Ortiz de Montellano Paul R.(Ed.) Cytochrome P450. Structure, Mechanism, and Biochemistry
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Induction of Cytochrome P450 Enzymes 345
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9
Hormonal Regulation of Liver
Cytochrome P450 Enzymes
David J. Waxman and Thomas K.H. Chang
1.
Introduction
Sex differences in hepatic drug metabolism
have been known for more than 30 years, based on
the early studies of Kato, Conney, Gillette, and
others^~^. In rats and certain other species, the rate
of drug metabolism is often several-fold higher
in males as compared to females as revealed by
in vivo pharmacokinetics and as demonstrated
in vitro by assaying prototypic phase I c5^ochrome
P450 drug substrates, such as ethylmorphine,
benzo[a]pyrene, or hexobarbital using isolated
liver microsome preparations (Figure 9.1). This
sex-dependence of P450 metabolism is most strik-
ing in the rat, where sex differences in metabolic
rates can be as high as 5-fold with some drug sub-
strates. This finding was initially unexpected,
because measurements of total liver P450 content
indicated that the overall P450 levels in rat liver
tissue are only —20% higher in males compared
to females (Figure 9.1). Research carried out dur-
ing the 1980s resolved this discrepancy with the
finding that there are multiple liver-expressed
P450 enzymes^' ^, each of which is encoded by a
separate gene, and only some of which are
expressed in a sex-dependent manner.
Based on the recently published human
genome sequence, we now know that there are
57 ftinctional human cytochrome P450 genes,
grouped into 17 distinct gene families^. Many of
these P450 enzymes catalyze the biosynthesis of
physiologically important endogenous substances,
such as steroid hormones, whereas others are pri-
marily involved in metabolism of environmental
chemicals and other xenobiotics, notably drugs.
From the perspective of foreign compound meta-
bolism, the most important cytochrome P450
("CYP") genes are those in the CYPl, CYP2, and
CYP3 families. These three families encompass
—
15-20 different cytochrome P450 enzymes, and
collectively carry out essentially all of
the
phase I
cytochrome P450 metabolic reactions in mam-
malian liver^^. As individual liver P450 enzymes
were purified and characterized, and subsequently,
when their genes were cloned from multiple
species, it became apparent that in certain species,
such as the rat and mouse, a subset of these drug-
metabolizing liver P450s is expressed in a sex-
dependent fashion and subject to endocrine
control^ ^ Human liver P450 enzyme levels and
their associated drug metabolism activities may
also be determined, in part, by age, sex, and hor-
mone status^^~^^. Studies of
the
underlying mech-
anisms governing the endocrine regulation of rat
liver P450 enzymes may thus be of general impor-
tance for our understanding of the hormonal
David J. Waxman • Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston,
MA. Thomas K.H. Chang • Faculty of Pharmaceutical Sciences, The University of British Columbia,
Vancouver, BC, Canada.
Cytochrome P450: Structure, Mechanism, and Biochemistry, 3e, edited by Paul R. Ortiz de Montellano
Kluwer Academic / Plenum Publishers, New York, 2005.
347
348 David J. Waxman and Thomas K.H. Chang
12
^
o
.^ fl 8
N ^
0
E
^H MALE
EZZ2 FEMALE
i li
1.
1
M
1
1
L
M
BP HB P450(10X)
Figure
9.1.
Sex-differences
in
rat hepatic microsomal
drug metabolism. Data shown are based on enzyme
assays in rat liver microsomes using the three indicated
xenobiotic substrates: ethylmorphine (EM)"*, benzo[a]
pyrene (BP)^, and hexobarbital (HB)^. Ethylmorphine
A^-demethylase and benzo[(3]pyrene hydroxylase
activities are expressed as nmol product formed per
minute per mg microsomal protein, whereas hexo-
barbital hydroxylase activity is expressed as nmol
product formed per 30 min per g liver. Also shown is
hepatic microsomal total cytochrome P450 content,
which is expressed as nmol per mg microsomal pro-
tein (values multiplied by 10)^. The data are shown as
mean ±
SD
for 4 or 5 rats, except for ethylmorphine
A^-demethylase and total P450 which are based on
a
pool
of
6
livers.
regulation of liver-expressed genes in both rodent
models and in man. Endocrine-regulated steroid
hydroxylase P450s contribute in an important way
to foreign compound metabolism in the liver, and
studies of their hormonal control may shed light
on the underlying basis for the influence of hor-
mone status on a broad range of P450-catalyzed
drug metabolism and carcinogen activation
reactions.
