| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Josai University, 1-1, Keyakidai, Sakado City 350-0295, Japan
Address all correspondence and requests for reprints to: Masahiko Ogihara, Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Josai University, 1-1, Keyakidai, Sakado City 350-0295, Japan. E-mail: ogiharam{at}josai.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
dose-dependently inhibited PGE2-
and 17-phenyl-trinor-PGE2-induced hepatocyte mitogenesis.
Treatment with the EP1 receptor agonists significantly
increased the secretion of TGF-, reaching a maximum within 5 min.
The increase in TGF- secretion was blocked by SC-51322,
U-73122, somatostatin, and verapamil and potentiated by ionomycin.
These results indicate that the proliferative mechanisms of action of
EP1 receptor agonists are mediated through an increase in
the autocrine secretion of TGF-, which is dependent on the
EP1 receptor/G-protein involved in PLC
regulation/PLC/Ca2+ system. The locally secreted
TGF-, in turn, acts as a complete mitogen that stimulates the
tyrosine kinase/MAPK pathway in these cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, rapidly induce DNA synthesis and proliferation in
serum-free primary cultures of adult rat hepatocytes
(1, 2, 3, 4). In addition, the action of these growth factors is
modulated differently by - and ß-adrenergic agonists.
According to the results of previous studies, two classes of mitogens
can be defined: 1) complete (direct or primary) mitogens
(e.g. EGF, HGF, PDGF, and TGF-), which, by themselves, in
serum-free conditions can stimulate DNA synthesis and proliferation in
quiescent hepatocyte populations; and 2) incomplete (secondary)
mitogens or comitogens (e.g. catecholamines and PGs), which,
by themselves, cannot stimulate proliferation in serum-free primary
hepatocyte cultures but appear to promote cell growth by acting
permissively in combination with complete mitogens (5, 6, 7, 8, 9).
The relative importance of each factor in vitro and in
vivo remains to be elucidated, and interactions between them are
little understood as yet. Among the substances classed as incomplete mitogens, PGs have diverse biological functions as chemical mediators for the maintenance of local homeostasis in nearly all mammalian tissues, including regulatory functions linked to inflammation, platelet aggregation, and contraction of smooth muscles (10). PGs are also thought to be involved in liver regeneration, because PGE2 in rat liver increases after partial hepatectomy and also because indomethacin, which is responsible for inhibiting DNA synthesis in the regenerating liver, prevents increases in PGE2 (11, 12). PGE2 is reported to be the main prostanoid secreted by sinusoidal endothelial cells in culture. Effects of exogenous PGs on hepatocyte DNA synthesis have also been studied in primary cultures of adult rat hepatocytes (13, 14, 15, 16, 17). Although the in vitro system has been useful for clarifying their mechanism of action, early studies examined the growth-promoting effects of PGs only in the presence of complete mitogens such as EGF and insulin. These primary mitogenic growth factors, which strongly influence hepatocyte DNA synthesis and proliferation by themselves, may in fact modulate the action of various PGs, even at lower concentrations. Therefore, little is known about the detailed dose-response relationships in PG-induced stimulation of hepatocyte growth responses and the cellular physiological mechanisms of PGs in the absence of exogenously added complete mitogens.
PGs produce this broad range of biological actions in various tissues
by binding to specific receptors on the plasma membrane. Recently,
various types of receptors for PGE2,
PGF2, PGI2,
PGD2, and thromboxane A2
have been characterized using the pharmacological approach
(10). PG receptors are members of the superfamily of G
protein-coupled receptors that appear to have a single subunit
structure containing seven membrane-spanning regions. Among them, there
is further subdivision of the prostaglandin E2 (EP)
receptors into four subtypes, recently defined at the molecular level:
EP1, EP2,
EP3, and EP4
(18, 19, 20, 21, 22). These subtypes are reported to differ somewhat
in their signal transduction mechanisms.
We have recently shown that PGs from different series alone can
stimulate DNA synthesis and proliferation in primary cultures of adult
rat hepatocytes (23). This was an unexpected finding, for
PGs are regarded as comitogenic growth factors that enhance the effects
of primary mitogens such as insulin and EGF (5, 6, 7, 8, 9). In
addition, the EP1 receptor subtype is not
reportedly involved in the proliferative actions of
PGE2 in primary cultured adult rat hepatocytes
(17). The main purpose of the present study, therefore,
was to investigate early stages in the growth-promoting effects of
several EP receptor agonists [naturally occurring
PGE2, its analog
17-phenyl-trinor-PGE2
(17-pt-PGE2), sulprostone, and
11-deoxy-PGE1] on DNA synthesis and
proliferation in primary cultures of adult rat hepatocytes and to
investigate the EP receptor subtype mediation of these prostanoids in
relation to the mechanism of intracellular signal transduction. Our
results showed that among the EP receptors, only the
EP1 receptor subtype was responsible for the
proliferative actions on primary cultured hepatocytes, which is in
direct contrast to the findings of the previous report
(17). As to the mechanism of intracellular signal
transduction, the EP1 receptor/G-protein involved
in PLC regulation (Gq)/PLC/Ca2+ pathway and the
tyrosine kinase/MAPK signaling pathway are closely implicated in the
effects of EP1 receptor agonists on hepatocyte
DNA synthesis and proliferation. In addition, we demonstrate that by
stimulating the EP1
receptor/Gq/PLC/Ca2+ pathway, the
EP1 receptor agonists induce secretion of
TGF-, which in turn increases hepatocyte mitogenesis via the
tyrosine kinase/MAPK pathway. These findings indicate a novel mechanism
of autocrine action for the indirect mitogen EP1
receptor subtype agonists PGE2 and
17-pt-PGE2 and, to a lesser extent,
sulprostone.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Hepatocyte isolation and culture
Rats were anesthetized by ip injection of sodium pentobarbital
(45 mg/kg). Hepatocytes were isolated from the normal liver by the
two-step in situ collagenase perfusion technique devised by
Seglen (24) to facilitate disaggregation of the adult rat
liver. In brief, dispersed hepatocytes were washed three times by
slow centrifugation (50 x g, 1 min) of the cell
suspension to remove cell debris, damaged cells, and nonparenchymal
cells. Viability as tested by Trypan blue exclusion was more than 97%.
