From the
The expression of CYP2C12 is liver-specific and
regulated at the transcriptional level by growth hormone (GH). In
attempts to elucidate the nature of signaling molecules mediating the
GH regulation of this gene in rat hepatocytes, a role for phospholipase
A
The cytochrome P450 gene superfamily (CYP) encodes
multisubstrate monooxygenases
(1) , catalyzing the metabolism of
various endogenous and exogenous hydrophobic
compounds
(2, 3, 4) . Members of the CYP2C subfamily encoding the rat liver specific enzymes P4502C7, 2C11,
2C12, and 2C13, are transcriptionally regulated by growth hormone
(GH),
The cellular responses to GH are initiated by the
binding of GH to its cell-surface receptor
(6, 7) which
belongs to the class I cytokine receptors (also known as hematopoietin
receptors)
(8, 9) . Several studies have demonstrated
that GH signal transduction involves a tyrosine kinase-mediated cascade
which leads to the phosphorylation of various cellular
proteins
(10, 11, 12, 13, 14) ,
and the tyrosine-specific protein kinase JAK2 was recently identified
as a primary signaling molecule in the GH signal transduction
pathway
(15) . The JAK-STAT signaling pathway, first described
for interferon
(16) , is used by several cytokine
receptors
(17, 18, 19, 20, 21, 22, 23) including the GH
receptor
(24, 25, 26) . Another signaling pathway
utilized in common by different cytokines is the activation of the
kinase cascade of raf, MAP kinase kinase and MAP
kinase
(27, 28) . Downstream events of MAP kinase
activation have recently been shown to include phosphorylation and
activation of a cytosolic high molecular weight form of phospholipase
A
Since MAP
kinase has been shown to become tyrosyl-phosphorylated and activated in
response to GH in various cell types
(34, 35, 36) including rat hepatocytes
(26) , it is conceivable
that GH could trigger the activation of cPLA
Levels of P4502C12 mRNA and IGF-I mRNA were
analyzed using specific
Data analysis was
performed using Student's t test or one-way analysis of
variance, followed by Fisher's least significant difference test,
when more than two samples were compared. Differences were considered
to be statistically significant at p < 0.05.
To investigate whether GH signaling in rat hepatocytes
involves MAP kinase and cPLA
To examine whether the observed
GH-dependent increase in eicosanoid production is involved in the GH
regulation of the CYP2C12 gene, the effect of mepacrine on
GH-induced steady state mRNA levels of P4502C12 was investigated. The
mRNA expression from another GH-regulated gene in hepatocytes, the
IGF-I gene
(39, 53) , was studied in parallel.
The time courses of P4502C12 and IGF-I mRNA induction, in the absence
or presence of mepacrine (40 µM), are shown in
Fig. 3A. After 9 h of GH treatment, the induced
expression of P4502C12 mRNA was reduced from 7.4-fold to 1.5-fold when
mepacrine was present. The GH-dependent increase in IGF-I mRNA was also
inhibited (from 5-fold to 2.5-fold). When dose-response studies were
performed, a slight difference in mepacrine sensitivity was observed
between the expression of P4502C12 and IGF-I mRNA
(Fig. 3B). Pretreatment of the cells with 30
µM mepacrine caused a 67% reduction of the P4502C12 mRNA
levels, whereas the IGF-I expression was reduced by 47%. As shown in
the insert of Fig. 3B, the GH-independent
induction of IGF-I mRNA by 8-bromo-cAMP
(37) was not affected by
mepacrine at concentrations up to 30 µM; however, at 40
µM, the mRNA levels were reduced by 82%. Thus, at the
highest dose of mepacrine (40 µM) other cellular
mechanisms than AA release might be affected.
