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Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104-6048
Address all correspondence and requests for reprints to: Dr. Bernard H. Shapiro, Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, Pennsylvania 19104-6048. E-mail: shapirob{at}vet.upenn.edu
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Abstract |
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10% of normal) to hypophysectomized
male rats and neonatal administration of monosodium glutamate (MSG),
producing a similar plasma GH profile, both result in an overexpression
(200–300%) of CYP2C11 messenger RNA (mRNA), the predominant
hepatic cytochrome P450 (CYP) drug-metabolizing enzyme in adult male
rats. Coincident with the severalfold elevation in transcript level is
a modest 10–30% overexpression of CYP2C11 protein and its catalytic
activities. Using hepatic tissue from adult, neonatally MSG-treated
rats, we have cloned a variant species of CYP2C11 mRNA containing all
of the essential elements of a full-length complementary DNA, including
initiating codon, termination codon, and polyadenylase tail. In
addition, the transcript contains a 742-bp intervening sequence
(identical to the complete terminal intron) between the last and
penultimate exons, and an intron-specific oligo probe for Northern
blotting demonstrates the presence of the variant transcript in liver
of MSG-treated rats. Associated with the overexpression and intron
retention of the transcript is a 50% reduction in the nuclear splicing
capacity of the liver for model precursor CYP2C11 mRNA. It is proposed
that this splicing defect may be a consequence of the mini-GH pulses
(secreted in otherwise normal masculine plasma profiles) signaling
abnormal processing of precursor CYP2C11 mRNA to produce a substantial
portion of intron retained, nontranslatable transcript. |
Introduction |
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An individual’s CYPs are broadly divided into constituent forms, or
so-called housekeepers, continuously expressed for normal physiological
function and the inducible forms whose expression is usually dependent
upon environmental inducers. Studies identifying the regulators of the
constituent CYPs have basically been limited to the rat and, to a
lesser extent, the mouse (2). In the case of the rat,
expression of most of the dozen or more constitutive hepatic CYPs is
sexually dimorphic and is under the regulatory control of the
gender-dependent circulating profiles of GH (3, 4). Male
rats secrete GH in episodic bursts (200–300 ng/ml plasma) every 3.5
to 4 h. Between the peaks, GH levels are undetectable. In females,
the hormone pulses are more frequent and irregular and are of lower
magnitude than those in males, whereas the interpulse concentrations of
GH are always measurable (2, 3). In spite of this clear
dimorphism in hormonal secretory patterns, GH regulation of
gender-dependent CYPs is rather complex. While expression of some of
the sexually dimorphic CYPs are dependent upon exposure to the
masculine plasma GH profile, others are regulated by the feminine GH
profile, while still others respond, albeit at different levels,
depending upon their gender, to both profiles and some sex-dependent
CYP isoforms are expressed in the absence of GH. In addition to their
inductive effects, the sexually dimorphic circulating GH profiles can
be suppressive. That is, some of the isoforms are suppressed by the
masculine GH profile, others by the feminine profile, and still others,
to different degrees, by both profiles (1, 2, 3). Another
layer of complexity is observed in the fact that each sex-dependent CYP
isoform appears to be expressed or suppressed by different signaling
elements in the GH profiles. These signals have been identified as the
amplitudes, frequencies, and/or durations of the GH pulse and
interpulse periods as well as the mean plasma concentration
(5, 6, 7). The cellular mechanism by which each CYP
"discriminator" recognizes its selective signal in the GH profiles
is unknown.
CYP2C11 is the predominant CYP isoform in male rats, accounting for at least 50% of the total content of hepatic CYP (8). Whereas exposure to the feminine plasma GH profile of continuous GH secretion completely suppresses CYP2C11 expression, elimination of GH from the circulation (i.e. hypophysectomy) permits a modest expression level of 20–25% of normal (5, 6). It is, however, exposure to the masculine profile of episodic GH secretion that is solely responsible for the high levels of CYP2C11 messenger RNA (mRNA) and protein expressed in male rat liver (3, 7). More specifically, it is a requisite periodic absence of GH from the circulation observed during the interpulse periods that signal hepatic CYP2C11 transcription (9). Although essential for male-like expression levels of CYP2C7 and 2A1 (7), the high amplitude pulses secreted every 3.5–4 h and so characteristic of the masculine GH secretory profile are not required for CYP2C11 expression (7). In fact, a reduction in the GH peak heights of 90–95% in an otherwise physiological masculine profile allows for near-normal CYP2C11 protein levels and catalytic activities (7, 10). Unexpectedly, however, exposure to these mini-GH pulses induces a >200% overexpression of apparently untranslated CYP2C11 transcript (7, 10).