Unless otherwise indicated, freshly isolated hepatocytes were plated
onto collagen-coated plastic culture dishes (Sumitomo Bakelite Co.,
Tokyo, Japan) at a density of 3.3 x 104
cells/cm2 (3.0 x 105
cells/35-mm dish) and allowed to attach for 3 h on collagen-coated
dishes in Williams’ medium E containing 5% newborn calf serum, 0.1
nM dexamethasone, 100 U/ml penicillin, 100
µg/ml streptomycin, and 0.10 µg/ml aprotinin in 5%
CO2 in air at 37 C. The medium was then replaced
by aspiration, and the cells were cultured further in serum- and
dexamethasone-free Williams’ medium E supplemented with PGs such as
PGE2, 17-pt-PGE2,
sulprostone, and 11-deoxy-PGE1. When appropriate,
the following agents were added: PGs with or without SC-51322, U-73122,
U-73343, sphingosine, metaproterenol, 8-bromo-cAMP, UK-14304, H-89,
2,4-dideoxyadenosine (DDA), ionomycin, A23187, verapamil,
diltiazem, somatostatin, and inhibitors of growth-related
signal-transducing elements (i.e. genistein, wortmannin,
PD98059, and rapamycin).
Measurement of DNA synthesis
Hepatocyte DNA synthesis was assessed by measuring the
incorporation of [3H]thymidine into
acid-precipitable materials. Briefly, after an initial attachment
period of 3 h, the hepatocytes were washed twice with serum-free
Williams’ medium E and cultured in a medium containing several PGs
with or without testing agents for an additional 4 h or 21 h.
The cells were pulsed at 1, 2, 3, or 19 h after PG stimulation for
2 h with [3H]thymidine (1.0 µCi/well)
followed by 10% trichloroacetic acid precipitation as described
previously (25). [3H]Thymidine
incorporation into DNA was counted using a scintillation counter as cpm
and normalized for cellular protein. Aphidicolin (10 µg/ml) was added
to some wells to establish the level of nonreplicative DNA synthesis.
Hepatocyte protein content was measured by a modified Lowry procedure
with BSA as a standard (26). Data are expressed as
dpm/h/mg cellular protein.
Counting nuclei
The number of nuclei rather than the number of cells was counted
using a modified version of the procedure previously described by
Nakamura et al. (27). Briefly, the primary
cultured hepatocytes were washed twice with 2 ml of Dulbecco’s PBS (pH
7.4). Then, isolated liver cell nuclei were prepared for quantitation
by exposure of the hepatocyte cultures to 0.25 ml of citric acid (0.1
M) containing Triton X-100 (0.1%) for 30 min at
37 C. An equal volume of the nucleus suspension was mixed with Trypan
blue (0.3%) in Dulbecco’s PBS (pH 7.4), and the number of nuclei were
counted in a hemocytometer. This procedure was performed because the
hepatocytes had firmly attached to the collagen-coated plates and were
not dispersed sufficiently by EDTA (0.02%)-trypsin (0.05%)
treatment.
Neutralization of endogenous growth factors
For experiments using the neutralizing antibodies, serum-free
primary cultured hepatocytes were treated with varying concentrations
of EP1 receptor agonists in the presence and
absence of monoclonal antibodies against EGF, HGF, IGF-I, and
TGF- (12.5, 25, 50, 75, and 100 ng/ml).
ELISA for TGF-
TGF- secretion into a culture medium after the addition of EP
receptor agonists was determined by ELISA kits for TGF-. Because
unknown ingredient(s) of Williams’ medium E or MEM interfere with the
ELISA assay system, we measured TGF- secretion in PBS (pH 7.4) that
contained 1.0 mM CaCl2 and 0.10
µg/ml aprotinin. In brief, hepatocytes were cultured in
serum-containing Williams’ medium E at a density of 6.0 x
104 cells/cm2. After
attachment for 3 h, the cultures were washed three times with PBS
(pH 7.4). The culture medium was replaced with 1.0 ml of PBS containing
1.0 mM CaCl2 and 0.10 µg/ml
aprotinin. Hepatocytes were preincubated in the conditioned medium for
5 min in humidified 5% CO2-95% air at 37 C and
then incubated with EP1 receptor agonists and/or
some pharmacological agents for various time periods. At each time
point, the conditioned medium (50 µl) was collected and the medium
samples were tested with an ELISA for TGF- according to the
manufacturer’s instructions (Calbiochem, Cambridge, MA).
Absorbances were determined at a wavelength of 490 nm using a
microplate reader (Bio-Rad Laboratories, Inc., Tokyo,
Japan). A standard curve was obtained in a linear range from 25–800
pg/ml at a minimum detectable limit of about 12.5 pg/ml.
Measurement of MAPK activity
Phosphorylated MAPK isoforms (ERK 1 and ERK 2) were identified
by Western blot analysis using anti-phospho-MAPK monoclonal antibody.
In brief, cultured hepatocytes were washed once with ice-cold PBS (pH
7.4) and 0.2 ml of lysis buffer was added, then the hepatocytes were
harvested. After centrifugation at 16,300 x g for 30
min at 4 C, the cell lysates were denatured in boiling water for 5 min.
Samples of the supernatant (50 µg of protein) were subjected to
SDS-PAGE using a 10% acrylamide resolving gel by the method of
Laemmli. After electrophoresis, proteins were transferred to
immobilon-P membranes (28).
For the detection of phosphorylated ERK1 [44 kDa (p44)] and ERK2 [42 kDa (p42)], the sheets were immersed in Tris-buffered saline containing 1% BSA. The sheets were then incubated with an antibody (1:2000 dilution) against phospho-MAPK and washed as described. Antibody binding was detected by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) with donkey antirabbit IgG conjugated to horseradish peroxidase (1:3000 dilution). Densitometric analysis was performed using the NIH Image program version 1.60 for Macintosh. The data were calculated in arbitrary units and expressed as mean ± SEM.
