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Original Studies |
-Hydroxylase/17,20-Lyase in Human Fetal Adrenal Cells via Protein Kinase C1
San Francisco Reproductive Endocrinology Center, University of California, San Francisco, San Francisco, California 94143
Address all correspondence and requests for reprints to: Robert B. Jaffe, M.D., Reproductive Endocrinology Center, University of California, San Francisco, San Francisco, California 94143-0556. E-mail: robert_jaffe{at}quickmail.ucsf.edu
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsDiscussionReferences |
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In primary cultures, CRH increased inositol trisphosphate. After CRH
treatment, inositol tris-, bis-, and monophosphates increased within 1
min, reaching maximal levels at 5 min. In contrast,
PGF2
, known to act via PKC, induced a sustained response
for up to 20 min. The response to CRH was dose dependent, maximal at 1
µmol/L at both 1 and 5 min. CRH increased DHEAS production, with a
much lesser effect on cortisol. CRH did not stimulate inositol
phospholipid in adult adrenal glands, suggesting that this pathway is
unique to the fetal adrenal. CRH increased messenger ribonucleic acid
encoding 17-hydroxylase/17,20 lyase (P450c17), but not
3ß-hydroxysteroid dehydrogenase/
4–5 isomerase.
However, 3ßHSD expression was stimulated by ACTH. PKC, but not PKA,
inhibitors blocked CRH-stimulated P450c17 induction, whereas PKA
inhibitors blocked ACTH-stimulated cortisol. Thus, CRH is coupled to
the phospholipase C-inositol phosphate second messenger system and
preferentially induces the expression of P450c17 and DHEAS, suggesting
a unique role of CRH regulating human fetal adrenal function via PKC.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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The source of DHEAS used by the placenta for estrogen production is the large, specialized inner compartment of the fetal adrenal gland, the fetal zone, which is unique to primates, including humans. The mechanism(s) by which DHEAS production is regulated in the fetal zone is incompletely understood. Growth and functional development of the fetal zone are primarily regulated by ACTH from the fetal pituitary gland (3, 7, 8). However, rapid growth of and DHEAS production by the fetal adrenal occur in the apparent absence of increasing ACTH concentrations (9). Thus, other factors specific to pregnancy may also play a role in human fetal adrenal steroidogenesis.
Placental CRH concentrations rise exponentially toward the end of human pregnancy (10). DHEAS in primate pregnancy also increases as term is approached (11). Therefore, CRH may serve as a secretagogue for fetal zone steroidogenesis. Placental CRH has been implicated in the timing of human parturition (1, 12, 13) and is a predictor of preterm labor (14). It is possible that CRH orchestrates a variety of events involved in the parturitional process, as CRH receptors also are present in the myometrium and may increase uterine contractility by decreasing cAMP production (15, 16). It has been suggested that CRH secretion into the fetal circulation may stimulate fetal pituitary ACTH, which could then stimulate adrenal cortisol secretion. As cortisol can stimulate placental CRH secretion (17), this could establish a feedforward loop, leading to the progressively increasing concentrations of placental CRH seen at the end of gestation.
In human fetal (18) and adult (19) pituitary glands, CRH acts via the classical adenylate cyclase-cAMP-protein kinase A (PKA) pathway; no alternative signaling pathways have been described. We explored whether CRH in the human fetal adrenal also acts through the PKA pathway. Further, as the fetal adrenal is a novel site for CRH action, and as other hypothalamic and pituitary peptides exert their actions via a phospholipase C (PLC)- protein kinase C (PKC) pathway (20, 21, 22), we also studied the PKC signaling pathway. Here we describe novel CRH signaling through the PLC-PKC pathway, and not the cAMP-PKA pathway, in the human fetal adrenal gland and the stimulation of steroidogenesis via this route.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Human fetal adrenal glands were obtained from second trimester fetuses (14–22 weeks gestation) after elective therapeutic termination of pregnancy by dilatation and evacuation. Gestational age was estimated by foot length. Glands were collected and immediately placed in ice-cold tissue culture medium. The experimental protocol was approved by the Committee on Human Research, University of California, San Francisco (UCSF).
ACTH-(1–24) (Cortrosyn) was obtained from Organon (West
Orange, NJ). CRH (human, rat synthetic) and
myo-[2-3H]inositol (SA, 20 Ci/mmol) were
obtained from Sigma Chemical Co. (St. Louis, MO).