In the present study we have examined the effects of subnormal pulse heights in the masculine GH secretory profile on CYP2C11 expression (mRNA, protein, and catalytic activity) in two different animal models and investigated a possible explanation for the apparent uncoupling of CYP2C11 transcription and translation induced by these minisecretory pulses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In another cohort, adult male and female Sprague Dawley rats [Crl:CD(SD)BR] were hypophysectomized at 8 weeks of age by the vendor (Charles River Laboratories, Inc., Wilmington, MA). Hypophysectomized rats exhibiting no significant weight gain for the next 5–6 weeks were used in the study. These animals had no pituitaries or fragments when necropsied at the end of the study.
Hormone replacement experiments with rat GH (rGH; 1.8 IU/mg) were begun when hypophysectomized male rats were around 13 weeks of age. Periodic injections via a chronic indwelling right atrial catheter implant and controlled by an external syringe pump (11, 12) were administered at six equal intervals per day as 3-min pulses at a dose of 4 µg rGH/kg BW·injection for 7 consecutive days. The amount of rGH in each pulse was chosen to replicate 10% of the normal masculine pulse parameters. To verify the effectiveness of the pumping apparatus as well as to determine the circulating GH profiles in MSG-treated and control rats, concurrent blood samples (25 µl) were obtained at 15-min intervals from at least four catheterized rats in each treatment group. Nine-hour plasma rGH profiles were determined using a RIA with a sensitivity of 2–3 ng/ml. Procedural details and statistical validation of the assay have been reported previously (13).
The atrial catheterizations were performed 4–5 days before initiation of the rGH treatments. At the time of surgery, all hypophysectomized rats were sc implanted with osmotic pumps (Alza Corp., Palo Alto, CA) set to continuously deliver, for 14 days, T4 at a dosage (0.8 µg/h·kg BW) that produced the euthyroidism (14) required to maintain normal concentrations of NADPH-cytochrome P450 reductase, a microsomal enzyme required for the expression of P450 catalytic activities (15). MSG-treated rats exhibit a basically selective GH deficiency (16).
Hypophysectomized rats were decapitated within 2 h of the last administered rGH pulse, whereas all other rats were similarly euthanized between 0900–1000 h. Livers were quickly removed and perfused with ice-cold saline. Each liver was quickly minced; a portion reserved for mRNA determination was plunged into liquid nitrogen and subsequently stored at -70 C. The remaining minced liver was used for microsomal preparation.
CYP2C11
Hepatic CYP2C11 mRNA and microsomal CYP2C11 protein were
isolated and quantified by Northern and Western blotting, respectively,
as described previously (5, 7). Hepatic microsomal
CYP2C11-dependent testosterone 2
-hydroxylase was assayed according
to methods we reported previously (17). Data were
subjected to ANOVA, and differences were determined with t
statistics and the Bonferroni procedure for multiple comparisons.
RT-PCR
Two micrograms of total hepatic RNA isolated by a single step
guanidinium thiocyanate method (18) were reverse
transcribed using random hexamers as primers and SuperScript II reverse
transcriptase (Life Technologies, Inc., Gaithersburg, MD).
Standard PCR reactions were performed using the equivalent of 500 ng
(250 ng for the MSG-treated animals) reverse transcribed RNA, 50 pmol
of each nucleotide as primers, and 1 U Taq polymerase
(Perkin-Elmer Corp., Foster City, CA), in the recommended
buffer in a total volume of 50 µl. The primer sequences were upstream
5'-GTATCGCTGTCATCCATAC-3' (1165–1183 bp; belonging to exon 8 of
CYP2C11) and 5'-CCCCATGGCTACAGGTC-3' (629–645 bp; belonging to the
junction of exons 4 and 5 of CYP2C11) and downstream
5'-GGAAATGGGGATATGTG-3' (1577–1560 bp; belonging to exon 9 of CYP2C11)
and 5'-ATCCACGTGTTTCAGCAGCAGCAGGAGTCC-3' (954–925 bp; belonging to
exon 6 of CYP2C11) as previously reported (19).
PCR reactions were carried out in a Perkin-Elmer Corp. thermal cycler, using melting, annealing, and extension cycling conditions of 94 C for 30 sec, 56 C for 1 min, and 72 C for 1 min. All amplifications were carried out for 23 cycles. Under these conditions, all complementary DNA (cDNA) fragment amplifications were found to produce single products within a linear range of 20–26 cycles (data not shown).