It was recently reported that cPLA
The
GH-stimulated release of AA from cultured hepatocytes does not per
se prove a GH-dependent activation of the cytosolic 85-kDa
PLA
cPLA
As discussed above, our results suggest that
GH activates a cPLA
PLA
The liver
has mainly been implicated in the inactivation of different eicosanoids
but there is also evidence for the formation of active
metabolites
(56) . The P450 enzyme system has been shown to
catalyze the formation of six regioisomeric
cistrans-conjugated hydroxyeicosatetraenoic acids, four
regioisomeric epoxyeicosatrienoic acids (EETs), as well as
Based on results obtained in
this study, we suggest that GH signaling in rat hepatocytes leading to
increased expression of CYP2C12 includes cPLA
We are indebted to Dr. G. Fürstenberger for the
generous gift of the cPLA
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(PLA
) as a transducer of GH-induced levels of
P4502C12 mRNA was investigated. GH was shown to induce
tyrosyl-phosphorylation of p42 and p44 microtubule-associated protein
(MAP) kinases and to reduce the electrophoretic mobility of a 100-kDa
protein, immunologically related to cPLA
. These events were
observed in parallel with GH-stimulated release of
[
H]arachidonic acid ([
H]AA)
from cellular phospholipids of rat hepatocytes labeled with
[
H]AA. These rapid effects of GH action, as well
as the GH-induced expression of CYP2C12, were inhibited in
cells treated with the tyrosine kinase inhibitor herbimycin A.
Similarly, when the GH-induced liberation of
[
H]AA was blocked by the PLA
inhibitor mepacrine or the Ca
channel blocker
verapamil, GH-induced accumulation of P4502C12 mRNA was absent. These
results suggest a correlation between PLA
activity and GH
regulation of the CYP2C12 gene. The inhibitory effect of
mepacrine on GH induction of P4502C12 mRNA was reversed by AA addition,
further supporting a role for eicosanoids in the regulation of
CYP2C12. Finally, inhibitors of P450-mediated AA metabolism,
SKF-525A and ketoconazole as well as eicosatetraynoic acid, blocked the
GH-mediated induction of P4502C12 mRNA, whereas more specific
inhibitors of cyclooxygenase or lipoxygenase metabolism did not. Based
on these results, we suggest that GH signaling in rat hepatocytes,
leading to increased expression of CYP2C12, involves PLA
activation and subsequent P450-catalyzed formation of an active
AA metabolite.
(
)
as demonstrated in vivo and in
primary cultures of adult rat hepatocytes (5). The components of the GH
signal transduction pathway and the nature of transcription factors
mediating the GH regulation of these CYP2C genes are, however,
largely unknown.
(cPLA
)
(29) . In line with this,
several cytokine receptors have been demonstrated to activate
cPLA
upon ligand
binding
(30, 31, 32, 33) .
. We have
previously demonstrated that protein kinases play an important role for
the GH-induced expression of P4502C12 mRNA in primary cultures of rat
hepatocytes
(37) . Here we have investigated whether PLA
transduces GH signaling to the CYP2C12 gene in rat
hepatocytes. We conclude that the GH induction of the CYP2C12 gene involves activation of PLA
and, furthermore,
depends on subsequent P450-catalyzed eicosanoid metabolism.
Animals and Materials
Sprague-Dawley rats (Alab,
Stockholm, Sweden), about 8 weeks of age, were maintained under
standardized conditions of light and temperature, with free access to
animal chow and water. The polyclonal anti-cPLA antibody
(32) was a generous gift from Dr. G. Fürstenberger (German
Cancer Research Center, Heidelberg, Germany). The mouse monoclonal
anti-phosphotyrosine (
PY-4G10) and anti-ERK2 antibodies were
purchased from Upstate Biotechnology Inc. (Lake Placid, NY) and
Transduction Laboratories (Lexington, KY), respectively. Polyvinylidene
diflouride membranes, molecular weight markers, and anti-rabbit and
anti-mouse IgG antibodies conjugated to horseradish peroxidase were
from Bio-Rad. Collagenase (type XI), insulin (24.4 units/mg),
arachidonic acid, mepacrine, A23187, indomethacin, and ETYA were
purchased from Sigma. Recombinant bovine GH was a generous gift from
American Cyanamid Co. (Princeton, NJ). Herbimycin A, verapamil,
SKF-525A, and 5,8,11-eicosatrienoic acid were obtained from Calbiochem,
[5,6,8,9,11,12,14,15-
H]arachidonic acid
(150-230 Ci/mmol), and the ECL detection system from Amersham
International plc (Aylesbury, UK), protein A-Sepharose from Pharmacia
LKB (Uppsala, Sweden), Proteinase K from Merck (Darmstadt, Germany),
glass-fiber filters (Whatman GF/C) from Whatman Ltd. (Madistone, UK),
RNase-A and RNase-T
from Boehringer-Mannheim, and
20-hydroxy-5,8,11,14-eicosatetraenoic acid from Cascade Biochem Ltd.
(Reading, UK). Reagents for in vitro transcription of cRNA
probes were obtained from Promega Biotech Inc.