-Helical CRH-(9–41) antagonist was obtained from Novabiochem (San
Diego, CA). Cell-permeable myristoylated PKA inhibitor (14–22 amide)
and myristoylated PKC inhibitor (19–27 amide) were obtained from
Calbiochem (La Jolla, CA) (23). The complementary DNA
(cDNA) encoding the human type II 3ßHSD enzyme was provided by Dr. J.
Simard, Centre de Recherche en Endocrinologie Moleculaire (Quebec,
Canada) (24), and the cDNA for P450c17 was provided by Dr. W. L.
Miller (UCSF) (25).
Fetal adrenal cortical cell culture
Primary cultures of midgestation fetal adrenal cortical
cells were prepared as previously described (26). Briefly, the capsule
with the adherent definitive zone was peeled away, and the fetal zone
cells were dispersed by enzymatic digestion with collagenase and then
plated onto 6-cm diameter plastic culture dishes (Falcon Plastics, Los
Angeles, CA). The culture medium used was Dulbecco’s Modified Eagles
H-16/Ham’s F-12 (1:1) containing nonessential amino acids and
antibiotics, supplemented with 10% FCS (UCSF Cell Culture Facility).
All plates were incubated in a humidified atmosphere of 95% air-5%
CO2 at 37C for 48 h before the first medium
change. Test substances, including PGF2 used as a
positive control and known to act through the PKC pathway, were then
added. Experimental conditions were varied depending on the objective
as described below. All experiments were replicated on adrenal cortical
cells obtained from at least three different fetuses.
Measurement of cAMP production and steroidogenesis
Cells were incubated with or without CRH (0.1 nmol/L to 10 µmol/L) or ACTH (0.1 or 1 nmol/L) for 10 min. Incubations were terminated by addition of an equal volume of 100 mmol/L sodium acetate (pH 4.0), followed by immersion in a water bath at 100 C for 10 min. The acidified and heat-treated samples were centrifuged at 1000 x g for 10 min, and cAMP content in the supernatant was measured by RIA. For steroidogenesis studies, cells were incubated for 24 h with or without CRH or ACTH, and media were collected for measurement of cortisol and DHEAS production by RIA, as described previously (1). In some experiments, inhibitors of PKA or PKC (100 µmol/L) were added 2 h before the addition of CRH or ACTH. Total RNA was extracted from the cells by the method of Chomczynski and Sacchi (27).
Isolation of [3H]inositol metabolites
Cells were incubated for 4–18 h with myo-[3H]inositol (10 µCi/mL), washed twice with medium containing 10 mmol/L inositol, and incubated in medium alone for 15 min. The majority of experiments were performed after labeling cells for 18 h. The 4-h studies were performed only with freshly isolated cells to investigate whether there was any difference in inositol phosphate production between freshly isolated vs. cultured cells. There were no significant differences in the degree of stimulation. The cells were incubated for an additional 15 min with LiCl (10 mmol/L). Test substances were then added, and incubations were terminated at different time points by the addition of ice-cold 10% trichloroacetic acid. The acid-precipitable proteins and lipids were removed by centrifugation. The acid-soluble fraction was extracted five times with diethyl ether to remove the tricholoroacetic acid. The samples were neutralized and loaded on columns of Bio-Rad AG 1-X8 ion exchange resin (formate form; Bio-Rad Laboratories, Inc., Hercules, CA) preequilibrated with 5 mmol/L inositol. Inositol metabolites were eluted from the column sequentially using the method described by Berridge et al. (28).
Effect of inhibitors of PKA and PKC on steroidogenesis
To define the role of CRH-stimulated phosphatidylinositol metabolism and subsequent activation of PKC, fetal adrenal cells were preincubated with cell-permeable PKA (100 µmol/L) and PKC (25–100 µmol/L) inhibitors for 2 h before the addition of either CRH (1 µmol/L) or ACTH (1 nmol/L). Supernatant media were collected for measuring DHEAS and cortisol production, and RNA was extracted from the cells for Northern analysis (see below). After incubation in the presence of either peptide inhibitor, 96% of the cells still excluded trypan blue dye, eliminating the possibility of a toxic effect. The total protein content of the cells also was measured.