Cloning and screening of full-length cDNA. From the hepatic
total RNA, we prepared poly-A RNA on oligo-dT columns, reverse
transcribed, ligated to SalI and NotI adapters, and cloned it into a
gt-22A vector by using the SuperScript system for cDNA synthesis
and cloning kit (Life Technologies, Inc.,
Gaithersburg, MD). The cDNA-library was screened by using a
32P-labeled 30 bp oligo from exon 6 of CYP2C11
described above, and the cDNA fraction from selected clones were
subcloned into a pSPORT- vector (Life Technologies, Inc.,
Gaithersburg, MD). Restriction endonuclease mapping was performed by
standard methods (20) and the nucleotide sequence of the
cDNA was determined in both the strands by the dideoxy method
(21). Sequence analysis was carried out using "DNASIS"
(Hitachi Software, Brisbane, CA).
Preparation of nuclear extracts
Nuclear extracts were prepared from individual, freshly excised
MSG treated and control rat livers using established procedure of
nuclei preparation (22), with inclusion of protease
inhibitors (23) and 3 mM
MgCl2 (24) in the homogenization
buffer. The final nuclear extracts were dialyzed in cassettes
(Pierce Chemical Co., IL) against 20 mM HEPES
(pH 7.9 at 4 C), 20% glycerol, 100 mM KCl, 0.2
mM EDTA, 0.2 mM phenylmethanesulfonylfluoride
and 0.5 mM DTT, and aliquots were snap frozen in liquid
nitrogen and stored at -70 C.
RNA splicing assays
A 1030-bp XbaI and AccI cut fragment containing the junctions of
exons 8 and 9 and the entire intervening 742-bp intron of CYP2C11 was
isolated from the cDNA, and subcloned in a pBS-II SK(-) vector
(Stratagene, La Jolla, CA) to produce sufficient substrate
for the splicing assay. The proteinase-K treated plasmid was
precipitated and in vitro transcribed using mMESSAGE
mMACHINE kit (Ambion, Inc., Austin, TX). The transcription
reaction mixture containing buffer (1x), nucleotides (1.5
mM), 32P-UTP (80 µCi, 0.1
nmol), linearized template DNA (1 µg) and RNA polymerase (1x) was
incubated at 37 C for 1 h and the template was treated with
DNase-I. The labeled RNA was purified on 1% agarose gels in MOPS and
extracted from gels using the "gene-capsule" extraction kit (Geno
Technologies, Inc., St. Louis, MO). Precursor RNAs were heated to 90 C
for 1 min in 1 mM Tris (pH 7.5) and 0.1
mM EDTA and then placed on ice to eliminate
aggregates. This was followed by subsequent incubation at 35 C for 30
min in the reaction buffer [HEPES 50 mM (pH
7.6), KC1 (50 mM), and 5 to 8
mM MgCl2] to permit
refolding. Splicing reactions were performed with a commercially
available kit (Promega Corp., Madison, WI) in a buffer
containing 5 mM HEPES, pH 7.9, 0.4
mM ATP, 20 mM creatine
phosphate, and 0.6% polyvinyl alcohol. Fifteen µg of protein from
the nuclear extracts were preincubated for 15 min in the presence of
RNase inhibitors, and splicing was carried out in the presence of 4 ng
of the intron containing labeled RNA (3000 cpm/ng) for 2 h at 30
C. The spliced products were extracted with phenol/chloroform/isoamyl
alcohol (25:24:1), precipitated with ammonium acetate and
electrophoresed on denaturing agarose gels
(20).3 The
efficiency of splicing was analyzed by quantitating scanned
autoradiographs. No detectable levels of splicing products were
observed in controls incubated without nuclear extracts suggesting no
basal level of nonenzymatic splicing.
Northern blot analysis for intron retained CYP2C11 in RNA
variant
To detect the presence of the MSG-induced intron retained
variant of CYP2C11 in liver, a synthetic oligo
(5'-GGGCATGTCAGAACCTTGCTTTGTCAATGGC-3'), selective for the retained
intron (BLAST search) was used to probe hepatic RNA by routine Northern
blot analysis (5, 7).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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200–300 ng/ml plasma) every 3.5
to 4 h. Between the peaks, GH levels were undetectable (Fig 1
). Episodic infusion of 4 µg rGH/kg
BW/pulse by our pumping apparatus produced masculine-like circulating
profiles of GH exemplified by a regular pulse every 4 h
interrupted by undetectable concentrations of the hormone for
approximately 3 h. The one distinction from the normal masculine
profile was a 90% reduction in the GH pulse amplitudes. Neonatal
administration of MSG resulted in a modified masculine secretory GH
profile in adulthood that was indistinguishable from the mini pulse
pattern in the rGH-infused hypophysectomized rats.