This chapter reviews studies leading to the
identification of the key endocrine regulatory
factors and the underlying mechanisms through
which these factors operate to control the expres-
sion of liver cytochrome P450 enzymes. Primary
emphasis is given to studies on the hormonally
regulated P450s expressed in rat liver, the best-
studied model system. Also discussed are environ-
mental and pathophysiological factors that can
perturb hormonal status and the impact of these
factors on the expression of sex-dependent
cytochromes P450.
2.
Steroid Hormones as
Substrates for Sex-Dependent
Liver P450s
The precise physiological function of the
endocrine-regulated rodent liver P450s is unclear;
however, steroid hormones are metabolized by
liver P450 enzymes with a higher degree of
regiospecificity and stereoselectivity than many
foreign compound substrates^ ^ suggesting that
these endogenous lipophiles serve as physiologi-
cal P450 substrates. The physiological require-
ments with respect to steroid hormone
hydroxylation differ between the sexes, and not
surprisingly, several steroid hydroxylase liver
P450s are expressed in a sex-dependent manner^
^' ^^.
Rat P450 enzymes CYP2C11 and CYP2C12 are
prototypic examples of sex-specific liver P450
enzymes, and they have been a major focus of
studies of the underlying endocrine factors, as
well as the cellular and molecular regulatory
mechanisms that govern sex-specific liver gene
expression. CYP2C11 is the major male-specific
androgen 16a- and 2a-hydroxylase in adult rat
liver, and is induced at puberty in males but not
females •^' ^^ under the influence of neonatal
androgenic imprinting (programming)^ ^ By
contrast, the steroid sulfate 15p-hydroxylase
CYP2C12 is expressed in a female-specific
manner in adult rats^'' ^^.
3. Developmental Regulation of
Sex-Dependent Rat Liver
P450S
Multiple rat liver cytochrome P450 enzymes
are expressed in a sex-dependent manner, sub-
ject to complex developmental regulation and
endocrine control (Table 9.1). CYP2C11, the
major male-specific androgen 2a- and 16a-
hydroxylase of adult liver, is not expressed in
immature rats but is induced dramatically at
puberty (beginning at 4-5 weeks of age) in male
but not female rat liver^^' ^^. A similar develop-
mental profile characterizes three other male-
specific rat liver cytochromes P450, CYP2A2
(refs [27], [36]), CYP2C13 (refs [28], [37]), and
CYP3A18 (refs [25], [38]). By contrast, another
Hormonal Regulation of Liver Cytochrome P450 Enzymes
349
Table 9.1.
CYP enzyme^
/. Male-specific
2A2
2C11
2C13
3A2
3A18
4A2
//. Female-specific
2C12
Hormonal Regulation of Sex-Dependent Rat Liver P450 Enzymes
///. Female-predominanf
2A1
2C7
3A9
5a-reductase
Testosterone
hydroxylase
activities^
15a
2a, 16a
6^, 15a
6P,2(3
6(3,2(3,
15P,
(see footnote
15P^
7a
16a
6P
—
16a
g)
Hormonal regulation^
Androgenic
imprinting'^
+ +
+ +
+ +
+ +
ND
ND
--
ND
ND
ND
Thyroid hormone^
+/-
+/-
ND
--
ND
-
4-/-
-
+ +
ND
+ +
''P450 gene designations are based on the systematic nomenclature of ref. [23]. Table is modified from ref [24].
^The major sites of testosterone hydroxylation catalyzed by the individual P450 proteins are shown. Testosterone
metabolites specific to the P450's activity in rat liver microsomal incubations are underlined. Based on refs [8], [11],
[21],
[25], [26] and references therein.
cu_|_
_)_»
in(jjcates a positive effect on adult enzyme expression, while " " indicates a suppressive effect. "—" indi-
cates a lesser degree of suppression, while "+/-" indicates no major effect. ND—^not determined in a definitive
manner.
'^For further details see refs [27]-[29].
^Based on refs [30]-[34].
^Purified CYP2C13 exhibits high testosterone hydroxylase activity in a reconstituted enzyme system, but this
enzyme makes only marginal contributions to liver microsomal testosterone hydroxylation^^.
