization, and
polymerase chain reaction. The onset of its expression coincides with
the morphological appearance of sebaceous glands in the neonatal rat. Recombinant CYP2B12 produced in Escherichia coli epoxidizes
arachidonic acid to 11,12- and 8,9-epoxyeicosatrienoic acids (80 and
20% of total metabolites, respectively). The identification of
arachidonic acid as a substrate for this skin-specific CYP
monooxygenase suggests an endogenous function in keratinocytes in the
generation of bioactive lipids and intracellular signaling.
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INTRODUCTION |
Mammalian CYP monooxygenases (CYP gene superfamily)
oxidatively metabolize small, hydrophobic compounds, including
steroids, sterols, fatty acids, fat-soluble vitamins, drugs, and toxins (1). These enzymes are expressed in most mammalian tissues, where they
function in the biosynthesis or catabolism of endogenous and exogenous
substrates. Members of the CYP2B gene subfamily are often
called phenobarbital-inducible cytochrome P450 enzymes because
phenobarbital treatment results in transcriptional activation of
certain hepatic CYP2B genes (2). A typical CYP2B
monooxygenase has 16-hydroxylase activity with androgenic steroids,
such as testosterone, and is expressed in the liver and organs such as kidney, lung, and testis (2).
CYP2B12 has long been known to be an unusual member of the
CYP2B gene subfamily. The gene encoding this enzyme
(CYP2B12) was discovered by Atchison and Adesnik (3).
Originally called gene 4, it was one of several partial genomic clones
that proved the multiplicity of the CYP2B gene subfamily.
Gene 4 expression was not detected in the liver or other organs but
rather in preputial glands (4), the large, paired sebaceous glands
beneath the genital skin in rodents. This finding led Friedberg
et al. (4) to isolate and characterize a full-length
cDNA corresponding to gene 4 from a preputial gland cDNA
library. More than 10 years after its discovery, the novel cytochrome
P450 named CYP2B12 is now recognized as the first identified
skin-specific cytochrome P450 enzyme.
CYP2B12 has remained enigmatic not only for its restricted
tissue-specific expression but also because its substrate was not identified. No activity could be demonstrated with preputial gland microsomes using typical CYP2B substrates, such as testosterone, androstenedione, benzphetamine, and ethoxy-, pentoxy-, and
benzoyresorufin (2, 4). Our preliminary investigations of a novel CYP2B in mouse skin1 having
sequence homology to rat CYP2B12 renewed interest in this cutaneous
P450 enzyme. We report here that CYP2B12 is a gene product unique to
sebocytes, a subset of differentiated keratinocytes, and that
arachidonate is a substrate for CYP2B12. It is hypothesized that the
epoxyeicosatrienoic acids
(EETs)2 produced by this
enzyme may function in signal transduction pathways essential for
establishing or maintaining the differentiated phenotype of
keratinocytes in cutaneous tissues.
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MATERIALS AND METHODS |
Analysis of CYP2B12 mRNA--
Total RNA was isolated by a
modification of the method of Chomczynski and Sacchi as described (5),
with the following changes: cutaneous tissues were frozen in liquid
nitrogen, pulverized with a mortar and pestle, dissolved in guanidinium
thiocyanate solution, and sheared with a 25 gauge needle. For Northern
analysis, total RNA was size-fractionated on formaldehyde-containing
1% agarose gels and transferred by capillarity to GeneScreen Plus
membranes (DuPont). Membranes were baked at 80 °C and stained with
methylene blue to assess RNA integrity. Hybridization was carried out
overnight with a 32P-labeled cDNA encoding
3'-untranslated sequences of CYP2B12 (570 bp; GenBank accession no.