Hepatocyte Isolation and Cell Culture
Hepatocytes
were isolated and cultured on matrigel in a modified Waymouth medium
containing 0.1 µg of insulin/ml as described
previously
(38, 39) . Treatment of the cells was carried
out after 66 h in culture. Details about treatments are described in
the figure legends. At harvesting of cells, the medium was first
aspirated, whereupon the plates were scraped with a rubber spatula in
ice-cold phosphate-buffered saline, and the suspended cells were
pelleted at 750 g for 5 min.
Immunoprecipitation of
cPLA
Hepatocytes were harvested in ice-cold
phosphate-buffered saline containing 5 mM EDTA, 50 mM
NaF, 30 mM NaP
O
, and 0.2
mM Na
VO
. The cell suspension was
incubated on ice for 1 h to dissolve the matrigel, pelleted by
centrifugation for 5 min at 1000
g, and lysed in a
buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM
NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 30
mM Na
P
O
, 0.2 mM
Na
VO
, 0.2 mM phenylmethanesulfonyl
fluoride, 5 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml
pepstatin. The cell lysate was cleared by centrifugation for 30 min at
15,000
g. Immunoprecipitation was performed by
incubation of 3 ml of lysate (8 mg of protein/ml) with 20 µl of a
polyclonal anti-cPLA
antibody overnight at 4 °C. The
immune complexes were precipitated by incubation with 50 µl of
protein A-Sepharose for 3 h.
Western Blotting
Whole cell lysates (100-200
µg of protein), prepared as described by Campbell et al.(35) and passed through a 23-gauge needle five times, or
immunoprecipitated proteins, prepared as described above, were resolved
by SDS-PAGE on 12 or 6% polyacrylamide gels and transferred to
polyvinylidene difluoride membranes with a Trans-Blot SD semi-dry
transfer cell (Bio-Rad). The membranes were blocked for 1 h in
Tris-buffered saline (TBS; 10 mM Tris, pH 8.0, 150 mM
NaCl) containing 0.05% (v/v) Tween-20 and 0.25% (w/v) gelatin (TBSTG),
incubated with either anti-phosphotyrosine, anti-ERK2, or
anti-cPLA antibodies in TBSTG for 1-2 h, washed three
times for 15 min with TBSTG, and incubated with secondary antibodies
for 1 h in TBSTG. After an additional washing step antibody binding was
visualized using an ECL detection system from Amersham. Analysis of [
H]Arachidonic Acid
Release-Hepatocytes were labeled for 24 h with 1 µCi/ml
[
H]arachidonic acid ([
H]AA)
in Waymouth medium containing 0.1% bovine serum albumin. The mean total
radioactivity incorporated at time 0 was 3.1 ± 0.4
10
dpm/dish. The labeling medium was withdrawn, and cells
were extensively washed and equilibrated for 5 min prior to addition of
GH. When the effects of herbimycin A, verapamil, or mepacrine were
investigated, the cells were incubated for 20 h (herbimycin A), 60 min
(verapamil), or 20 min (mepacrine) in the presence of the inhibitor
prior to GH addition. Aliquots (0.2 ml) of medium (containing 0.1%
bovine serum albumin) were subsequently removed, without replacement,
at the indicated times. The samples were centrifuged, to clear the
medium from cells withdrawn with the media, prior to quantification of
released [
H]AA and its metabolites by
scintillation counting. At the conclusion of the experiment, the cells
were harvested and solubilized for determination of total
[
H]AA incorporation. Counts were corrected for
volume and total incorporation and, after subtracting the time 0
values, [
H]AA release was expressed as
disintegrations/min/dish.
DAG Mass Assay
The total amount of extractable DAG
was analyzed by using a DAG mass assay system from Amersham, based on a
method described by Preiss et al.(40) . The assay was
performed according to the manufacturer, with lipid extracts prepared
by the procedure of Bligh and Dyer
(41) .
Solution Hybridization
Total nucleic acids were
prepared from pooled cells from three to five culture dishes, as
described previously
(39) . The concentration of nucleic acids in
total nucleic acid samples was measured spectrophotometrically, and the
DNA concentration was quantified using a fluorometric
assay
(42) .