Materials
The following reagents were obtained from Sigma
(St. Louis, MO): aphidicolin, ionomycin, A23187, UK-14304
(5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline),
metaproterenol hemisulfate, 8-bromo-cAMP
(8-bromoadenosine-3'-5'-cyclophosphate sodium), dexamethasone,
somatostatin, verapamil hydrochloride, diltiazem
hydrochloride and aprotinin. 17-pt-PGE2,
PGE2, and SC-51322
(2-[3-[(2-furanylmethyl)-thiol]-1-oxopropyl]hydrazide) were
obtained from BIOMOL Research Laboratories, Inc. (Plymouth
Meeting, PA). 11-Deoxy-PGE1 and sulprostone
were from Cayman Chemical Co. (Ann Arbor, MI). U-73122
(1-[6-[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]hexyl]-1H-pyrol-2,5-dione),
U-73343
(1-[6-[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]hexyl]-2,5-pyrrolidine-dione),
sphingosine, DDA, and H-89
(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
dihydrochloride) were obtained from BIOMOL Research Laboratories, Inc., as were genistein, wortmannin, and rapamycin. PD98059
(2'-amino-3'-methoxyflavone) was obtained from
Calbiochem-Behring (La Jolla, CA). Monoclonal antibodies
against EGF, HGF, IGF-I, and TGF- were obtained from BIOMOL Research Laboratories, Inc. Recombinant human TGF- was
obtained from PeproTech, Inc. (Rocky Hill, NJ). Williams’ medium E and
newborn calf serum were purchased from Flow Laboratories (Irvine,
Scotland). Collagenase (type II) was obtained from Worthington Biochemical Corp. Co. (Freehold, NJ).
[methyl-3H]Thymidine (20
Ci/mmol) was purchased from NEN Life Science Products
(Boston, MA). The ELISA kit for TGF- was obtained from
Calbiochem. Anti-phospho-p44/42 MAPK monoclonal antibody
was obtained from New England Biolabs, Inc. (Beverly, MA).
All other reagents were of analytical grade.
Statistical analysis
Data are expressed as means ± SEM. Group
comparisons were made by the ANOVA for unpaired data followed by post
hoc analysis using Dunnett’s multiple comparison test. Differences at
P < 0.05 were considered to be statistically
significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The reason why
cultured hepatocytes initiate DNA synthesis in such a short period
(2–2.5 h) is that the cells have already been primed during the 3-h
attachment period (i.e. culture conditions consisting
of a low cell density in 5% serum and a low dose of
dexamethasone-containing medium). The mitotic activity of the
hepatocytes in PGE2- and
17-pt-PGE2-treated cultures peaked at 4.0 and
3.5 h, respectively (Fig. 1B). Maximal stimulation for hepatocyte
DNA synthesis and proliferation seen with these agents was
approximately 6.0- and 1.3-fold, respectively. On the other hand, the
DNA synthetic activity of the
EP1/EP3 receptor subtype
agonist sulprostone (10-6
M) reached a plateau at about 4 h and was
sustained for 21 h. The number of nuclei induced by sulprostone
increased significantly at about 4.0 h after the addition (Fig. 1). In contrast, the
EP2/EP4 receptor subtype
agonist 11-deoxy-PGE1
(10-6 M) produced no
significant stimulation of hepatocyte DNA synthesis and proliferation
during the early (4 h) and late phases (21 h) of culture (Fig. 1).
|
). DNA synthesis stimulated by
17-pt-PGE2 was also dose dependent and reached a
plateau at 10-9 M, with an
ED50 of 1.8 x 10-10
M (Fig. 2A). 17-pt-PGE2 is about 2
orders of magnitude more potent than PGE2 in
stimulating hepatocyte DNA synthesis. Although
PGE2 was less potent than
17-pt-PGE2, the maximal response induced by
PGE2 was nearly the same. The relative magnitude
of the proliferating effects of PGE2 and
17-pt-PGE2 roughly corresponded to their
activities as stimulators of DNA synthesis, with
ED50 values of 2.5 x
10-8 M and 1.7 x
10-10 M, respectively (Fig.
2B). Sulprostone induced dose-dependent stimulation of hepatocyte
DNA synthesis, with an ED50 of 2.6 x
10-8 M (Fig. 2A). The maximal
induction by sulprostone was seen at a concentration of 2.8 x
10-7 M. The maximal responses
induced by sulprostone were significantly lower than those induced by
PGE2 and 17-pt-PGE2. The
relative magnitude of the proliferating effects of sulprostone
roughly corresponded to the stimulatory activity for DNA synthesis,
with an ED50 of 2.8 x
10-8 M (Fig. 2B). On the other hand,
the DNA synthetic and proliferative effects of
11-deoxy-PGE1 on cultured hepatocytes were
negligible, with concentrations ranging between
10-10 and 10-6
M (Fig. 2). Therefore, among the PGs we tested,
17-pt-PGE2 had the most potent DNA synthetic
and proliferative effects, followed by PGE2,
sulprostone, and 11-deoxy-PGE1 in that
order.
|
). SC-51322, when added alone, did not
affect hepatocyte DNA synthesis or proliferation. The results confirmed
that the EP1 subtype receptor is closely involved
in the proliferative actions of PGE2 and
17-pt-PGE2. Because the effects of the
EP1/EP3 receptor subtype
agonist sulprostone (10-6 M) on
hepatocyte DNA synthesis and proliferation were also blocked completely
by SC-51322 (10-7 to 10-6
M), the results suggest that the EP3
subtype of EP receptors contributed less than the
EP1 subtype to the proliferative action of
sulprostone on these cells. The very weak effects of the
EP2/EP4 receptor subtype
agonist 11-deoxy-PGE1
(10-6 M) on hepatocyte DNA synthesis
and proliferation were not influenced by SC-51322
(10-8 to 10-6
M). These results are in complete agreement with the
effects being mediated via EP1 receptor
subtypes.
|
2- and ß2-adrenergic
agonist, 8-bromo-cAMP, DDA, and H-89 on the hepatocyte DNA synthesis
and proliferation induced by PGE2 or
17-pt-PGE2),
suggesting that adenylate cyclase and PKA may not contribute to
hepatocyte mitogenesis induced by the EP1
receptor agonists. We also examined the effects of the
2- and ß2-adrenergic
agonists on EP1 receptor agonist-induced
hepatocyte DNA synthesis and proliferation during 4 h of culture.
