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Experimental Studies |
Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556
Address all correspondence and requests for reprints to: Dr. Robert B. Jaffe, Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556.
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Abstract |
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-hydroxylase/17,20-lyase (P450c17), and 3) basal
and ACTH-stimulated steady state abundance of mRNA encoding the ACTH
receptor. Basal cortisol (23.93 ± 1.20 pmol/105
cells·24 h) and DHEA-S (548.87 ± 43.17 pmol/105
cells·24 h) productions were significantly (P <
0.05) increased by IGF-I (2.3- and 1.8-fold, respectively) and IGF-II
(2.8- and 1.8-fold, respectively). As expected, ACTH, forskolin, and
cAMP markedly increased the production of cortisol by 26-, 10-, and
13-fold, respectively, and that of DHEA-S by 5.4-, 4.6-, and 5.5-fold,
respectively, compared with basal levels. IGF-II (100 ng/mL)
significantly (P < 0.001) increased ACTH-,
forskolin-, and cAMP-stimulated production of cortisol by 2.4-, 4.3-,
and 3.2-fold, respectively, and that of DHEA-S by 1.4, 1.6-, and
1.4-fold, respectively. IGF-I (100 ng/mL) had similar effects as IGF-II
and significantly (P < 0.001) increased ACTH-,
forskolin-, and cAMP-stimulated production of cortisol by 2.8-, 3.9-,
and 3.1-fold, respectively, and that of DHEA-S by 1.3-, 1.6-, and
1.4-fold, respectively. The similar potencies of IGF-I and IGF-II
suggest that the actions of these factors were mediated via a common
receptor, most likely the type I IGF receptor. The effects of IGF-II
on ACTH-stimulated steroid production were dose-dependent
(EC50, 0.5–1.0 nmol/L), and IGF-II markedly increased the
steroidogenic responsiveness of fetal zone cells to ACTH. With respect
to cortisol production, IGF-II shifted the ACTH dose-response curve to
the left by 1 log10 order of magnitude. IGF-II also
increased ACTH-stimulated abundance of mRNA encoding P450scc (1.9-fold)
and P450c17 (2.2-fold). Basal expression of P450scc was not affected by
IGF-II. In contrast, basal expression of P450c17 was increased 2.2-fold
by IGF-II and IGF-I in a dose-responsive fashion. Neither IGF-I nor
IGF-II affected basal or ACTH-stimulated abundance of mRNA encoding the
ACTH receptor, suggesting that the increase in ACTH responsiveness was
not mediated by an increase in ACTH-binding capacity. Taken together,
these data indicate that activation of the type I IGF receptor
increases ACTH responsiveness in fetal zone cells by modulating ACTH
signal transduction at some point distal to ACTH receptor activation.
These data also indicate that locally produced IGF-II modulates fetal
adrenal cortical cell function by increasing responsiveness to ACTH and
possibly (based on its direct stimulation of P450c17 expression)
augmenting the potential for adrenal androgen synthesis. Thus,
activation of the type I IGF receptor on adrenal cortical cells may
play a pivotal role in adrenal androgen production, both
physiologically in utero and at adrenarche, and in
pathophysiological conditions of hyperandrogenemia, such as the
polycystic ovary syndrome. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The growth and function of the fetal zone and the specific ontogenetic differentiation of the transitional and definitive zones, are primarily regulated by ACTH secreted by the fetal pituitary gland (5, 6, 7, 8). We have examined the mechanism by which ACTH regulates fetal zone growth and hypothesized that, as it is not a mitogen per se, its growth stimulatory effects are mediated by locally produced growth factors which then act in an autocrine/paracrine mode (9, 10). In support of this concept, we have found that some growth factors that are mitogenic for fetal zone cells also are expressed by fetal zone cells in response to ACTH (10, 13).