S-UTP-labeled cRNA probes in
solution hybridization assays, as described
previously
(39, 43) . Quantification of the mRNA species
was achieved by comparison with standard curves obtained from
hybridizations to liver total nucleic acids, calibrated to known
amounts of in vitro synthesized mRNA. Samples were analyzed in
triplicate, and the results are expressed as attomoles of mRNA per
µg of DNA. The interassay variations were controlled by using
internal total nucleic acids standards prepared from normal livers. The
interassay variation averaged 10%.
Statistical Analysis
All experiments were
performed two to five times, with cells obtained from different rats.
Results are expressed as the average of two experiments or, when more
than two identical experiments were carried out, as the mean ±
S.E. Ranges (broken error bars) are given where the results are
expressed as the average of two experiments.
, GH dependent phosphorylation
of these enzymes was studied by immunoblotting of proteins from rat
hepatocytes cultured in the absence or presence of GH. Western blot
analysis with phosphotyrosine antibody revealed GH stimulated
tyrosyl-phosphorylation of proteins with the apparent molecular masses
of 42 (p42) and 44 (p44) kDa (Fig. 1A), indicating
phosphorylation and activation of MAP kinases (ERK2 and ERK1,
respectively). Indeed, these proteins comigrated with 42 and 44 kDa
proteins which were recognized by an anti-ERK2 antibody (data not
shown). This observation might implicate GH-mediated activation of
signal transducers downstream of these kinases, such as
cPLA
. When immunoblot analysis of cPLA
was
performed, a 100-kDa protein (p100) was detected which showed reduced
electrophoretic mobility in GH treated cells (Fig. 1B).
Decreased electrophoretic mobility of cPLA
has previously
been observed in agonist treated cells
(33, 44) , or upon
in vitro phosphorylation of the cPLA
protein by
p42 MAP kinase
(29, 45) . The predicted molecular mass of
cPLA
is 85 kDa
(46, 47) ; however, the
apparent size determined by gel electrophoresis has been reported to be
slightly higher (95-110
kDa)
(31, 32, 44, 48, 49, 50) .
The GH-dependent decrease in electophoretic mobility of p100 was
blocked in the presence of the tyrosine kinase inhibitor herbimycin A
(Fig. 1B), previously shown to block GH-dependent
tyrosyl-phosphorylation of the GH receptor
(14) . The
GH-stimulated tyrosyl-phosphorylation of p42 and p44 was also inhibited
(Fig. 1A).
Figure 1:
GH
mediated tyrosyl-phosphorylation of MAP kinases and mobility shift of
cPLA in rat hepatocyte. Rat hepatocytes were cultured in
the absence or presence of GH (50 ng/ml) for 30 min with or without
herbimycin A (2 µM) (H) pretreatment, as indicated in the
figure. A, cells were harvested in SDS-PAGE loading buffer,
proteins subjected to SDS-PAGE (12%), transferred to polyvinylidene
difluoride membrane, immunoblotted with a monoclonal phosphotyrosine
antibody, and developed by using the Amersham ECL system. B,
cells were harvested and lysed as described in the materials and
methods section, immunoprecipitated with a polyclonal cPLA
antibody, subjected to SDS-PAGE (6%), and immunoblotted using the
same cPLA
antibody.
Decreased electrophoretic mobility of
cPLA has been shown to be associated with increased
enzymatic activity
(44) . As demonstrated in
Fig. 2A, GH stimulated the release of
[
H]AA from rat hepatocytes labeled with
[
H]AA. A significant increase in the liberation
of [
H]AA from cellular phospholipids was evident
2 min after GH addition, and a 6-fold induction was observed after 20
min. The release of [
H]AA into the medium was
inhibited by herbimycin A (Fig. 2B), indicating a
correlation between inhibited tyrosyl-phosphorylation of p42 and p44,
blocked electrophoretic mobility shift of p100, and blocked PLA
activity. Moreover, the GH-induced liberation of
[
H]AA was blocked when the hepatocytes were
pretreated with the PLA
inhibitor mepacrine
(51) (Fig. 2B).
Figure 2:
Release of [H]AA
from hepatocytes treated with GH or A23187, or GH in the absence or
presence of herbimycin A, verapamil, or mepacrine. Primary cultures of
rat hepatocytes were labeled for 24 h with 1 µCi/ml
[
H]AA prior to the different treatments.