As shown in Fig. 4, hepatocyte DNA synthesis and proliferation induced
by 10-6 M PGE2
or 10-9 M
17-pt-PGE2 was potentiated by the
2-adrenergic agonist UK-14304
(10-7 M), whereas it was inhibited
by the ß2-adrenergic agonist metaproterenol
(10-7 M) or a direct stimulator of
PKA, 8-bromo-cAMP (10-7 M). The
2-adrenergic agonist,
ß2-adrenergic agonist, and 8-bromo-cAMP by
themselves did not significantly influence hepatocyte DNA synthesis or
proliferation during 4 h of culture (data not shown).
|
). Neither U-73343
(10-6 M), a close structural analog
of U-73122 that has no such inhibitory action on PLC, nor sphingosine
(10-6 M), a PKC inhibitor,
significantly affected PGE2- or
17-pt-PGE2-induced hepatocyte DNA synthesis and
proliferation during 4 h of culture. These inhibitors on their own
did not show any significant effects on hepatocyte DNA synthesis and
proliferation during 4 h of culture (data not shown).
|
). In addition, somatostatin
(10-7 M), which inhibits the release
of certain gastrointestinal and pancreatic hormones, presumably by
affecting decreasing cytosolic Ca2+, strongly
inhibited hepatocyte DNA synthesis and proliferation induced by these
EP1 receptor subtype agonists. These inhibitors
and stimulators on their own did not influence hepatocyte DNA synthesis
and proliferation during 4 h of culture (data not shown). Our
results suggest that EP1 agonists induce
hepatocyte DNA synthesis and proliferation through the
receptor-mediated activation of PLC and an extracellular
Ca2+-dependent mechanism sensitive to calcium
channel blockers but that this process is independent of the activation
of PKC.
Effects of specific inhibitors of growth-related signal-transducing
elements on hepatocyte DNA synthesis and proliferation induced by
PGE2 or 17-pt-PGE2
We next investigated whether the mitogenic responses of primary
cultured hepatocytes to the EP1 receptor agonists
were mediated by signal transducers, such as receptor tyrosine kinase,
PI3K, MAPK kinase, and ribosomal protein S6 kinase (p70 S6K), by using
corresponding specific inhibitors of the signal transducers, namely
those of genistein, wortmannin, PD98059, and rapamycin. As shown in
Fig. 6, hepatocyte DNA synthesis and
proliferation induced by PGE2
(10-6 M) and
17-pt-PGE2 (10-9
M) was almost completely blocked by genistein (5 x
10-6 M), wortmannin
(10-7 M), PD98059
(10-6 M), and rapamycin (10 ng/ml).
These inhibitors on their own did not affect the hepatocyte DNA
synthesis and proliferation during 4 h of culture (data not
shown). In terms of the intracellular signal transduction mechanisms of
action of the EP1 receptor agonists, these
pharmacological characterizations suggest that receptor tyrosine
kinase, PI3K, MAPK kinase, and p70 S6K are closely involved in
hepatocyte DNA synthesis and proliferation.
|
or IGF-I on
hepatocyte DNA synthesis and proliferation induced by PGE2
or17-pt-PGE2 and 6 show that EP1 receptor
agonist-induced hepatocyte DNA synthesis and proliferation are
mediated through both the EP1
receptor/Gq/PLC/Ca2+ pathway and the receptor
tyrosine kinase/MAPK cascade. However, how these associate with each
other remains to be elucidated. Because PGs on their own have been
reported to act as comitogens in vivo and in
vitro, we hypothesized that the EP1 receptor
agonists would selectively induce the secretion of primary mitogen(s)
in an autocrine manner to stimulate hepatocyte mitogenesis. TGF-
or IGF-I are potential candidates for the primary growth factor,
because hepatocytes express messages for TGF- and IGF-I and the
cells can synthesize and store these primary growth factors. To examine
the possibility that TGF- or IGF-I mediates
EP1 agonist-induced hepatocyte DNA synthesis
and proliferation in the primary culture system, we examined the
effects of neutralizing monoclonal antibodies against TGF- and IGF-I
and compared the results with those of EGF and HGF. Fig. 7 shows that the addition of a
neutralizing monoclonal antibody against TGF- dose-dependently
inhibited the growth-promoting effect of PGE2
(10-6 M) and
17-pt-PGE2 (10-9
M) on hepatocyte DNA synthesis and proliferation.
The median inhibitory concentration values for such synthesis
and proliferation at 4 h of culture were 28 and 36 ng/ml,
respectively. In contrast, the DNA synthetic and proliferative effects
of EP1 agonists were not affected significantly
by the treatment of hepatocytes with various concentrations of
monoclonal antibody against IGF-I (1–100 ng/ml), EGF (1–100
ng/ml; data not shown), and HGF (1–100 ng/ml; data not shown). These
monoclonal antibodies on their own did not significantly influence
hepatocyte DNA synthesis and proliferation during 4 h of culture
(data not shown). The results indicate that this elimination of the
effects of PGE2 (10-6
M) and 17-pt-PGE2
(10-9 M) was specific for
antibody against TGF-. Accordingly, a novel model can be proposed in
which the mitogenicity of the EP1 receptor
agonists is mediated by the autocrine secretion of TGF- and the
subsequent stimulation of DNA synthesis and proliferation in primary
cultures of adult rat hepatocytes.
|
secretion
by primary cultured hepatocytes: time course study and dose-response
relationship. After
PGE2 or 17-pt-PGE2
treatment, there was a rapid and marked increase in medium TGF-
levels compared with the control (Fig. 8A). A time course study showed that a
detectable increase began within 3 min of treatment with
PGE2 (10-6 M)
or 17-pt-PGE2 (10-9
M). The levels remained significantly increased for the
duration of the experiment. TGF- secretion stimulated by
17-pt-PGE2 (10-9
M) increased more rapidly and was significantly higher than
that induced by PGE2 (10-6
M) during a 5-min period (Fig. 8A). The medium TGF-
levels after stimulation with sulprostone (10-6
M) were significantly lower than those induced by
PGE2 (10-6 M)
or 17-pt-PGE2 (10-9
M) within a 30-min period.