^CYP4A2 catalyzes fatty acid co-hydroxylation, but does not catalyze testosterone hydroxylation.
^15P-hydroxylation of steroid sulfates. CYP2C12 also catalyzes weak testosterone 15a- and la-hydroxylase
activities.
'Liver expression of these enzymes is readily detectable in both male and female rats, but at a 3-10-fold higher level
in females as compared to males.
adult male-specific liver P450, the steroid 6|3-
hydroxylase CYP3A2, is expressed in prepubertal
rat liver at similar levels in both sexes, but is selec-
tively suppressed at puberty in females^
^' ^^"^^
The
steroid sulfate ISP-hydroxylase CYP2C12 is
expressed at a moderate level in both male and
female rats at 3^ weeks of age. Beginning at
puberty, however, CYP2C12 levels are further
increased in females while they are fully sup-
pressed in males^^' '^^. Several other female-pre-
dominant liver enzymes have also been shown to
increase at puberty in adult female rats. These
include: CYP2C7 (refs [37], [42]), which catalyzes
retinoic acid 4-hydroxylation'^^; CYP3A9 (ref
[44]),
which catalyzes steroid 6p-hydroxylation^^;
and steroid 5a-reductase, which is not a
cytochrome P450 enzyme but plays an important
role in steroid metaboHsm in adult female
rats^^'
^^.
Finally, CYP2A1 is a female-predominant steroid
7a-hydroxylase that is expressed in both sexes
shortly afler birth, but is suppressed at puberty to a
greater extent in male than in female rat liver^^'
^^' ^^
(Table 9.1). Each of these sex-dependent P450
enzymes is primarily expressed in liver, although
low-level extrahepatic expression may occur in
some cases"^^^^. Studies of liver P450 expression
during senescence have revealed a general loss of
gender-dependent enzyme expression that reflects
a decrease in male P450 levels and an increase in
expression of female-predominant P450 enzymes
350
David J. Waxman and Thomas K.H. Chang
in aging male rats^^'
^^.
This appears to be related
to the age-dependent reduction in the secretion of
growth hormone (GH) releasing factor and the
changes in plasma profile of GH^^, which is a
major regulator in the sex-dependent expression of
liver CYP enzymes (see Section 4.2).
4. Hormonal Control of Liver
P450 Expression
4.1.
Regulation by Gonadal
Hormones
Initial studies on the endocrine regulation of the
liver P450 enzymes demonstrated that the gonadal
steroids play an important role in regulating
enzyme expression, but only indirectly. Gonadal
steroids do not act directly on the liver to confer
the sex-dependent pattern of hepatic steroid and
drug metabolism and P450 expression, but rather,
act indirectly via the hypothalamus, which regu-
lates the pituitary gland and its secretion of the
polypeptide hormone, GH (see Section 4.2).
4.1.1.
Testosterone
4.1.1.1.
Distinct effects of neonatal
androgen and adult androgen. Gonadal hor-
mones play an essential role in determining the
expression of the major sex-specific rat liver P450
forms at adulthood. In the case of testosterone,
there are two distinct postnatal developmental
periods of hormone production, neonatal and post-
pubertal, and each period makes a distinct con-
tribution to the expression of the sex-dependent
liver P450s at adulthood. Castration of male rats at
birth eliminates both periods of androgen produc-
tion, and this in turn abolishes the normal adult
expression of each of the male-specific P450s:
CYP2A2 (ref [27]), CYP2C11 (refs [20], [21], [29],
[37],
[53]), CYP2C13 (ref [28]), and CYP3A2
(refs [21], [29], [53]). CYP2C13 mRNA levels^^
are also abolished in birth-castrated rats, indicat-
ing that enzyme expression is regulated at a pre-
translational step. Treatment of birth-castrated
male rats with testosterone during the neonatal
period leads to a partial restoration of the expres-
sion of these male-specific P450 forms at adult-
hood^^'
^^' ^^.