A, release of [
H]AA and its metabolites
from control (
) and GH-treated (50 ng/ml) (
) cells was
measured at different time-points. B, release of
[
H]AA from control (C) cells and cells
treated for 20 min with A23187 (10 µM) and GH (50 ng/ml)
in the absence or presence of herbimycin A (2 µM),
verapamil (150 µM), or mepacrine (40 µM) was
determined. The cells were pretreated for 20 h with herbimycin A
(H), for 60 min with verapamil (V), and for 20 min
with mepacrine (M) prior to the addition of GH. A23187 and
herbimycin A were dissolved in Me
SO, and verapamil and
mepacrine were dissolved in ethanol. Inset, dose-response
curve of mepacrine on GH-stimulated liberation of
[
H]AA. Data are given as disintegrations/min
released [
H]AA per dish. Each point is the mean
± S.E. of triplicate determinations in five (A) or four
(B) separate experiments.
The requirement of
Ca for full activity of most PLA
enzymes
is well
documented
(29, 44, 47, 49, 52) .
The effect of the Ca
channel blocker verapamil on the
GH-induced liberation of [
H]AA was therefore
investigated. As shown in Fig. 2B, pretreatment of the
cells with verapamil inhibited the GH-stimulated phospholipase
activity, whereas treatment of the hepatocytes with the
Ca
-mobilizing agent A23187 increased the levels of
[
H]AA in the medium. Taken together, these
results show that GH stimulates release of eicosanoids from primary
cultures of rat hepatocytes, in a tyrosine kinase- and
Ca
-dependent manner, which could be due to increased
cPLA
activity.
Figure 3:
Steady-state mRNA levels of P4502C12 and
IGF-I in GH-treated hepatocytes in the absence or presence of
mepacrine. A, Time course induction of P4502C12 ( and
) and
IGF-I (
and
) mRNA expression by GH, in the absence
(
and
) or presence (
and
) of mepacrine (40
µM). B, dose-response curves of mepacrine on
GH-stimulated P4502C12 (
) and IGF-I (
) mRNA levels after 8
h of treatment. Inset, dose-response curve of mepacrine on
8-bromo-cAMP-stimulated IGF-I mRNA levels. Mepacrine, dissolved in
ethanol, was added 20 min prior to GH or 8-bromo-cAMP (100
µM) treatment. Data are expressed as fold induction
compared to control cells receiving vehicle only, and represent
(A) average ± ranges, or (B) mean ±
S.E. of triplicate determinations in two or four separate experiments,
respectively.
Since higher doses of
mepacrine have been demonstrated to inhibit the actions of both
phospholipases A and C, by forming complexes with the
phospholipid substrate
(51) , we investigated the effect of
mepacrine on phospholipase C activity. The dose of mepacrine (40
µM), which completely blocked the effect of GH on
[
H]AA release and P450 mRNA induction, had no
significant effect on phospholipase C activity, as judged from the
unaffected GH stimulation of DAG production (Fig. 4).
Figure 4:
Time course of induction of DAG formation
by GH in the absence or presence of mepacrine. The production of DAG in
control cells () and cells treated with GH (50 ng/ml) in the
absence (
) or presence (
) of mepacrine (40 µM)
was analyzed at different time points. Results are expressed as
nanomoles of DAG per dish. Each point is the mean ± S.E. of
duplicate determinations in three separate
experiments.
If
PLA activation is involved in the GH regulation of P4502C12
or IGF-I, the inhibitory effect of mepacrine should be reversed by the
product of PLA
activity, AA. As shown in Fig. 5, the
mepacrine-dependent inhibition of GH-stimulated P4502C12 mRNA
expression was partially rescued by the addition of 10 µM
AA to the cells. This was not observed if AA was substituted with
linoleic acid or docosahexaenoic acid (data not shown). A complete
restoration of the GH effect was observed with repetitive additions of
AA to the cells, 100 nM added every 3 h over the 9-h treatment
time. AA by itself, regardless of how it was added, did not affect the
expression of CYP2C12 (data not shown). This may be due to
activation of more than one signaling pathway by GH, which somehow
converge on the CYP2C12 gene. The rescuing effect seen after
AA addition to the cells was specific for P4502C12 since IGF-1 mRNA
expression was not restored (Fig. 5). This may be interpreted to
mean that the inhibitory effect of mepacrine on IGF-I mRNA expression
involves other mechanisms in addition to PLA
inhibition.
Taken together, these results suggest an important role for PLA
and AA in the GH-mediated regulation of P4502C12, whereas the GH
induction of IGF-I might be more dependent on other signaling
molecules.