11-Deoxy-PGE1 had no significant effects on
TGF- secretion. As shown in Fig. 8B, PGE2 and
17-pt-PGE2 induced a significant increase in the
medium TGF- levels in a dose-dependent manner, with
ED50 values of 2 x
10-8 M and 5 x
10-10 M, respectively. The treatment
of hepatocytes with 17-pt-PGE2
(10-11 to 10-7
M) resulted in a more potent increase in medium TGF-
levels than that of PGE2
(10-9 to 10-6
M). The addition of sulprostone
(10-9 to 10-6
M) to the culture mildly increased the medium levels of
TGF-, whereas the addition of 11-deoxy-PGE1
(10-9 to 10-6
M) had no significant effect. These findings provide direct
evidence for the DNA synthetic and proliferative role of
EP1 receptor agonists in primary cultured
hepatocytes.
|
by
primary cultured hepatocytes: effects of inhibitors or stimulators of
the adrenergic receptor/adenylate cyclase pathway and the
EP1 receptor/PLC pathway levels, we examined the effects of 2-
and ß2-adrenergic agonists on
EP1 receptor agonist-induced increase in medium
TGF- levels. As shown in Fig. 9A, the
increase in medium levels of TGF- induced by
10-6 M PGE2 or
10-9 M
17-pt-PGE2 was not affected significantly by the
2-adrenergic agonist UK-14304
(10-7 M), the
ß2-adrenergic agonist metaproterenol
(10-7 M), or 8-bromo-cAMP
(10-7 M). To test the possibility
that EP1 receptor agonists stimulate the increase
in medium levels of TGF- via an adenylate cyclase/PKA pathway, we
also investigated whether a direct inhibitor of adenylate cyclase, DDA,
and an inhibitor of protein kinase A, H-89, have inhibitory effects on
the medium levels of TGF- induced by PGE2
(10-6 M) or
17-pt-PGE2 (10-9
M). DDA (10-6 M) and
H-89 (10-7 M) did not affect
EP1 receptor agonist-induced increase in the
medium levels of TGF- (Fig. 9A); 10-6
M PGE2- or
10-9 M
17-pt-PGE2-induced secretion was not affected by
the PKC inhibitor sphingosine (10-6
M). Neither DDA, H-89, sphingosine,
2-adrenergic agonist,
ß2-adrenergic agonist, nor 8-bromo-cAMP alone
significantly influenced the medium levels of TGF- at 30 min of
culture (data not shown).
|
, PGE2
(10-6 M)-induced increase in the
medium levels of TGF- was completely blocked by the
EP1 receptor antagonist SC-51322
(10-7 M). This indicates that
TGF- is released from primary cultured hepatocytes as a result of
EP1 receptor stimulation. U-73122
(10-6 M) completely inhibited the
PGE2 (10-6
M)-induced increase in medium TGF- levels, suggesting
that PLC is closely involved in TGF- secretion by hepatocytes.
U-73343 (10-6 M), a close structural
analog of U-73122, which has no such inhibitory action on PLC, did not
significantly affect PGE2
(10-6 M)-induced increase in medium
TGF- levels.
Next, we evaluated the effects of somatostatin, which inhibits the
release of certain gastrointestinal and pancreatic hormones, presumably
by affecting decreasing cytosolic Ca2+ levels and
attenuating cAMP levels, on PGE2
(10-6 M)-induced increase in the
medium levels of TGF- and examined the signal transduction mechanism
involved. Treatment of cultured hepatocytes for 10 min with
somatostatin (10-7 M) caused a
marked inhibition of PGE2-induced TGF-
secretion. In addition, PGE2-induced TGF-
secretion was also blocked by verapamil (10-6
M) or diltiazem (10-6 M;
data not shown). On the other hand, this increased TGF- level was
potentiated by ionomycin (10-7 M) as
well as A23187 (10-6 M; data not
shown). These inhibitors or Ca2+ ionophores did
not affect the basal TGF- levels independently (data not shown). The
pharmacological profiles of the effects of SC-51322, U-73122,
somatostatin, Ca2+ channel blockers, and calcium
ionophores on 17-pt-PGE2
(10-9 M)-stimulated medium levels of
TGF- were very similar to those of PGE2 (Fig. 9B). The results suggest that activation of the
EP1 receptor/Gq/IP3 pathway and mobilization of
intracellular Ca2+ led to a rapid secretion of
TGF- from primary cultured hepatocytes into the conditioned medium.
These observations also indicate the need for influx of extracellular
calcium in the process of EP1 receptor
agonist-induced hepatocyte DNA synthesis and proliferation, which is
mediated through TGF- secretion.
EP1 receptor agonist-stimulated TGF- secretion by
primary cultured hepatocytes: effects of specific inhibitors on
growth-related signal transducers
To investigate the signal-transducing mechanisms that
mediate EP1 agonist-induced TGF- secretion, we
investigated whether or not this process is mediated by growth-related
signal transducers such as receptor tyrosine kinase, PI3K, MAPK kinase,
and p70 S6K by using corresponding specific inhibitors of the signal
transducers, namely genistein, wortmannin, PD98059, and rapamycin.
PGE2 (10-6 M)
and genistein (5 x 10-6 M),
when added in combination, caused a rapid increase in the medium levels
of TGF- (Fig. 10).
PGE2 (10-6
M)-induced TGF- secretion was unaffected by wortmannin
(10-7 M), PD98059
(10-6 M), and rapamycin (10 ng/ml).