A brief period of neonatal androgen
exposure is thus sufficient to "imprint" or irre-
versibly program the male rat to express these
P450 enzymes later on in adult life. These effects
of neonatal androgen on male-specific P450
enzymes are very similar to the androgenic
imprinting effects observed in earlier studies of
liver microsomal steroid hydroxylase activities'^'
54,
55^ several of which can be associated with
specific liver P450 forms^^
However, neonatal testosterone given to birth-
castrated male rats only partially restores
CYP2C11 (refs [21], [53]) or CYP2C13 (ref [28])
to normal adult male levels, indicating that neona-
tal androgen alone is insufficient for full adult
expression of these male-specific P450s. Con-
sistent with this observation, the combination
of neonatal androgen treatment with adult andro-
gen exposure results in complete restoration of
normal adult male expression of the male-specific
P450s (ref [28]). Moreover, testosterone treat-
ment of adult male rats that were castrated either
neonatally or prepubertally, can substantially
increase the expression of CYP2C11 (refs
[29],
[53],
[56],
[57]) and CYP2C13 (ref [28]). However, in
contrast to the irreversible imprinting effects of
neonatal androgen treatment, the effects of adult
androgen exposure are likely to be reversible, as
evidenced by the partial loss of CYP2C11 in male
rats castrated at adulthood^^' ^' and by the reversal
of this loss by the synthetic androgen methyl-
trienolone^^. Similarly, the continued presence of
testosterone at adulthood is also required to main-
tain normal adult expression of CYP3A2, since
castration at 90 days of age reduces hepatic
CYP3A2 mRNA levels by >80%, but this can be
restored by subsequent administration of testos-
terone to the adult rat^^. Thus, while neonatal
testosterone imprints the rat for expression of the
male-specific P450 enzymes beginning at
puberty, a time when the demand for P450-
dependent liver steroid metabolism is increased,
the additional presence of androgen during the
pubertal and postpubertal periods is required to
maintain full enzyme expression during adult
life^O'^i.
4.1.1.2. Testosterone suppression of
female enzymes. In contrast to the positive reg-
ulation by testosterone of the male-specific
enzymes, testosterone suppresses expression of
the female-specific CYP2C12 as well as the
female-predominant enzymes CYP2A1 and
Hormonal Regulation of Liver Cytochrome P450 Enzymes
351
steroid 5a-reductase. Hepatic CYP2C12 content
is reduced in intact, adult female rats exposed
chronically to testosterone^^ or to the synthetic
androgen methyltrienolone^^. Similarly, treatment
of neonatally or prepubertally ovariectomized rats
with testosterone, either neonatally or pubertally,
results in a major decrease in microsomal steroid
5a-reductase activity^^'
^^.
Birth castration of male
rats increases the adult levels of hepatic CYP2A1,
but testosterone administration to these animals
re-masculinizes (i.e., decreases) the levels of
this P450 (ref [62]). Androgens thus exert a sup-
pressive effect on liver CYP2A1 expression.
Studies of the effect of testosterone on the expres-
sion of the female-predominant CYP2C7 are
inconclusive^^' ^^.
4.1.1.3. Mechanisms of testosterone
regulation. The mechanisms by which neonatal
testosterone imprints the expression of liver P450
enzymes during adulthood are only partially
understood. Testosterone's primary effects on liver
P450 profiles are mediated by the hypothalamic-
pituitary axis^"^ and its control of the sex-depend-
ent pattern of pituitary GH secretion^^' ^^.
Consistent with this conclusion, testosterone has
only minor effects on liver enzyme profiles in
hypophysectomized rats in most^^ but not all^^'
^^
instances. As discussed in Section 4.2, pituitary
GH secretory patterns play a key role in regulating
the expression of
the
sex-dependent P450 forms.
4.1.2.
Estrogen
Whereas testosterone has a major positive reg-
ulatory influence on the male-specific P450
forms,
estrogen plays a somewhat lesser role in
the expression of the female-specific and the
female-predominant liver P450 enzymes. Ovari-
ectomy at birth reduces, but does not abolish,
expression of hepatic CYP2C7, CYP2C12, and
steroid 5a-reductase in adult female
rats^^'
^^' ^^
and
normal adult enzyme levels can be restored by
estrogen replacement. Ovariectomy during adult-
hood^^ or neonatal administration of an estrogen
receptor antagonist, tamoxifen^ \ reduces hepatic
CYP3A9 expression in adult female rats. The
reduced expression of CYP3A9 in ovariectomized
rats can be restored by estrogen^^. By contrast,
estradiol suppresses hepatic CYP2C11 in both
intact and castrated male rats^^' ^^. However, the
absence of CYP2C11 in adult female rats is not
due to a negative effect of estrogen. Thus, ovari-
ectomy alone does not lead to CYP2C11 expression
in female
rats^^'
^^' ^^.