X63545) or CYP2B15 (260 bp; GenBank accession no. D17349). These
fragments were generated by reverse transcription-polymerase chain
reaction (RT-PCR) from rat skin RNA using oligonucleotides 1 and 2 for
CYP2B12 or oligonucleotides 3 and 4 for CYP2B15 (Table
I). Hybridized membranes were washed stringently and exposed to autoradiographic film. For RT-PCR, an RNA
PCR kit (Perkin-Elmer) was used according to the manufacturer's instructions, with total RNA as template. Oligonucleotides contained in
exons 4 and 9 (Table I, oligonucleotides 5 and 6) were used to
determine the tissue distribution of CYP2B12 transcripts. Thus, products amplified from RNA (920 bp) or genomic DNA could be
distinguished. The PCR products were size-fractionated on agarose gels
and visualized with ethidium bromide. To survey expression of putative
CYP epoxygenases in sebaceous tissues by RT-PCR, the following primers
(Table I) were used: 1) CYP1A subfamily-specific (oligonucleotides 7 and 8); 2) CYP2B subfamily-specific (oligonucleotides 9 and 10); and 3)
CYP3A subfamily-specific (oligonucleotides 11 and 12). Two primer pairs
were used in the same reaction to achieve consensus for mammalian CYP2C
subfamily members (oligonucleotides 13-16). Finally, two pairs of
oligonucleotides were used to detect expression of CYP2E1
(oligonucleotides 17 and 18) and CYP2J3 (oligonucleotides 19 and
20).
In Situ Hybridization--
Sprague-Dawley rats were obtained
from Harlan Sprague-Dawley and handled according to protocols approved
by Vanderbilt University. Whole skin samples were immersion-fixed for
24 h in 4% buffered paraformaldehyde. Tissue preparation and
in situ hybridization were done as described (5).
[35S-UTP]cRNAs were produced by in vitro
transcription from a 570-bp fragment of CYP2B12 (described above).
Specific hybridization was distinguished by comparing silver grain
development produced by sense and antisense cRNAs applied to
consecutive sections on the same slide.
Expression of CYP2B12 in Escherichia coli--
A CYP2B12
cDNA (4) (pBS2B12) was modified and subcloned into pKK233-2
(Amersham Pharmacia Biotech) to make the expression construct pKK2B12,
similar to the method of John et al. (6), who used pKK233-2
successfully to express several CYP2B enzymes in E. coli.
Briefly, a 1.6 kb fragment of pBS2B12, containing all but 83 bp of
5'-coding sequence, was isolated by restriction with XbaI,
digestion with Klenow fragment of DNA polymerase I to produce a blunt
end, and then restriction with NcoI. This fragment was
purified and ligated into pKK233-2. Before replacing the missing 83 bp
5'-end of CYP2B12, the native N-terminal sequence MEFGVLL was changed
to MALLLAV by PCR mutagenesis as described (6, 7), using
oligonucleotides 21 and 22 (Table I). The PCR product containing this
N-terminal modification was restricted with NcoI, producing
the missing 83-bp fragment, and ligated into pKK2B12 construct above.
By DNA sequence analysis, this final pKK2B12 expression construct
contained the entire open reading frame of CYP2B12, the MALLLAV
N-terminal modification, and 40 bp of untranslated sequence beyond the
stop codon. Competent E. coli cells (Topp3 strain;
Stratagene) were transformed with pKK2B12. Media and conditions for
expression were as described (8). The cultures were harvested after
48-72 h at 30 °C at 200 rpm. Typical yields were
500 nmol of
P450/liter of culture by whole cell spectral assay. Whole cells or
cellular fractions were diluted in 50 mM potassium
phosphate buffer containing 20% glycerol to measure CO difference
spectra (9). Bacterial membranes solubilized with CHAPS detergent were prepared as described (6) for metabolism studies.