These inhibitors did not affect the basal TGF- levels when added
alone. The pharmacological profiles of these inhibitors for
17-pt-PGE2 (10-9
M)-induced TGF- secretion were very similar to those of
PGE2 (Fig. 10), demonstrating that these
inhibitors of the signal transducers inhibited
PGE2- or 17-pt-PGE2-induced
hepatocyte DNA synthesis and proliferation without affecting TGF-
secretion.
|
and
EP1 receptor agonists can stimulate MAPK
activity. In addition, we examined the effects of the MAPK kinase
inhibitor PD98059 on MAPK activity induced by EP1
receptor agonists or TGF-. Fig. 11A shows that exogenously added
TGF- (human recombinant) caused an initial rapid increase in the
phosphorylation of MAPK isoform p42 MAPK, peaking about 3.0-fold at 5
min after the addition, followed by a sharp decrease.
PGE2 (10-6 M)
and 17-pt-PGE2 (10-9
M) stimulated the phosphorylation of p42 MAPK, peaking
about 3.0-fold at about 25 and 20 min, respectively, followed by slow
decreases. Incubation of hepatocytes with sulprostone
(10-6 M) caused MAPK activity to
increase slowly to a plateau that was 1.6-fold greater than the
control. In contrast, both TGF- and the EP1
receptor agonists did not significantly affect MAPK isoform p44 MAPK
activity (data not shown). Fig. 11B shows that PD98059
(10-6 M) completely abolished the
TGF- or EP1 receptor agonist-induced increase
in p42 MAPK activity. In addition, p42 MAPK activation induced by
EP1 receptor agonists was abolished by genistein
(5 x 10-6 M) or wortmannin
(10-7 M) treatment (data not shown).
These results demonstrate that TGF- and EP1
receptor agonist-induced hepatocyte DNA synthesis and proliferation is
actually mediated through the activation of p42 MAPK. Furthermore, the
results suggest that stimulation of hepatocyte DNA synthesis and
proliferation induced by the EP1 receptor
agonists was mediated through the activation of the tyrosine
kinase/PI3K/MAPK pathway.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and 2, we demonstrated that PGE2 and a synthetic
PGE2 analog, 17-pt-PGE2
(a specific EP1 receptor subtype agonist),
significantly induced DNA synthesis and proliferation in primary
cultures of adult rat hepatocytes in the absence of exogenously added
primary growth factors. Moreover, we demonstrated that sulprostone (an
EP1/EP3 subtype agonist)
was weakly mitogenic for primary cultured hepatocytes.
11-Deoxy-PGE1 (an
EP2/EP4 subtype agonist)
had no such effects. SC-51322, an EP1 receptor
subtype-specific antagonist (29), almost completely
blocked this EP1 receptor agonist-induced
hepatocyte DNA synthesis and proliferation (Fig. 3), confirming that
the EP1 receptor agonists apparently act as
complete mitogens in primary cultures of adult rat hepatocytes. These
results are in direct contrast to those of another report, which showed
EP3 receptor mediation, but not
EP1 receptor mediation, of hepatocyte DNA
synthesis and proliferation (17). That report also stated
that PGs act as comitogenic growth factors through the
EP3 subtype coupled with the Gi protein in
primary cultured adult rat hepatocytes. A possible reason for this
discrepancy may be related to differences in culture conditions
(e.g. hormonal condition and initial plating density). In
addition, we observed that the cell density-independent nature of the
EP1 receptor agonists for hepatocyte mitogenesis
at high plating densities (1.0 x 105
cells/cm2) are similar to those of insulin
(2), PDGF (3), IGF-II (30), and
TGF- (31) (data not shown). However, postreceptor
mechanisms responsible for the proliferative action of the
EP1 receptor agonists as well as intracellular
signal transduction mechanisms remain to be clarified.
It has been reported that signal transduction mechanisms appearing to
serve as mediators for prostanoid receptors include 1) stimulation of
adenylate cyclase via the Gs protein, 2) inhibition of adenylate
cyclase via the Gi protein, 3) stimulation of phosphatidylinositol-PLC
via Gq, and, possibly 4) increased intracellular
Ca2+ through a phosphatidylinositol-dependent
process (10). Accordingly, we investigated
pharmacologically which signal transduction pathways are mediated by
EP1 subtype receptor stimulation.
EP1 receptor agonist-stimulated hepatocyte
mitogenesis was inhibited by the PLC inhibitor U-73122
(32) but not by the inactive analog of U-73122
(i.e. U-73343) (Fig. 5). Therefore, the functional
EP1 receptors contained in hepatocytes in primary
culture appear to stimulate phosphatidylinositol-PLC and increase
Ca2+ mobilization by PGE2
or 17-pt-PGE2 as a primary mechanism in
EP1 receptor agonist-induced hepatocyte DNA
synthesis and proliferation. If this is true, then these responses
could also be stimulated by the Ca2+ ionophore
ionomycin or A23187, which increase Ca2+ influx
into cultured hepatocytes. Conversely, the Ca2+
channel blockers verapamil and diltiazem could inhibit such synthesis
and proliferation. The results shown in Fig. 5 are consistent with this
notion. On the other hand, it may be unlikely that Gq/PLC/PKC or
adenylate cyclase/PKA contributes to EP1
receptor agonist-induced mitogenesis, because an inhibitor of PKC,
sphingosine (33), a direct inhibitor of adenylate cyclase,
DDA (34), and an inhibitor of PKA, H-89 (35),
did not affects such DNA synthesis and proliferation (Fig. 4). However,
there seems to be cross-talk between the EP1
subtype pathway and an adenylate cyclase/cAMP pathway, because
EP1 receptor agonist-induced hepatocyte DNA
synthesis and proliferation was potentiated by UK-14304
(31) and inhibited by metaproterenol and 8-bromo-cAMP.