In male rats, the suppression
of CYP2C11 by estradiol may be irreversible, as
demonstrated by the major loss of this P450 in
adult male rats exposed to estradiol during the
neonatal period or at puberty. However, this effect
is not a consequence of
a
direct action of estradiol
on the liver, since estradiol does not impact on
hepatic CYP2C11 levels in hypophysectomized
rats^^.
Rather, the effects of estradiol on liver P450
expression involve the hypothalamo-pituitary
^^jg64,72^
^j^^
j^Qg^
likely result from an estrogen-
dependent increase in the interpeak baseline levels
of plasma
GH^"^'
^^.
This effect of estradiol may be
sufficient to alter the sex-specific effects of the
GH secretory pattern since, as discussed in greater
detail below, recognition of a "masculine" GH
pulse by hepatocytes requires an obligatory recov-
ery period during which there is no plasma GH
and hence no stimulation of hepatocyte GH recep-
tors (GHRs)^^. In addition, estrogen may antago-
nize the induction of
CYP2C11
by testosterone as
suggested by the absence of androgen imprinting
of this P450 in intact female rats treated with
neonatal or pubertal testosterone^^'
^^.
Indeed, the
stimulatory effect of testosterone on pulsatile GH
secretion can be blocked by the presence of intact
ovaries in female rats^^. Interestingly, prepubertal
treatment of intact (i.e., non-ovariectomized)
female rats with tamoxifen enhances the induction
of CYP2C11 and CYP3A2 protein expression by
pubertal and postpubertal androgen^^. The precise
neuroendocrine mechanisms responsible for the
antagonistic effects of estrogen on androgen
imprinting remain to be elucidated.
4.2.
Regulation by Growth
Hormone
4.2.1.
Sex-Dependent GH Secretory
Profiles
In many species, the pituitary gland secretes
GH into plasma in a highly regulated temporal
fashion that differs between males and females.
This sex-dependent secretion of GH is most strik-
ing in rodents^"*"^^, but key features are conserved
in humans^^"^^. In the rat, GH is secreted by the
pituitary gland in adult males in an intermittent, or
352
David J. Waxman and Thomas K.H. Chang
pulsatile, maimer that is characterized by high (Figure 9.2(A)). By contrast, in the adult female
peaks of hormone in plasma (150-200 ng/ml) rat, GH is secreted more frequently (multiple pitu-
each 3.5-4 hr followed by a period of very low or itary secretory events per hour) and in a manner
undetectable circulating GH (< 1-2 ng/ml) such that the plasma GH pulses overlap and the
Females
9:00 11:00 13:00 15:00 17:00
I I I
I"
I
I I I I I I I I' I I I' I I I I I I I I I I I I I I I I I I
9:00 11:00 13:00 15:00 17:00
Figure 9.2. Sex-dependence of plasma GH profiles in adult rats. Shown are plasma GH profiles measured during
the course of an 8 hr day in unrestrained and unstressed male (panel A) and female rats (panel B). Data shown are
fromref [73].
A GH Pulse Induction oiCYPICll: B Continuous GH Feminization
Pulse Frequency Dependence
Male Hx + GH
M Hx 6P 2P F
2C11-
mRNA
fir
12 3 4 5 6 7 8
Female Hx + GH
F Hx 6P 2P M
2C11_
mRNA
Mrtl
M+ M +
M F hGH rGH M
^/;'
W^M
4A2
I
12345 6789
2C11
W 3A2
2C12
Tubulin
12345678 9 10 11
Figure 9.3. Role of GH secretory profiles in the expression of sex-dependent rat hepatic GYP mRNAs. Shown are
Northern blots probed with oligonucleotide probes specific to each of the indicated GYP RNAs. Panel A shows
the male (M) specificity of
GYP2G11,
its absence in hypophysectomized rats (Hx) and its induced expression in
either male or female (F) hypophysectomized rats given either 2 or 6 pulses (P) of GH/day for 7 days. Data based
on ref [73]. Panel B shows the effects of continuous rat (r) or human (h) GH infusion in male rats (lanes 6-10) on
the expression of CYPs 4A2, 2G11, and 3A2 (all male-specific; lanes 1, 2, 11 vs lanes 3-5), as well as the induction
of
GYP2G12.
Tubulin RNA is shown as a loading control. Data based on ref [32].