Figure 5:
Effect of AA on mepacrine inhibited
GH-dependent P4502C12 and IGF-I mRNA expression. P4502C12 (black
bars) and IGF-I (gray bars) mRNA levels were determined
in cells treated for 8 h with GH and with combinations of mepacrine (40
µM) and AA, as indicated in the figure. Data are expressed
as -fold induction over appropriate control, and represent the mean
± S.E. of four experiments. *p < 0.05, ***p < 0.001 compared to mepacrine/GH-treated
cells.
Since the GH-stimulated release of
[H]AA was found to be inhibited by the tyrosine
kinase inhibitor herbimycin A or the Ca
channel
blocker verapamil, these agents should also affect the GH-induced
accumulation of P4502C12 mRNA if PLA
is involved in the GH
regulation of P4502C12. The GH- mediated increase in P4502C12 mRNA
levels was dose-dependently inhibited by both herbimycin A
(Fig. 6A) and verapamil (Fig. 6B).
Interestingly, GH-stimulated expression of the IGF-I gene
appeared more sensitive toward tyrosine kinase inhibition than the
CYP2C12 gene, whereas the opposite was observed after blockage
of Ca
channels. Addition of 1 µM
herbimycin A, prior to GH treatment of the cells, totaly inhibited the
induction of IGF-I expression and reduced the CYP2C12 expression by 70%. Similar results were obtained with the tyrosine
kinase inhibitor tyrphostin. The presence of herbimycin A (1
µM) did not affect the 8-bromo-cAMP-induced expression of
IGF-I mRNA in the cells, only the GH-dependent induction was inhibited
(data not shown).
Figure 6:
Effects of herbimycin A and verapamil on
GH-induced mRNA levels of P4502C12 and IGF-I. Dose-response curves of
A, herbimycin A and B, verapamil on GH-stimulated
P4502C12 () and IGF-I (
) mRNA levels after 8 h of
treatment. Herbimycin A (dissolved in Me
SO) was added 20 h
prior to GH treatment, and verapamil (dissolved in ethanol) was added
60 min prior to GH treatment. Data are presented as -fold induction
over appropriate control. Results are expressed as mean ± S.E.
of triplicate determinations in three separate
experiments.
Pretreatment of the cells with 200 µM
verapamil almost completely blocked the GH induction of P4502C12 mRNA,
whereas the IGF-I expression was reduced by only 50%. To further
investigate the role of Ca in the GH regulation of
P4502C12 and IGF-I, GH dose-response studies were performed in the
absence or presence of the Ca
-mobilizing agent
A23187. As shown in Fig. 7, a dose-response shift in induction of
CYP2C12 expression by GH was obtained by addition of A23187.
P4502C12 mRNA was equally well induced by GH at 1 ng/ml in the presence
of A23187 as by GH at 10 ng/ml in the absence A23187, suggesting a role
for Ca
in GH signaling to the CYP2C12 gene.
Consistent with the observed difference in verapamil sensitivity
between the GH induced expression of P4502C12 and IGF-I mRNA,
respectively, no effect of A23187 on GH induced IGF-I mRNA levels could
be observed (Fig. 7).
Figure 7:
Effect of A23187 on GH induced P4502C12
and IGF-I mRNA expression. Dose-response curves of GH on P4502C12
( and
) and IGF-I (
and
) mRNA expression in
the absence (
and
) or presence (
and
) of
A23187 (0.2 µM). Results are expressed as average ±
ranges of triplicate determinations in two separate
experiments.
AA is known to be metabolized to a
spectrum of biologically active eicosanoids, which are thought to serve
as regulators of intracellular events. Three distinct pathways of AA
metabolism are known: the cyclooxygenase, the lipoxygenase
(54, 55) and the cytochrome P450 pathways
(56, 57) .