Specific inhibitors of growth-related signal-transducing elements such as genistein (36), wortmannin (37), PD98059 (38), and rapamycin (39) attenuated EP1 receptor agonist-stimulated hepatocyte DNA synthesis and proliferation (Fig. 6). This demonstrates that mitogenic signaling through the EP1 receptor agonist pathway also requires the activation of tyrosine kinase, PI3K, MAPK kinase, and p70 S6K. Other studies have reported that growth factors such as PDGF, EGF, and insulin all evoke autophosphorylation of tyrosine residue on their respective receptors, which leads to the activation of tyrosine kinase toward other cellular components, including PI3K, MAPK, and p70 S6K, which further results in DNA synthesis and subsequent cell proliferation (40, 41, 42). However, the complete cascade of sequential phosphorylation events remains to be definitively established.
Although both the EP1
receptor/Gq/PLC/Ca2+ signaling pathway and the
tyrosine kinase signaling cascade are critically involved in
EP1 receptor agonist-induced hepatocyte DNA
synthesis and proliferation, the links between the
EP1 receptor/Gq/PLC/Ca2+
pathway and the tyrosine kinase signaling cascade have not been
characterized clearly. We reported previously that an
1-adrenergic receptor- or vasopressin
receptor-mediated pathway, which uses the
PLC/Ca2+ pathway, has no significant effects on
hepatocyte DNA synthesis and proliferation but only modulates the
growth-promoting effects of primary growth factors such as EGF and HGF
(1, 4). Moreover, there is little evidence as yet that
EP1 receptor/Gq/PLC/Ca2+
pathways directly stimulate (or phosphorylate) elements of the tyrosine
kinase-signaling pathway to induce cell proliferation
(10). Therefore, any such association between the
EP1 receptor/Gq/PLC/Ca2+
pathway and the receptor tyrosine kinase system is speculative at this
time. To this end, therefore, we hypothesized that the
EP1 receptor/Gq/PLC/Ca2+
pathway strongly stimulates secretion of a certain mitogen by cultured
hepatocytes in an autocrine manner, which, in turn, induces the
hepatocyte DNA synthesis and proliferation through stimulation of the
downstream tyrosine kinase pathway. Two potential mitogens that could
fulfill this requirement are TGF- and IGF-I. TGF- and IGF-I are
reported to be cytokines that are synthesized and stored in
parenchymal hepatocytes, and they are among the most active growth
factors stimulating hepatocyte DNA synthesis and proliferation
(5, 6, 7, 8, 9). To verify this hypothesis, we examined the
effects of a monoclonal antibody against the putative growth factors
TGF- and IGF-I on EP1 receptor agonist-induced
hepatocyte DNA synthesis and proliferation (Fig. 7) and compared the
effects with those of monoclonal antibodies against HGF or EGF
(data not shown). PGE2- or
17-pt-PGE2-induced hepatocyte DNA synthesis
and proliferation was almost completely inhibited by a monoclonal
antibody against TGF- but not by those against IGF-I, HGF (data not
shown), or EGF (data not shown) (Fig. 7). Therefore, the present
results suggest that cytokine TGF- is stored within the parenchymal
hepatocytes and is triggered to secrete to the extracellular side via
EP1 receptor stimulation. The TGF- signaling
system, which we have been studying extensively in primary cultures of
adult rat hepatocytes (31), is mediated mainly through the
tyrosine kinase/PI3K/MAPK/p70 S6K pathway. In addition, if the
proliferative effects of EP1 receptor agonists
are mediated through the secretion of TGF-, the cell
density-independent nature of the EP1 receptor
agonists (data not shown) is consistent with that of TGF-
(31).
To confirm this hypothesis, it is important to demonstrate that
EP1 receptor agonist treatments actually lead to
TGF- secretion into culture medium. As shown in Fig. 8, we have
demonstrated for the first time that PGE2 and
17-pt-PGE2 rapidly stimulated TGF- secretion
into a conditioned medium; the maximal TGF- level was about 0.025
ng/ml. In contrast, we have previously shown that exogenously added
TGF- (human recombinant) can stimulate DNA synthesis and
proliferation in primary cultures of adult rat hepatocytes: 0.5 ng/ml
TGF- in the culture medium sufficiently stimulated DNA synthesis and
proliferation (31). Therefore, the levels of TGF- in
the culture medium are about 1 order of magnitude lower than those of
exogenously added TGF- to stimulate hepatocyte DNA synthesis and
proliferation. However, it is reasonable to consider the possibility
that local TGF- concentrations around the hepatocyte plasma membrane
may become transiently much higher than in other extracellular spaces
in the culture medium during times of autocrine secretion. Furthermore,
the results in Fig. 8B demonstrate that EP receptor agonists in the
order 17-pt-PGE2 > PGE2 >
sulprostone >> 11-deoxy-PGE1 secrete TGF-
into the conditioned medium. This may be one of the main reasons why
17-pt-PGE2, with considerably less
EP1/EP3 receptor agonist
sulprostone or EP2/EP4
receptor agonist 11-deoxy-PGE1, was more potent
than PGE2 in stimulating hepatocyte DNA synthesis
and proliferation (Fig. 2). The TGF--releasing action of the
EP1 receptor agonists, which is not a direct
proliferating action, sufficiently explains the phenomenon of increased
DNA synthesis and proliferation in primary culture of adult rat
hepatocytes.
To confirm links between EP1 receptor
agonist-induced TGF- secretion and hepatocyte mitogenesis, we
investigated the regulatory mechanisms associated with the rapid
TGF- secretion by EP1 receptor agonists.
TGF- secretion by primary cultured hepatocytes may be regulated by
the EP1
receptor/Gq/PLC/Ca2+ pathway (Fig. 9B), because
the stimulatory effects of these EP1 receptor
agonists on medium levels of TGF- were also antagonized by SC-51322,
inhibited by U-73122, verapamil, and diltiazem, and potentiated by
ionomycin. Blockade of EP1 receptor
agonist-induced TGF- secretion confirmed this hypothesis. In
addition, somatostatin strongly inhibited EP1
receptor agonist-induced hepatocyte DNA synthesis and proliferation
by inhibiting TGF- secretion (Fig. 9B). Together, these observations
show that Ca2+-mediated selective TGF-
secretion is an essential step in the stimulation of
EP1 receptor agonist-induced hepatocyte DNA
synthesis and proliferation. Within 5 min of treatment, hepatocytes
started to release TGF-, suggesting that TGF- increase in the
conditioned medium is not caused by de novo synthesis of
TGF- protein by hepatocytes. These results support the notion that
PGE2, 17-pt-PGE2, and, to a
lesser extent, sulprostone selectively act as TGF- releasers to
affect the hepatocyte’s acute phase response and that TGF-, in
turn, plays an essential role in the stimulation of hepatocyte DNA
synthesis and proliferation.