Northern blot analysis
In the above experiments, after removal of supernatant medium for steroid measurement, total RNA was extracted, and 5–10 µg were subjected to electrophoresis on a 1.2% agarose gel. Subsequent probe preparation and hybridization were performed as described previously (29). The relative abundance of mRNA was measured with scanning densitometry using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). All data were normalized to the abundance of transcripts encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is constitutively expressed.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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In the first set of experiments, the activation of the classical
adenylate cyclase-cAMP-PKA pathway was investigated (Fig. 1
). Incubation of adrenal cortical cells
with ACTH (0.1 and 1 nmol/L) for 10 min caused a 10- to 14-fold
increase in cAMP production at a dose of 1 nmol/L. However, treatment
with CRH at increasing doses of 0.1 nmol/L to 10 mmol/L had no effect
on cAMP production.
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on inositol phosphate
accumulation
Treatment of adrenal cortical cells with CRH (1
µmol/L) for 5, 10, and 20 min rapidly increased inositol
trisphosphate (IP3) accumulation (Fig. 2). This increase was transient and was
maximal at 5 min. Inositol bisphosphate (IP2) and
monophosphate (IP) accumulation showed a similar trend, although of
lesser magnitude. In contrast, treatment with 1 µmol/L
PGF2 (Fig. 2, inset) caused a rapid, but
sustained, accumulation of all three metabolites,
IP3, IP2, and IP, over a
period of 20 min. Similar results were seen with accumulation of the
metabolite glycerophosphoinositol (data not shown). Levels of free
[3H]inositol were unchanged over the relatively
short times of incubation. However, great variability was seen in the
levels of incorporation of [3H]inositol, which
could be attributed to differences in gestational age (14–22 weeks) of
the fetal adrenals. Most of the inositol phosphate studies were
performed with fetal adrenals between 18–22 weeks gestation. Apart
from differences in basal levels of incorporation, there was no
difference in the degree of response. A consistent 5- to 7-fold
increase in IP3 production was noted. We do not
know whether changes in PLC activity would be seen at younger
gestational ages. Cumulative data (expressed as a percentage of the
control value) from two to four experiments using adrenals from three
fetuses of different gestational ages are shown in Table 1. The specificity of the response to CRH
was established using the CRH antagonist, -helical CRH-(9–41) at a
concentration of 100 µmol/L. A 50–60% inhibition of accumulation of
inositol metabolites was seen with subsequent CRH treatment (IP, 56%;
IP2, 57%; IP3, 48%).
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. Cells
were treated with CRH (10 nmol/L to 1 µmol/L) for 1 min (Fig. 3A) and
also at doses of 1 nmol/L to 1 µmol/L for 5 min (Fig. 3B). Both sets
of experiments showed a linear dose-dependent increase in accumulation
of all three metabolites. However, CRH failed to stimulate inositol
phospholipid accumulation in human adult adrenals under the same
circumstances. Table 2 shows the
effects of CRH and ACTH on inositol phosphate, cAMP production, and
steroidogenesis in two adult adrenal glands obtained from the UCSF
organ transplant service. These glands were dispersed with collagenase,
cultured, and treated under identical conditions as the fetal adrenals.
There was no effect on IP3,
IP2, or IP production with either CRH or ACTH
treatment. However, ACTH stimulated cAMP and cortisol production in
these cells. DHEAS levels remained unchanged with CRH treatment.
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To define the role of CRH-stimulated phosphatidylinositol metabolism and subsequent activation of PKC, fetal adrenal cells were treated with cell-permeable PKC and PKA inhibitors.
The inhibitors, when added by themselves, produced a 30% reduction in
total protein content over an incubation period of 24 h (data not
shown). Hence, all data were normalized to protein content. Figure 4 shows a 3-fold increase in DHEAS
production in response to CRH. This effect was blocked by the PKC
inhibitor at a dose of 100 µmol/L. The PKA inhibitor had no effect on
the CRH-stimulated response. Neither inhibitor when added alone
affected basal DHEAS production. A dose-dependent inhibition of
CRH-stimulated DHEAS production was seen with the PKC inhibitor (Fig. 5). Figure 5 also shows that treatment
with ACTH stimulated DHEAS production 6-fold and that this stimulation
was blocked by the PKA inhibitor. Figure 5 (inset)
illustrates cortisol production at the corresponding doses. CRH
treatment had minimal effect on cortisol production, with no
significant effect of PKC inhibitors. However, ACTH-stimulated cortisol
production 7- to 8-fold, and the PKA inhibitor blocked this response by
80%. Experiments on the steroidogenic activity of these cells were
performed with fetal adrenals at 14–19 weeks gestation. The difference
noted in gestational ages were that at younger gestational ages the
basal and stimulated levels of cortisol were lower, whereas the reverse
trend was noted in DHEAS levels.