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-hydroxylase activity were as expected (6, 10),
reduced to 20 to 25% of normal. Restoration of an otherwise masculine
plasma GH profile as observed in the rGH-infused hypophysectomized rats
and the neonatally MSG treated rats in which episodic GH pulses were
secreted at 10% the physiologic amplitudes, resulted in a 2 to 3-fold
overexpression in CYP2C11 mRNA (Fig. 1
). In contrast, exposure to the
mini GH pulses increased CYP2C11 protein and CYP2C11-dependent
testosterone 2-hydroxylase as well as CYP2C11-dependent testosterone
16ß-hydroxylase (the latter not reported) a mere 10 to 30% above
normal. Restoration of the masculine plasma GH profile at physiologic
pulse amplitudes results in normal expression levels of hepatic CYP2C11
mRNA, protein and catalytic activities that are indistinguishable from
intact rats (7, 10).
Since the overexpressed transcript levels were greater in the MSG than
the rGH-infused rats, and the former is a more robust and more easily
produced model, we chose to use the neonatally MSG-treated rat to
further investigate the mini GH pulse induced overexpressed CYP2C11
mRNA. Prolonged electrophoresis followed by Northern blot analysis with
a 32P-oligo specific CYP2C11 probe [954 bp-925
bp (19)] revealed more than one CYP2C11 species in the
MSG- exposed rats; i.e. the apparently normal CYP2C11
mRNA (1.8 kb) and a larger species > 1.8 kb (Fig. 2.1A). To identify the presence of an
intervening sequence(s) in the putative larger size mRNA variant, we
used primer sequences complementary to at least the four exon regions
of CYP2C11 and amplified by reverse transcriptase PCR (RT-PCR) hepatic
RNA obtained from both control and neonatal MSG-treated adult male
rats. In contrast to the control animals, the RT-PCR products from the
MSG-treated animals showed an additional and larger molecular weight
product when using the primers from the last exon (#9) and penultimate
exon (#8) (Fig. 2.1B).
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.1C). To determine the exact size of the 2.6 kb mRNA,
and to exclude the presence of mutational differences, the clone was
sequenced and analysis showed complete homology to the CYP2C11 cDNA
(19), with the exception of an additional 742 bp
intervening sequence representing the last intron between exons 8 and 9
(25). The consensus DNA sequence deduced from this
independent cDNA clone contained a single 5' open reading frame
beginning with a translational initiation codon and extending for 2243
bp before interruption by an originally reported (19, 25)
ochre (UAA) termination codon (Fig. 3).
An additional in-frame termination codon was contained in the 742 bp
intervening sequence. At 311 bp downstream from the last termination
codon, concluding exon 9, was a polyadenylation signal (AATAAA),
followed 20 bp [instead of the initially reported 17 bp
(25)], and ending with a polyadenylase tail. Northern
blot analysis of total liver RNA using an oligo probe selective for the
retained intron detected an RNA species of approximately 2.6 kDa in the
neonatally MSG-treated adult rats (blot not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A survey of mammalian splice site mutations characterized in disease gene DNA describe four phenotypes: exon skipping (51%), activation of a cryptic splice site (32%), creation of a pseudo exon within an intron (11%), and intron retention (6%) (33). Regarding the latter, administration of the highly potent inducing agent, Aroclor 1254, to rats has been reported to induce the expression of both wild-type and alternatively spliced forms of CYP2B2 (34) and CYP1A2 (35), in which a partial intron is incorporated into a mature transcript. Moreover, variant forms of constituent CYP2B (36, 37) and CYP2D (33) mRNAs with complete or partial introns retained have been found in normal human liver, suggesting that endogenous factors can be responsible for aberrant splicing of precursor transcripts. Accordingly, this raises the question as to whether the expression of the intron retained CYP2C11 variant is a direct result of MSG action on the neonatal liver or some mediating factor. We propose that the evidence supports the latter view. If neonatal exposure to MSG directly interfered with differentiation of hepatic mechanisms transcribing CYP2C11, it is difficult to explain why increased neonatal exposure to the amino acid (i.e. additional injections on days 5, 7, and 9 of life) results in a complete repression of adult CYP2C11, not to mention the expected overexpression (13, 38). It seems more likely that the episodic mini-GH pulses secreted in the MSG-treated rats are responsible for the incomplete splicing of the CYP2C11 transcript. In support of this conclusion are findings of a dramatic overexpression of CYP2C11 mRNA associated with normal or slightly above normal levels of CYP2C11 protein and catalytic activities in neonatally MSG-treated adult rats secreting mini-GH pulses (10, 38), hypophysectomized rats in which 5–20% of physiological pulse amplitudes in otherwise normal masculine plasma GH profiles are restored (7, 10), and dwarf rats secreting substantially subnormal GH pulse heights (3).