Conversely, inhibitors of growth-related signal-transducing
elements, such as genistein, wortmannin, PD98059, and rapamycin, had no
significant effects on EP1 receptor
agonist-induced increase in the medium levels of TGF- (Fig. 10).
Consequently, the suppression of EP1 receptor
subtype signaling by these specific inhibitors of growth-related
signal-transducing elements may occur downstream of TGF- secretion.
These inhibitors only blocked EP1 receptor
subtype signaling by inhibiting corresponding signal transducer
activities [e.g. tyrosine kinase and MAPK activity (Fig. 11)], thereby blocking hepatocyte DNA synthesis and proliferation
(Fig. 6). Together, our findings confirm that the ability of the
EP1 receptor agonists to promote hepatocyte
mitogenesis is attributable to an indirect autocrine effect of the
secreted TGF-. EP1 receptor agonist-induced
TGF- secretion is regulated by the EP1
receptor/Gq/PLC/Ca2+ pathway. This is a novel
mechanism of action compared with that of classic growth factors,
including insulin, EGF, and HGF, and a similar indirect mechanism on
cell growth has been reported in the human prostate cancer cell line
TSU-Pr1, in which the proliferative effects of TGF-ß are mediated
through the autocrine secretion of PDGF (43).
In conclusion, we provide here evidence that the
EP1
receptor/Gq/PLC/Ca2+-dependent autocrine
secretion of TGF- occurring in response to EP1
receptor activation is an essential step in the stimulation of DNA
synthesis and proliferation in primary cultures of adult rat
hepatocytes. The
EP2/EP3/EP4
receptor subtypes play only a minor role in this process. In addition,
our findings indicate the usefulness of culture conditions to the study
of the autocrine loop in the proliferation of adult rat hepatocytes.
Because PGE2 is the main prostanoid secreted by
sinusoidal endothelial cells (11, 12), we have proposed
that the hepatocyte proliferation regulation involves locally produced
PGE2, generated in response to hepatic injury,
acting as an inducer of hepatic regeneration in vivo.
TGF- is subsequently secreted by hepatocytes under the influence of
EP1 receptor stimulation to play a fundamental
role in the initiation of mitogenic responses. Further research on
these mechanisms involving PGs may shed light not only on growth
regulation of the liver but also on the therapeutic application of
prostanoids for hepatic injury.
|
Footnotes |
|---|
Received March 7, 2001.
Accepted for publication June 27, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
:
additivity with the effect of norepinephrine, and synergism with
epidermal growth factor. J Cell Physiol 159:35–40[CrossRef][Medline]
of DNA synthesis and proliferation of
adult rat hepatocytes in primary cultures: modulation by - and
ß-adrenoceptor agonist. J Pharmacol Exp Ther 291:171–180This article has been cited by other articles:
 |
|
 |
|
  C. Han, G. Li, K. Lim, M. C. DeFrances, C. R. Gandhi, and T. Wu Transgenic Expression of Cyclooxygenase-2 in Hepatocytes Accelerates Endotoxin-Induced Acute Liver Failure J. Immunol., December 1, 2008; 181(11): 8027 - 8035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Meisdalen, O. F. Dajani, T. Christoffersen, and D. Sandnes Prostaglandins Enhance Epidermal Growth Factor-Induced DNA Synthesis in Hepatocytes by Stimulation of E Prostanoid 3 and F Prostanoid Receptors J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1044 - 1050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. L. Lopes, J. Desmarais, S. Ledoux, N. Y. Gevry, P. Lefevre, and B. D. Murphy Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison J. Biol. Chem., August 25, 2006; 281(34): 24602 - 24611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ishikawa and P. L. Morris A Multistep Kinase-Based Sertoli Cell Autocrine-Amplifying Loop Regulates Prostaglandins, Their Receptors, and Cytokines Endocrinology, April 1, 2006; 147(4): 1706 - 1716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mendez and M. C. LaPointe PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2111 - H2117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-N. Cho, J. Y. Choi, C.-H. Kim, S. J. Baek, K. C. Chung, U. Y. Moon, K.-S. Kim, W.-J. Lee, J. S. Koo, and J.-H. Yoon Prostaglandin E2 Induces MUC8 Gene Expression via a Mechanism Involving ERK MAPK/RSK1/cAMP Response Element Binding Protein Activation in Human Airway Epithelial Cells J. Biol. Chem., February 25, 2005; 280(8): 6676 - 6681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Suganami, K. Mori, I. Tanaka, M. Mukoyama, A. Sugawara, H. Makino, S. Muro, K. Yahata, S. Ohuchida, T. Maruyama, et al. Role of Prostaglandin E Receptor EP1 Subtype in the Development of Renal Injury in Genetically Hypertensive Rats Hypertension, December 1, 2003; 42(6): 1183 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Fajas, S. Miard, M. R. Briggs, and J. Auwerx Selective cyclo-oxygenase-2 inhibitors impair adipocyte differentiation through inhibition of the clonal expansion phase J. Lipid Res., September 1, 2003; 44(9): 1652 - 1659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Walch, E. Clavarino, and P. L. Morris Prostaglandin (PG) FP and EP1 Receptors Mediate PGF2{alpha} and PGE2 Regulation of Interleukin-1{beta} Expression in Leydig Cell Progenitors Endocrinology, April 1, 2003; 144(4): 1284 - 1291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Battersby, S. C. Boddy, H. O. D. Critchley, and H. N. Jabbour Expression and Localization of Endothelial Monocyte-Activating Polypeptide II in the Human Endometrium across the Menstrual Cycle: Regulation of Expression by Prostaglandin E2 J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3928 - 3935. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