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To investigate whether DHEAS production by the
CRH-stimulated PKC pathway was a result of specific induction of
enzymes involved in the steroidogenic pathway, Northern analysis of two
key enzymes, P450c17 and 3ßHSD, was performed. Figure 6 shows that CRH treatment of fetal
adrenal cells produced a 2- to 3-fold increase in P450c17 mRNA levels.
This induction was blocked by the PKC inhibitor (100 µmol/L), whereas
the PKA inhibitor had no effect. The two peptide inhibitors, when added
to cells by themselves, had no significant effect (data not shown).
Levels of 3ßHSD mRNA were not significantly altered by the
treatments. Constitutively expressed GAPDH mRNA levels, shown in the
lower panel, served as normalization controls. All
steroidogenesis data were obtained from the same set of cells from
which media were collected before RNA extraction. These studies
confirmed similar results obtained previously in our laboratory with
CRH and ACTH (1).
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Studies in bovine, ovine, and human adrenocortical cells have shown
that second messenger systems play an important role in the
differential regulation of steroidogenic enzyme activity (30, 31, 32). The
integrated effects of PKA and PKC pathways result in modulation of the
expression and activity of the key steroidogenic enzymes, P450c17 and
3ßHSD (32). ACTH, acting via the cAMP pathway, can stimulate both of
these enzymes, a response that is consistent across species (32). The
PLC-PKC-Ca2+ pathway also has been implicated in
differential regulation of these enzymes. Angiotensin II, by activating
this pathway in a human adrenal cell line, can stimulate 3ßHSD
expression to a greater extent than ACTH (31), but affects P450c17
expression only marginally. The net effect of the two pathways in the
adult human adrenal is to reduce stimulation of steroidogenesis through
the 5 pathway, resulting in lower levels of
DHEAS and an increase in cortisol bioynthesis.
In the human fetal adrenal, the pattern of steroidogenesis varies with
gestational age, and expression of enzymes is zone specific (3, 33, 34). Recently, we showed that CRH directly and preferentially
stimulates DHEAS production in the human fetal adrenal (1), suggesting
a direct role for placental CRH in fetal adrenal function. In these
earlier studies, there was corroborative evidence that CRH treatment of
fetal adrenal cells stimulated a 4- to 5-fold increase in P450c17 mRNA
levels (1). This resulted in a marked increase in DHEAS production (1),
suggesting an increase in activity of the induced enzyme. The present
study not only confirms these observations, but demonstrates that
treatment of fetal adrenal cortical cells with CRH results in a rapid
accumulation of inositol metabolites, particularly
IP3, indicating the activation of PLC. The
production of diacylglycerol, which accompanies hormone-stimulated
inositol phospholipid hydrolysis, stimulates PKC. Our data indicate
that PKC activation by CRH can specifically induce the steroidogenic
enzyme P450c17 that drives steroidogenesis from pregnenolone toward
DHEAS production. In contrast, there is no effect of CRH on 3ßHSD.
Hence, unlike the effects of ACTH, glucocorticoid production is not
markedly stimulated. The specificity of the response and the time and
dose relationships indicate that the effects are mediated by a specific
CRH receptor, which is present in the human fetal adrenal (1).
Surprisingly, CRH did not activate the adenylate cyclase-cAMP pathway
in fetal adrenal cells, explaining the lack of effects of the PKA
inhibitor on CRH-stimulated events. The inhibitors, when added by
themselves at maximal concentrations, did not affect basal
steroidogenesis. However, the PKA inhibitor was effective in blocking
ACTH-mediated responses, including cortisol production, establishing
the specificity of the actions of the inhibitors on functional
responses of steroidogenic enzymes in these cells. The antagonists used
in the current study are synthetic pseudosubstrate peptides, which,
unlike previous inhibitors such as staurosporine and H7, were devised
to be more specific and have the natural function of keeping kinases in
their inactive state. The pseudosubstrate sequence on which the
myr-
-PKC was based is derived from the sequence of PKC and -ß
(23)
Several hypothalamic and pituitary trophic hormones are known to exert
their actions via multiple signaling pathways, including the
phosphoinositol metabolic pathway. An increase in
IP3 levels has been demonstrated with GnRH (20),
LH (21), and ACTH (22). In addition, PGF2 activates
the PLC-PKC pathway in several systems (35, 36). CRH also enhances
PGF2 production by the placenta (37), forming a
positive feedforward loop. Therefore, we added PGF2
to the fetal adrenal cells and found rapid and sustained accumulation
of IP3 in response. The exact role of
PGF2 in the fetal adrenal gland remains to be
elucidated. It is possible that it could synergize with CRH.