The mechanism by which nominal GH pulses may interfere with normal intron splicing is suggested by our finding of a reduction in nuclear splicing activity directed toward deletion of the 3'-terminal intron in CYP2C11 mRNA. However, there are many steps between GH activation of its membrane receptor and CYP2C11 transcription. While episodic plasma GH profiles have been reported to regulate CYP expression by activating the hepatic JAK-2/Stat signal transduction pathway (23), there is no evidence to suggest that mini-GH pulses can interfere with this pathway or, for that matter, whether the JAK-2/Stat pathway actually functions at the level of transcript splicing.
In summary, we propose that hepatic exposure to nominal GH pluses
(10% of physiological concentration) in otherwise normal
circulating masculine profiles produces a severalfold overexpression of
CYP2C11, the predominant CYP-dependent drug-metabolizing enzyme in the
male rat. A substantial portion of the elevated transcript, at levels
similar to those observed in control liver, is normally processed mRNA,
explaining our finding of a modest 10–30% increase in CYP2C11 protein
and its catalytic activities. However, a larger portion of the mature
CYP2C11 mRNA will remain incompletely processed, retaining the
3'-terminal intron in an otherwise normally sequenced transcript. In
this regard, the very presence of the final intron may have been
sufficient to block transport of the mRNA variant to cytosolic
translation sites, thus explaining the disproportionately lower levels
of CYP2C11 protein observed in affected livers. In addition, the
presence of a premature termination codon in the retained intron could
have resulted in the translation of a truncated protein highly
vulnerable to swift degradation. Lastly, associated with the
overexpression of CYP2C11 mRNA was a 50% decline in nuclear capacity
to splice the 3'-terminal intron from the adjoining exons, which could
explain the build-up of intron-retained mature mRNA.
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Acknowledgments |
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Footnotes |
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2 Present address: Scripps Research Institute, La Jolla, California
92037.
3 Due to their large size, the products were more
effectively separated on agarose compared with acrylamide gels.
Received May 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase,
bile acid 6ß-hydroxylase, and growth hormone-responsive steroid
hormone hydroxylase. J Steroid Biochem Mol Biol 43:1055–1072[CrossRef]
-hydroxylase cytochrome P-450 apoprotein in the
rat. J Biol Chem 260:11895–11898This article has been cited by other articles:
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  A. S. Verma, R. N. Dhir, and B. H. Shapiro Inadequacy of the Janus Kinase 2/Signal Transducer and Activator of Transcription Signal Transduction Pathway to Mediate Episodic Growth Hormone-Dependent Regulation of Hepatic CYP2C11 Mol. Pharmacol., March 1, 2005; 67(3): 891 - 901. [Abstract] [Full Text] [PDF]
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 A. K. Agrawal and B. H. Shapiro Constitutive and Inducible Hepatic Cytochrome P450 Isoforms in Senescent Male and Female Rats and Response to Low-Dose Phenobarbital Drug Metab. Dispos., May 1, 2003; 31(5): 612 - 619. [Abstract] [Full Text] [PDF]
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A. Bhathena, C. Lee, and D. S. Riddick Suppression of Cytochrome P450 2C11 by Aromatic Hydrocarbons: Mechanistic Insights from Studies of the 5'-Flanking Region of the CYP2C11 Gene Drug Metab. Dispos., December 1, 2002; 30(12): 1385 - 1392. [Abstract] [Full Text] [PDF]
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 A. Kaufhold, P. K. Nigam, R. N. Dhir, and B. H. Shapiro Prevention of Latently Expressed CYP2C11, CYP3A2, and Growth Hormone Defects in Neonatally Monosodium Glutamate-Treated Male Rats by the N-Methyl-D-Aspartate Receptor Antagonist Dizocilpine Maleate J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 490 - 496. [Abstract] [Full Text] [PDF]
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