Metabolism Studies--
CYP2B12 activity was assayed as
described (10). Briefly, CHAPS-solubilized E. coli membranes
were incubated at 30 °C with purified rat NADPH-cytochrome P450
reductase (molar ratio of P450:reductase, 1:5) in the presence of
L-
-dilauroyl-sn-glycero-3-phosphocholine and
a NADPH regenerating system. After 1 min equilibration at 30 °C,
[1-14C]arachidonic acid was added (50 µCi/µmol; final
concentration, 50-75 µM), and the reaction was initiated
with NADPH (final concentration, 1 mM). At various time
points, the reaction mixtures were extracted with ethyl ether, and the
organic extracts were resolved by reversed phase HPLC with on-line
liquid scintillation detection (Radiomatic Flo-One
-detector;
Radiomatic Instruments). For product structural analyses, CYP2B12 was
reconstituted in the presence of NADPH and 70 µM
[1-14C]arachidonic acid (0.2-0.4 µCi/µmol).
Radioactive fractions with retention times corresponding to those of
authentic 5,6-, 8,9-, and 11,12-EET were collected, and individual EET
regioisomers were resolved by normal phase HPLC as described (10, 11). Purified EET regioisomers were converted to the corresponding pentafluorobenzyl (PFB) esters, and their optical antipodes were resolved by chiral phase HPLC as described (11, 12). Aliquots of
purified EET-PFB regioisomers were analyzed by negative ion chemical
ionization-gas chromatography-mass spectroscopy (NICI/GC/MS) as
described (11). Endogenous EET was measured in freshly isolated rat
preputial and clitoral glands. Homogenized tissue was extracted in the
presence of triphenylphosphine and analyzed as described by NICI/GC/MS
(11). The tissue content of 8,9-, 11,12-, and 14,15-EET was estimated,
but 5,6-EET is too labile to be detected (11).
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RESULTS |
Skin-specific Expression of CYP2B12--
CYP2B12 is a cutaneous
monooxygenase expressed constitutively in rat preputial gland (the
tissue source from which a full-length CYP2B12 cDNA was isolated)
(4). Among cutaneous tissues, the single most enriched source of
CYP2B12 mRNA is the pair of preputial (male) or clitoral (female)
(Fig. 1) glands in the rat. These large,
paired sebaceous glands in the genital skin are readily dissected free
of surrounding cutaneous tissue and subcutaneous fat. As such, they are
composed nearly exclusively of keratinocytes at different stages of
differentiation. Although CYP2B12 transcripts were detectable in all
cutaneous tissues examined, transcript abundance varied. Among samples
of full-thickness skin, tail and anogenital skin showed a relatively
high level of expression. Lower levels were found in the abdominal
skin, ear, and foot pad. For these latter samples, visible bands of the
expected molecular size appeared after longer exposure times than that
shown in Fig. 1. The differences in hybridization signal intensity
among cutaneous tissues appeared to be proportional to the relative
size and abundance of sebaceous glands observed morphologically.

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Fig. 1.
CYP2B12 transcripts in cutaneous tissues of
adult rats detected by Northern blot analysis. RNA was isolated
from whole skin (ventral abdominal, foot pad, tail, ear, and
anogenital), liver, and isolated clitoral (sebaceous) and adrenal
glands. Longer exposure times were required to visualize bands in the
lanes showing ventral abdominal skin, ear, and foot pad RNA samples. No
signal was detected for liver or adrenal RNAs. The positions of 18S and
28S ribosomal RNAs are indicated.
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By Northern blot analysis, CYP2B12 mRNA was undetectable in the
liver, lung, kidney, testis, adrenal gland, and intestinal mucosa (4)
(Fig. 1). The tissue-specificity of CYP2B12 gene expression
was investigated further by the highly sensitive RT-PCR technique.
Using total RNA as template, no product of the expected size could be
detected in the following noncutaneous tissues of the rat: liver,
kidney, lung, heart, adipose, spleen, uterus, ovary, adrenal glands,
and tongue (not shown). The positive control, clitoral gland RNA, was
amplified only when reverse transcriptase was included in the reaction
mixture. These results indicate that CYP2B12 is truly a cutaneous
monooxygenase.