Hormonal Regulation of Liver Cytochrome P450 Enzymes
353
hormone is continually present in circulation at
significant levels (~15^0ng/ml) at nearly all
times^''
(Figure 9.2 (B)). Hypophysectomy and
GH replacement experiments carried out in sev-
eral laboratories have demonstrated that these sex-
dependent plasma GH profiles are, in turn,
responsible for establishing and for maintaining
the sex-dependent patterns of liver P450 gene
expression in rats^^' ^^' ^^' ^^'
^^
and mice^"^'
^^
(for
earlier reviews, see refs [24], [86]). Clinical stud-
ies in humans also demonstrate a role for GH^^~^^
and its sex-dependent plasma secretory patterns^^
in regulating P450-dependent drug metabolism.
Studies in the rat model reveal three distinct
responses of liver P450s to plasma GH profiles
(Figure 9.3).
(1) Continuous plasma GH, a characteristic
of adult female rats, stimulates expression of female
specific enzymes, such as CYP2C12 and steroid
5a-reductase^^' ^^, and female-dominant liver
enzymes, such as
CYP2A1,
CYP2C7, and CYP3A9
(refs [31], [62], [93], [94]). Hepatic levels of
CYP2C12 and steroid 5a-reductase are unde-
tectable in hypophysectomized female rats but can
be restored to near-normal female level by continu-
ous GH replacement^^'
^^~^^.
This restoration can be
achieved with as little as 12-25% of the physiologi-
cal levels of
GH^^.
Higher levels of GH are required
to induce expression of CYP2C12 and steroid 5a-
reductase in hypophysectomized male rats^^.
(2) Intermittent plasma GH pulses, which
are characteristic of adult male rats, induce
expression of the male-specific liver enzyme
CYP2C11 (Figure
9.3(A))
and its associated testos-
terone 2a-hydroxylase activity^^'
^^' ^^' ^^.
The stim-
ulatory effects of intermittent GH stimulation on
this "class I" male P450 enzyme can be distin-
guished from the effects of GH pulses on a larger,
second group
("class
II") male-specific liver P450s
(CYP2A2, CYP2C13, CYP3A2, CYP3A18, and
CYP4A2). In contrast to the class
ICYP2C11,
class
II male P450s are not obligatorily dependent on GH
pulses, as judged by their high level of expression in
the absence of GH, as shown in hypophysectomized
rats of both
sexes27'28,38,4i,97,98,100,101.
Nevertheless,
liver expression of the class II enzymes CYP2A2
and CYP3A2 is induced when intermittent GH
pulses are given to adult male rats that are depleted
of circulating GH by neonatal monosodium gluta-
mate (MSG) treatment^^^.
(3) Continuous GH exposure exerts major
negative regulatory effects on liver P450 enzyme
expression, as revealed by the marked suppression
of each of the class I and class II male-specific rat
P450s following continuous GH treatment of
intact male rats (Figure 9.3(B)). In some cases this
effect can be achieved at fairly low GH levels, cor-
responding to only 3-12% of the physiological
GH levels present in intact female
rats^^.
The high
level expression of class II P450 mRNAs seen in
the absence of GH pulses, that is, in hypophysec-
tomized male rats, is also suppressed by continu-
ous GH, indicating that continuous GH actively
suppresses P450 gene expression, and does not
simply abolish the stimulatory pulsatile plasma
Table 9.2. Response of Sex-Specific Rat CYPs to GH
GYP
F
-
_
Intact rats
M
+ +
+ +
M
+
GH_,
-
_
Hypophysectomized rats
F M
-
+ + + +
M
+
GH,„,
+
4-
+ +
M
+
GH,,„,
-
+/-
MSG-treated rats
M
-
_
M
+
GHi„,
+ +
+
4-
CYP2C1F
(Male class I)
CYP2A2^
(Male class II)
CYPC12^
(Female-specific)
+ +
F,
female; M, male;
GH^^j^^,
continuous GH;
GHj^^j,
intermittent (pulsatile) GH.
"+ +" indicates a positive effect, "—" indicates a suppressive effect, or absence of expression, and "+/—" indicates no major effect.
''Data are based on refs [20], [73], [97], [98],
[100], [101],
[104]-[110].
^Data are based on refs [27], [97], [98],
[100], [101], [104],
[109]-[111].
'^Data are based on refs [95], [97], [98],
[104], [107],
[109]-[111].