By using different inhibitors of these pathways we investigated the
need for active AA metabolizing enzymes in the GH-induced expression of
P4502C12 and IGF-I mRNA. As demonstrated in Fig. 8A,
ETYA, a nonselective inhibitior of AA metabolism
(58) dose-dependently decreased the GH induced accumulation
ofP4502C12 and IGF-I mRNA. Neither the cyclooxygenase inhibitor
indomethacin, nor the lipoxygenase inhibitor 5,8,11-eicosatrienoic
acid, shown to block the synthesis of prostaglandins
(59) and
leukotrienes
(60) , respectively, blocked the GH-induced
accumulation of these mRNA species. Instead, a small increase of the
GH-induced P4502C12 mRNA levels was observed both in the presence of 10
µM indomethacin (from 5.1 ± 0.9 to 6.9 ±
0.5, when expressed as -fold induction over control) and 1
µM 5,8,11-eicosatrienoic acid (from 5.9 ± 0.5 to
7.1 ± 0.8). Pretreatment of the hepatocytes with the P450
inhibitor SKF-525A caused a dose-dependent decrease in the
GH-stimulated P4502C12 and IGF-I mRNA expression
(Fig. 8B). Again, a difference in sensitivity toward
inhibition of AA metabolism was observed, with P4502C12 expression
being more sensitive than that of IGF-I. Similar results were obtained
with the P450 inhibitor ketoconazole, whereas metyrapone was less
effective (data not shown). As shown in Fig. 9, the rescuing
effect of AA on the mepacrine inhibited GH induction of P4502C12 mRNA
(cf. above and Fig. 5) was blocked by ETYA or SKF-525A,
indicating that the rescuing effect of AA is mediated by an eicosanoid
formed via P450-catalyzed metabolism of AA.
Figure 8:
Dose-response of ETYA and SKF-525A on GH
induced P4502C12 and IGF-I mRNA expression. P4502C12 () and IGF-I
(
) mRNA levels were determined in hepatocytes treated with GH for
8 h in the presence or absence of different concentrations of
A, ETYA or B, SKF-525A, both dissolved in ethanol.
The inhibitors were added 20 min prior to GH treatment. Data are
expressed as -fold induction over control and represent mean ±
S.E. of three experiments.
Figure 9:
Effects of ETYA and SKF-525A on AA induced
P452C12 mRNA expression. P4502C12 mRNA levels were determined in cells
treated for 8 h with GH alone or in combination with mepacrine (40
µM), AA (10 µM), ETYA (50 µM),
or SKF-525A (50 µM) as indicated in the figure. Results
are expressed as -fold induction over appropriate control and represent
the average of two experiments with range.
is
serine-phosphorylated and activated by p42 MAP kinase in
agonist-treated cells
(29) . The GH-increased
tyrosyl-phosphorylation of MAP kinase in rat hepatocytes
(26) suggests that GH signaling in hepatocytes include MAP
kinase, as well as activation of signal transducers downstream of these
kinases, such as cPLA
. Results obtained in this study
demonstrate simultaneous GH-dependent tyrosyl-phosphorylation of p42
MAP kinase, reduced electrophoretic mobility of a 100-kDa protein
immunologically related to cPLA
, and liberation of
[
H]AA from labeled cells, indicating a role for
cPLA
in GH signal transduction in rat hepatocytes.
. The increase in [
H]AA release
could result from any PLA
activity but also from the
sequential enzymatic actions of PLA
and lysophospholipase,
or from phospholipases C or D and diglyceride lipase. The PLA
route of AA formation was favored by the observation that agents
known to block the enzymatic activity of PLA
, such as
mepacrine and verapamil, inhibited the GH-stimulated release of AA from
the cells. The most compelling evidence for cPLA
involvement was the demonstration that the GH-stimulated
liberation of AA was paralleled by a GH-induced mobility shift of a
protein with the expected molecular mass of cPLA
(95-110 kDa) in Western blotting with an anti-cPLA
antibody.
has been shown to translocate from
cytosol to membranes in a Ca
-dependent
manner
(61) , and therefore Ca
-dependent
activation of cPLA
presumably occurs through enhanced
enzyme-substrate interaction. This is in line with our observation that
liberation of AA could be induced by the
Ca
-mobilizing agent A23187 and inhibited by the
Ca
channel blocker verapamil. Similarly, the
GH-induced expression of CYP2C12 was augmented by A23187 and
blocked by verapamil. Thus, a correlation between Ca
levels, eicosanoid production, and GH signaling to the
CYP2C12 gene seems to exist. Interestingly, Schwartz et
al.(62) have shown that GH increases intracellular levels
of Ca
in fat cells, an effect which has been
suggested to be due, at least partially, to the activation of
Ca
channels of the L-type. These authors
have also shown that verapamil inhibits metabolic effects of GH (63,
64). Whether GH affects Ca
influx in hepatocytes has
yet to be determined.
in rat hepatocytes. However, it should
be mentioned that a 14-kDa type II PLA
has been cloned from
rat liver, and the mRNA encoding this enzyme is detected in freshly
isolated hepatocytes
(65) . The type II 14-kDa PLA
appears to function both as a cell-associated enzyme and
extracellularly when released in response to proinflammatory mediators.