Cell-specific Expression of CYP2B12 in Differentiated
Keratinocytes--
In situ hybridization was used to
identify the cutaneous cell type(s) expressing CYP2B12. CYP2B12
antisense cRNA hybridized specifically to preputial gland acini
containing sebaceous keratinocytes. Acini are shown in Fig.
2, A and B,
surrounding a central collecting duct, which directs the flow of sebum
to the skin surface. At higher magnification (not shown), silver grains
localized specifically to sebocytes, the terminally differentiated
keratinocytes in this gland, but not to undifferentiated keratinocytes
at the periphery of individual acini or any other cutaneous tissue.
Reproducible epidermal expression was not observed in situ,
although a very low level of expression in epidermal keratinocytes
cannot be ruled out. CYP2B12 transcripts also localized to sebocytes in
other types of sebaceous glands, for example, the Meibomian glands in the eyelid (not shown), anal glands in anogenital skin (not shown), and
sebaceous glands associated with hair follicles. In hair follicles (Fig. 2, C and D), CYP2B12 antisense cRNA
localized to sebocytes but not to keratinocytes making up the hair
follicle root sheath, epidermis, or other cutaneous structures. During
postnatal development, the onset of expression of CYP2B12 coincides
with the morphological appearance of well-developed sebaceous glands in
both sexes (not shown). These data demonstrate that expression of this
cutaneous cytochrome P450 enzyme is restricted to a subset of
differentiated keratinocytes found in sebaceous glands.

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Fig. 2.
CYP2B12 transcripts localized in
situ to the differentiated keratinocytes of sebaceous
glands. [35S]cRNA encoding CYP2B12 hybridized with
rat anogenital skin. Tissue morphology is shown in brightfield
(A and C). Specific hybridization with antisense
cRNA is represented by silver grains, which appear white in
darkfield (B and D); results for sense cRNA are
not shown. A and B, preputial (sebaceous) gland
from an 11-day-old male is indicated (arrow) beneath the
dermis. C and D, sebaceous glands associated with
hair follicles from an adult female. The follicle on the
left shows hair fiber and hair root sheath but only the
periphery of the associated sebaceous gland (arrow). The
follicle on the right shows the major profile of a sebaceous
gland (arrow), whereas the associated hair fiber and root
sheath are largely beneath the plane of section. Magnification:
A and B, × 25; C and D, × 50.
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Arachidonic Acid Is a Substrate for CYP2B12--
Differentiated
keratinocytes are responsible for producing the unusual lipids found in
the epidermal water permeability barrier and sebum (13). Because
CYP2B12 is expressed constitutively in lipid-laden sebocytes, it seemed
plausible that its endogenous substrate(s) would be lipids. To test
this hypothesis, recombinant CYP2B12 was produced in E. coli
after its first seven amino acids were replaced with the sequence
MALLLAV (7). The DNA sequence of this bacterial expression construct,
pKK2B12, was compared with the CYP2B12 cDNA in GenBank (4). Except
for the deliberate N-terminal modification, only one difference was
found in the nucleotide sequence (ATT
AGT) that would lead to an
amino acid change at residue 476 (Ile
Ser). At this residue, the
sequence of pKK2B12 agreed with that obtained by sequencing RT-PCR
products amplified from rat preputial gland RNA.
Reconstituted, recombinant CYP2B12 was active with
[14C]arachidonic acid as substrate. Shown in Fig.
3A, metabolism generated, in a
NADPH-dependent manner, a major radioactive fraction
eluting at 24 min. This fraction had a retention time similar to that of authentic 11,12-EET; radioactivity eluting at 34 min represents unmetabolized substrate. Production of the metabolites contained in
this fraction was specific for pKK2B12 transformants because no
metabolites were observed with membranes isolated from untransformed E. coli cells (Fig. 3B). CYP2B12 substrate
specificity was also demonstrated by its lack of catalytic activity
toward the short chain fatty acid laurate (C12:0) (not shown).