354
David J. Waxman and Thomas K.H. Chang
GH pattern. GH suppression is also a key deter-
minant of the lower responsiveness of female rats
to phenobarbital induction of CYP2B1 (refs
[102],
[103]), and probably also the lower respon-
siveness of the females to the induction of CYP4A
enzymes by peroxisome proliferators such as
clofibrate^^.
The response of the class II male P450 genes
to hypophysectomy of female rats, which de-
represses, that is, increases liver enzyme levels to
near-normal intact male liver levels, demonstrates
that the class II male liver P450s are subject to neg-
ative pituitary regulation in female rat liver, where
their expression is strongly repressed by the near
continuous pattern of plasma GH exposure. These
patterns of hormonal regulation are summarized in
Table 9.2, which presents the responses of repre-
sentative sex-specific liver CYPs to continuous and
intermittent GH treatment applied to intact, hypo-
physectomized and neonatal MSG-treated rats.
4.2.2.
Transcriptional Effects of GH on
CYP Genes
GH regulates liver P450 steady-state mRNA
levels (e.g.. Figure 9.3) in parallel with P450
protein and P450 enzyme activity levels, all but
ruling out major regulation by translational and
post-translational mechanisms, such as GH regu-
lation of P450 protein turnover. Induction of
CYP2C12 mRNA by continuous GH requires
ongoing protein synthesis^
*^,
suggesting either an
indirect induction mechanism or a requirement for
one or more protein components that may have a
short half-life. Analysis of liver nuclear RNA
pools demonstrates that unprocessed, nuclear
CYP2C11 and CYP2C12 RNAs respond to circu-
lating GH profiles in a manner that is indistin-
guishable from the corresponding mature,
cytoplasmic mRNAs^^^. Consequently, transport
of CYP2C11 and CYP2C12 mRNA to the cyto-
plasm, and cytoplasmic P450 mRNA stability are
unlikely to be important GH-regulated control
points for sex-specific P450 expression. More-
over, nuclear run-on transcription analyses have
established that GH regulates the sex-specific
expression of the CYP2C11 and CYP2C12
genes at the level of transcript initiation^^^' ^^^.
Transcription is also the major step for regulation
of the male class II CYP2A2 and CYP2C13
mRNAs^^^'
^^^,
whose male-specific expression is
primarily a consequence of the suppressive effects
of continuous GH exposure in adult female rats^^.
Thus,
transcription initiation is the key step at
which the three distinct effects of GH outlined in
Section 4.2.1 are operative: stimulation of 2C11
expression by pulsatile GH, suppression of both
class I and class II male-specific P450s by contin-
uous GH, and stimulation of CYP2C12 expres-
sion by continuous GH^^^.
Consistent with the finding that GH regulates
sex-dependent liver CYPs by transcriptional
mechanisms, the 5'-flanking DNA segments of
the CYP2C11 (ref [115]) and CYP2C12 genes^^^
both contain specific DNA sequences that interact
in a sex-dependent and GH-regulated manner with
DNA-binding proteins (putative transcription fac-
tors) that are differentially expressed in male vs
female rat liver nuclei^^^' ^^^. These DNA
sequences are hypothesized to include GH res-
ponse elements that contribute to the sex-specific
transcription of the CYP2C11 and CYP2C12
genes.
Two negative regulatory elements
("silencer elements") were also identified in the
CYP2C11 promoter; however, their significance
with respect to GH regulation and sex-specific
P450 expression is as yet unclear^
^^.
Functional
studies of the sex-specific CYP promoters
and their interactions with liver-enriched and
GH-responsive transcription factors are discussed
below.
4.2.3.
Cellular Mechanisms of
GH Signaling
The cellular mechanisms whereby pituitary GH
secretory profiles differentially regulate expres-
sion of the sex-dependent liver P450s are only
partially understood. GH can act directly on the
hepatocyte to regulate liver P450 expression, as
demonstrated by the responsiveness of primary rat
hepatocyte cultures to continuous GH-stimulated
expression of CYP2C12 mRNA; however, these
effects do not involve IGF-I, a mediator of several
of GH's physiological effects on extrahepatic
tissues'^^' ''^. Discrimination by the hepatocyte
between male and female plasma GH profiles is
likely to occur at the cell surface, where a higher
level of GHRs (see below) is found in female as
compared to male rats'^^. This sex difference in