It is well known that the catalytic activity of this secreted enzyme is
dependent on Ca
(61) . It can therefore not be
excluded that also other types of PLA
enzymes than
cPLA
contribute to the increased AA release after A23187 or
GH treatment of hepatocytes.
enzymes catalyze the
hydrolysis of the sn-2-acyl chain of phospholipid substrates
to yield fatty acids and lysophospholipids. This reaction is of
particular significance when the fatty acid is AA, since AA and its
metabolites have been shown to act as first and second messengers
affecting a number of cellular processes, including blood clotting,
inflammation, vascular tone, renal function, and
reproduction
(55, 56, 66, 67) .
Furthermore, eicosanoids have recently been implicated in the
regulation of various genes, such as the murine stearoyl-CoA desaturase
gene
(68) , the rat CYP4A1(69) , and human heat
shock genes (70). AA and eicosanoids, derived mainly through the
cyclooxygenase and lipoxygenase pathways, have been shown to stimulate
the expression of proto-oncogenes in various cell
systems
(71, 72, 73, 74, 75) .
Neither products of the the cyclooxygenase nor the lipoxygenase pathway
appears to be involved in the regulation of CYP2C12, since
inhibitors of these pathways did not reduce GH-stimulated levels of
P4502C12 mRNA. However, SKF-525A and ketoconazol, as well as ETYA,
inhibitors of P450 dependent oxidation of AA into epoxidated and
hydroxylated metabolites
(76, 77) , significantly
decreased the GH-induced expression of CYP2C12.
and
-1 alcohols
(57) . Some of the constitutively expressed
P450 forms in rat liver which have been shown to metabolize AA (see
Ref. 78 and references therein) are expressed in primary rat
hepatocytes when cultured as in this study
(79) . Biological
effects exerted by eicosanoids derived through the P450 pathway have
mainly been studied in extrahepatic tissues. However, 14,15-EET has
been shown to increase cytosolic Ca
concentrations
and to stimulate glycogenolysis in rat hepatocytes
(80) . In
attempts to identify the eicosanoid(s) that mediates the GH signaling
to the CYP2C12 gene, various eicosanoids that could be formed
by P450-catalyzed metabolism of AA were added to the hepatocytes.
Different concentrations of the four regioisomeric EETs, 5,6-, 8,9-,
11,12-, and 14,15-EET, the 14,15-dihydroxyeicosatrienoic acid, or
20-hydroxyeicosatetraenoic acid were added according to the protocols
used for AA treatments (cf. above). In some but not all
experiments, the 11,12- and the 14,15-EETs were found to have a similar
effect as AA on the P4502C12 mRNA expression (data not shown). The lack
of consistent results could be due to rapid inactivation of these
compounds when added to the cells, and further work is required to
resolve what eicosanoid is involved.
activation and subsequent P450-catalyzed formation of an active
AA metabolite. This PLA
- and P450-dependent signaling
molecule was shown to be necessary but not sufficient for GH regulation
of P4502C12 mRNA levels, indicating the importance of other
GH-activated factors, yet to be identified. Possible candidates for
such factors include members of the family of STAT proteins, recently
shown to be tyrosyl-phosphorylated and activated in response to
GH
(24, 25, 26) . The observation that GH
regulation of IGF-I expression was more dependent on tyrosine kinase
activity, and less sensitive toward inhibition of eicosanoid production
or Ca
influx, indicates that GH signaling leading to
increased expression of the IGF-I gene is different from
signaling to the CYP2C12 gene. It is obvious that GH can
activate various signaling molecules and that the nature and importance
of different signaling events have to be identified for each specific
target gene. The existence of different GH receptor signal transduction
mechanisms could be a prerequisite for the broad range of physiological
actions exerted by GH.
, phospholipase A
;
cPLA
, cystolic phospholipase A
; AA, arachidonic
acid; PAGE, polyacrylamide gel electrophoresis; IGF-I, insulin-like
growth factor I; ETYA, eicosatetraynoic acid; EET, epoxyeicosatrienoic
acid; DAG, diacylglycerol.
antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.