Metabolites eluting at 24 min (Fig. 3A) were pooled and
resolved by normal phase HPLC into two radioactive fractions having the
same retention times as authentic 11,12- and 8,9-EET (Fig.
4). These two EETs were the sole reaction
products observed, with 11,12- and 8,9-EET accounting for 80 and 20%
of total metabolites, respectively. Table
II shows the chiral analysis of these
products. Both EETs were produced in an enantioselective fashion. The
predominant enantiomers generated by CYP2B12 were
11(S),12(R)- and
8(R),9(S)-EET. The stereochemistry of 11,12-EET
in Table II is similar to that found endogenously in rat liver (14),
further evidence that the function of CYP2B12 is metabolism of
arachidonic acid.

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Fig. 3.
Arachidonic acid metabolites generated by
recombinant CYP2B12, resolved by reversed phase HPLC.
CHAPS-solubilized membranes were prepared from E. coli
transformed with pKK2B12 (A) or untransformed cells
(B) and incubated with [14C]arachidonic acid
for 20 min at 30 °C. The radioactivity eluting at 34 min represents
unmetabolized [14C]arachidonic acid. The arrow
in A indicates radioactive metabolites having the same
retention time as authentic 11,12-EET standard. No metabolites were
observed with membranes isolated from untransformed E. coli
cells (B).
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Fig. 4.
Resolution of EET regioisomers generated by
recombinant CYP2B12. The metabolites eluting at 24 min in Fig.
3A were resolved by normal phase HPLC into two EET
regioisomers that eluted with retention times identical to authentic
11,12- and 8,9-EET standards.
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Table II
Regio- and stereochemical composition of epoxyeicosatrienoic acids
produced from [14C]arachidonic acid by recombinant CYP2B12
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Structures of the 11,12- and 8,9-EET metabolites were confirmed by
NICI/GC/MS. A mass spectral fragmentation pattern for the 11,12-EET-PFB
is shown (Fig. 5). Major negative ions
were as follows: m/z 319 (loss of the PFB from the
molecule), m/z 321 (loss the PFB from the isotopically
labeled molecule), and m/z 301 and 303 (loss of PFB,
H2O, and oxygen). Similar results were obtained for the
purified 8,9-EET produced by CYP2B12 (not shown). The fragmentation
patterns are consistent with those of authentic EETs (11). These data
demonstrate for the first time that arachidonic acid is a substrate for
CYP2B12 and that the regiospecific metabolism by CYP2B12 is unlike most
arachidonate (CYP) epoxygenases, which typically produce
several EET regioisomers as major products (14).

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Fig. 5.
Mass spectral fragmentation pattern for
11,12-EET generated by recombinant CYP2B12. For product structural
analysis, CYP2B12 was reconstituted in the presence of NADPH and 70 µM [1-14C]arachidonic acid (0.2-0.4
µCi/µmol). Purified 11,12-EET-PFB was analyzed by NICI/GC/MS. The
major anion m/z 319 is diagnostic for loss of PFB from the
molecule; that at m/z 321 is derived by loss of PFB from the
isotopically labeled molecule.
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Production of Endogenous EET in Sebaceous Tissue--
Endogenous
EETs were measured in freshly isolated rat preputial and clitoral
glands using a quantitative mass spectral assay (11). From 5.7 g
of sebaceous tissue, 202 ng of EET were recovered; 49% (98 ng) was
14,15-EET, 25% (51 ng) was 11,12-EET, and 26% (52 ng) was 8,9-EET.
These results indicate the presence of another epoxygenase(s) because
recombinant CYP2B12 produced 11,12-EET nearly exclusively. Although the
fraction of EETs generated by CYP2B12 in sebaceous tissue cannot be
determined, one possibility is that CYP2B12 contributes a major
fraction of 11,12-EET. However, another epoxygenase(s) must be
responsible for generating a major fraction of total EETs.