Recently, a new member of the CRH family of peptides, urocortin, has been identified and characterized (38). It has been suggested that urocortin may represent an endogenous ligand for the type 2 CRH receptor (39). Furthermore, it has been shown that urocortin is produced by the human placenta (40) and that it plays a potential role in placental vasodilation, myometrial quiescence, and coordination of fetal maturation (41, 42, 43). However, recent studies by Glynn et al. (44) have suggested that urocortin, by virtue of binding to CRH-binding protein, is unlikely to be responsible for the high levels of free CRH circulating in maternal plasma at term. It is possible that urocortin played a role in our studies in activating the PLC pathway. However, as the type 2 CRH receptor has not been identified in the human fetal adrenal gland, CRH itself or another member of the CRH-related peptide family, as yet unidentified, is more likely to have played a role in the present study.
The data presented in this study support an important and novel role of
CRH, which is produced in increasing amounts by the placenta with
advancing gestation (10), in the development and function of the fetal
adrenal gland. Previous studies have focused on the effects of CRH on
human fetal membranes (37) and maternal myometrium, where CRH receptors
have been localized (15, 16). Although CRH does not have a direct
ionotropic effect on the myometrium, it enhances myometrial
contractility in response to PGF2 (45) and oxytocin
via the cyclooxygenase pathway (46). In the pregnant human myometrium,
CRH receptors increase in affinity toward term (14) and mediate
its actions via adenylate cyclase activation. However, at term
there is a decrease in cAMP production (15), suggesting that the
effects of CRH could be mediated either by different subtypes of the
CRH receptor or by coupling to a different G regulatory protein than
Gs. Our studies raise the possibility that the
CRH receptor may be coupled to the subunit of the
Gq family of G proteins, which then couples to
PLC (PLCß) (47). Activation of PLCß would then lead to hydrolysis
of phosphatidylinositol-4,5-bisphosphate and IP3
production. IP3 production mobilizes
Ca2+. The effects of Ca2+
mobilization on function of the fetal adrenal are beyond the scope of
the present study. We chose to investigate signaling events further
downstream triggered by PKC activation. Our data demonstrate specific
involvement of PKC in mediating CRH effects. This leads us to
hypothesize that functional changes in the fetal adrenal developmental
process are orchestrated by modulation of second messenger systems, and
that placental CRH may play an important role in this process.
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Acknowledgments |
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Footnotes |
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2 Present address: Mothers and Babies Research Centre, John Hunter
Hospital, Newcastle, 2310 New South Wales, Australia.
Received March 3, 1999.
Revised June 14, 1999.
Accepted June 29, 1999.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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5-4 isomerase in
human fetal adrenals and gonads. Mol Endocrinol. 5:1147–1157.[CrossRef][Medline]
-hydroxylase/17,20 lyase): cloning of human adrenal and testis
cDNAs indicates the same gene is expressed in both tissues. Proc Natl
Acad Sci USA. 84:407–411.-hydroxylase and 3ß-hydroxysteroid
dehydrogenase expression in human adrenocortical H295R cells. J
Clin Endocrinol Metab. 81:2171–2178.[Abstract]
-hydroxylase and 3ß-hydroxysteroid dehydrogenase in the
integration of gonadal and adrenal steroidogenesis via the -5 and
-4 pathways of steroidogenesis in mammals. Biol Reprod. 56:789–799.[CrossRef][Medline]
-hydroxylase/17,20-lyase, and 3ß-hydroxysteroid dehydrogenase
isomerase steroidogenic enzymes in the human and rhesus fetal adrenal
gland: reappraisal of functional zonation. J Clin Endocrinol
Metab. 77:1184–1189.
. Endocrinology. 119:12–18.[Abstract]
stimulates
phosphatidylinositol 4,5-bisphosphate hydrolysis and mobilizes
intracellular calcium in bovine luteal cells. Proc Natl Acad Sci USA. 84:3728–3732. activity on human myometrium in
vitro. Am J Obstet Gynecol. 171:126–131.[Medline]
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