The identity of other CYP epoxygenases in rat preputial/clitoral glands
was explored using RT-PCR and DNA sequencing, whereas the relative
abundance of specific transcripts was evaluated by Northern blot
analysis or in situ hybridization. CYP1A and CYP3A subfamily-specific oligonucleotides yielded no PCR product. CYP2C subfamily-specific oligonucleotides yielded very faint bands of predicted size, negligible compared with the large amount of product from CYP2B subfamily-specific primers (not shown). CYP2C PCR products were not sequenced because of their low abundance. Sequencing of CYP2B
PCR products indicated the presence of CYP2B1/2B2 mRNAs, but the
levels in rat skin were too low to be detected by in situ hybridization. In situ hybridization is more sensitive than
Northern analysis. In addition to CYP2B12, which is a major transcript (Fig. 1), and low levels of CYP2B1/2B2, CYP2B15 (15) was the third
transcript detected in rat sebaceous RNA. CYP2B15 is a major transcript
(Fig. 6) that appears to be expressed at
levels comparable to CYP2B12, in the same tissues. In situ
hybridization studies (not shown) show that CYP2B15 is co-expressed
with CYP2B12 in sebocytes.

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Fig. 6.
CYP2B15 transcripts in cutaneous tissues of
adult rats detected by Northern blot analysis. The membrane
represented here was prepared in parallel with that in Fig. 1, using
the same RNA samples. These included whole skin (ventral abdominal,
foot pad, tail, ear, and anogenital), liver, and isolated clitoral
(sebaceous) and adrenal glands. Longer exposure times were required to
visualize bands in the lanes showing ventral abdominal skin, ear, and
foot pad RNA samples. No signal was detected for liver or adrenal RNAs.
The positions of 18S and 28S ribosomal RNAs are indicated.
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Although the activity of CYP2B15 is not known, we believe that this
enzyme will prove to be an epoxygenase. This hypothesis is based on
unpublished data1 suggesting that CYP2B15 is orthologous to
murine CYP2B19, a novel keratinocyte-specific CYP2B in mouse skin
having epoxygenase activity with arachidonic acid. Hence, we predict
that CYP2B12 and CYP2B15 will be the major epoxygenases in rat
sebaceous tissues.
Two other CYP2 monooxygenases (CYP2E1 and CYP2J3) having epoxygenase
activity (Table III) were detected in
preputial/clitoral gland RNA by RT-PCR. Sequence analysis revealed the
presence of authentic CYP2E1 and CYP2J3 mRNAs, as well as a novel
CYP2J sequence that was 83% identical to both CYP2J3 (16) and CYP2J4
(17) (not shown). By Northern analysis, CYP2E1 and CYP2J3 mRNAs
were not detected in clitoral gland RNA after membranes were exposed to
film for 2 days (compared with <20 h for Figs. 1 and 6), indicating a
low level of constitutive expression (not shown).
In summary, these data demonstrate that CYP2B12 is a major epoxygenase
in rat sebaceous tissue, but at least one other major epoxygenase must
be present. We predict that the second epoxygenase will prove to be
CYP2B15. Furthermore, CYP1A, CYP2C, and CYP3A transcripts are low or
undetectable in rat sebaceous glands. CYP2B1/2B2, CYP2E1, and CYP2J3
transcripts are detectable, but constitutive expression is very low
compared with that of CYP2B12 and CYP2B15.
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DISCUSSION |
The expression pattern of CYP2B12 is highly unusual
compared with other CYP2B genes. First, it shows high,
constitutive expression in an extrahepatic tissue. Second, it is
expressed in a single cell type: differentiated keratinocytes. As shown
by in situ hybridization, it is restricted to a subset of
differentiated keratinocytes called sebocytes. As demonstrated by
Northern blot analysis, in situ hybridization, and RT-PCR,
the singular cell type specificity of this cutaneous cytochrome P450
has few parallels in the CYP gene superfamily. Third, the
ontogeny of CYP2B12 gene expression during postnatal
development coincides with the morphological appearance of the
sebaceous tissues in which the gene is expressed.
The function of CYP2B12 has remained obscure, not only because of its
cell type specificity but also because no substrate for CYP2B12 was
identified. Preputial gland microsomes showed no enzymatic activity
with substrates metabolized by other CYP2B enzymes (4), providing no
clues as to the physiological functions of CYP2B12. In situ
hybridization studies led us to identify sebocytes as the source of
CYP2B12 in skin, providing new insight on potential substrates and
endogenous functions. A role in some aspect of cutaneous lipid
metabolism seemed plausible because an important function of
differentiated keratinocytes in the epidermis and sebaceous glands is
the biosynthesis of specialized lipids (13). Cutaneous lipids are
essential to prevent excessive transepithelial water loss and are
important for thermoregulation in response to stress (13, 18, 19).
The successful expression of CYP2B12 in E. coli allowed the
identification of the first known substrate for this enzyme.
Recombinant CYP2B12 catalyzes NADPH-dependent epoxidation
of arachidonic acid, an important precursor of many lipid mediators
(14). Other CYP2B arachidonic acid epoxygenases are known: rabbit lung
CYP2B4 (20), a putative CYP2B4 orthologue in guinea pig lung (21), and
rat liver CYP2B1 and CYP2B2 (22). Unlike CYP2B12, these CYP2B
epoxygenases are expressed in multiple tissues, and their activities
are less regioselective. CYP2B12 produces a single major EET
regioisomer, mainly 11,12-EET, from arachidonic acid. This degree of
regioselectivity during arachidonic acid epoxidation is quite unusual
(23) (Table III). An exception is rabbit lung CYP2B4, which generates
5,6-EET as the predominant EET (20). Another distinction is that
CYP2B12 catalyzes only epoxidation of arachidonic acid, whereas CYP2B4 (20), CYP2E1 (24), CYP2J3 (16), and CYP2J4 (17), for example, generate
/
-1 alcohols or other eicosanoids as major products. Importantly,
lauric acid is not a CYP2B12 substrate.
The catalysis of eicosanoid oxidation by CYP2B12 suggests that this
enzyme may participate in signal transduction mechanisms in
keratinocytes. Although CYP2B12 exhibits a relatively high level of
constitutive expression, EET generation may be regulated by cellular
phospholipases and arachidonic acid release (14, 23). Another source of
EETs might include EETs esterified into cellular glycerophospholipids
(25, 26). Although in vivo functions for endogenous EETs
produced by CYP2B12 remain to be proven, clues may be obtained from
studies of other arachidonic acid epoxygenases. The biological
activities of EETs or their hydration products, dihydroxyeicosatrienoic
acids, involve changes in membrane permeability to Ca2+,
Na+, K+, and H+, as well as
stimulation of peptide hormone secretion and vasomodulation (14, 23,
27). In epidermal keratinocyte cultures, increases in extracellular
Ca2+ concentrations lead to increases in intracellular
Ca2+ concentration and result in keratinocyte
differentiation (28, 29). The correlation also exists in mouse
epidermis in situ. The greatest concentrations of inter- and
intracellular Ca2+ localize cytochemically to the upper
epidermal cell layer (30), the stratum granulosum, which contains the
most terminally differentiated, nucleated keratinocytes. By analogy,
sebocytes are the most terminally differentiated keratinocytes in
sebaceous glands. We hypothesize that the CYP2B12-dependent
production of 11,12-EET may be involved in establishing the
differentiated phenotype or maintaining a differentiated function of
keratinocytes in sebaceous tissues.
We thank Drs. Yasuna Kobayashi and James
Halpert for assistance in expression and purification of CYP2B12 and
Dr. Jorge Capdevila for assistance in arachidonic acid metabolism
studies and critical interpretation of the data. Support and technical
assistance from the Skin Diseases Research Center at Vanderbilt are
greatly appreciated, especially the contributions of Drs. Lillian
Nanney and Lloyd E. King, Jr., in data interpretation and consultations
in cutaneous biology.