Of the growth factors examined, insulin-like growth factor II (IGF-II) is particularly interesting as 1) it is highly expressed by the human fetal adrenals (11, 12, 13); 2) its expression by human fetal adrenal cortical cells is positively regulated by ACTH (13, 14, 15); 3) it is mitogenic for fetal zone cells and acts cooperatively with other fetal zone mitogens, such as basic fibroblast growth factor and epidermal growth factor (13); 4) it is an exclusive fetal adrenal cortical growth factor, i.e., soon after birth its expression by adrenal cortical cells decreases to undetectable levels and becomes refractory to ACTH stimulation (16); 5) the closely related peptide, IGF-I, modulates the growth and function of cultured human adult adrenal cortical cells (17, 18); and 6) the actions of IGF-I and IGF-II on fetal adrenal cortical cells appear to be mediated via a common receptor, the type I IGF receptor, which has been localized in the human fetal adrenal cortex (19). Thus, IGF-II is likely to play a major role in the regulation of fetal zone development. The present studies were undertaken to further characterize the effects of IGF-II on the differentiated function of fetal zone cells. We hypothesized that in the developing human fetal adrenal cortex, IGF-II, acting via the type I IGF receptor, not only affects the proliferation of fetal zone cells, but also modulates their differentiated function and may influence adrenal androgen synthesis. In the present study, we tested this hypothesis using primary cultures of fetal zone cells derived from midgestation human fetal adrenals. We examined the effects of IGF-II on basal and stimulated steroidogenic activities and the expression of key steroid-metabolizing enzymes. For comparison, we also examined the effect of IGF-I on the same parameters.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal adrenal glands were obtained from second trimester fetuses (16–22 weeks; gestation estimated by foot length; n = 20) after elective termination of pregnancy by dilatation and evacuation. Glands were collected immediately after pregnancy termination and placed in ice-cold tissue culture medium (see below). Pairs of adrenals from individual fetuses were pooled and treated separately from adrenals derived from other fetuses. The study protocol was approved by the Committee on Human Research of the University of California-San Francisco (UCSF).
Human recombinant IGF-II was obtained from Upstate Biotechnology (Lake
Placid, NY). Human recombinant IGF-I was a generous gift from Chiron
Corp. (Emeryville, CA). Human ACTH-(1–24) (Cortrosyn) was obtained
from Organon (West Orange, NJ). Forskolin and 8-bromo-cAMP were
obtained from Sigma Chemical Co. (St. Louis, MO). The full-length
complementary DNAs (cDNAs) encoding human cytochrome P450 side-chain
cleavage (P450scc) and cytochrome P45017 hydroxylase/17,20 lyase
(P450c17) were provided by Dr. W. L. Miller, UCSF (20, 21). The
full-length cDNA encoding the human ACTH receptor was provided by Dr.
R. Cone, Vollum Institute, Oregon Health Sciences University (Portland,
OR) (22). The full-length cDNA encoding human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from
American Type Culture Collection (Rockville, MD).
Preparation of fetal adrenal cortical cultures
Primary cultures of midgestation human fetal adrenal cortical cells were prepared as previously described (23). Briefly, the adrenal capsule with adherent definitive/transitional zones was carefully dissected away from the underlying cortical tissue, and the remaining fetal zone was dispersed by enzymatic digestion with collagenase. Cells were plated onto 6-cm diameter plastic culture dishes (Falcon Plastics, Los Angeles, CA) at a density of 5 x 105 cells/dish or onto 48-well culture dishes (Nunc, Naperville, IL) at a density of 1 x 105 cells/well. In each case, the culture medium was DMEM H-16-Ham’s F-12 (1:1) medium containing nonessential amino acids and supplemented with 10% fetal calf serum (FCS), 2 mmol/L glutamine, and 50 µg/mL gentamicin (Cell Culture Facility, UCSF). All plates were incubated in a humidified environment at 37 C in 95% air-5% CO2. After 48 h, media were changed to a medium containing 2% FCS, and test substances were added for various times. Media were then collected and stored at -20 C for subsequent assay of cortisol and DHEA-S. The remaining cells were either collected after trypsinization and counted in a particle counter or processed for total ribonucleic acid (RNA) isolation.
RNA analysis
Northern blot, slot blot, and ribonuclease protection analyses were used to assay the abundance of specific messenger RNA (mRNA) transcripts. Total RNA was extracted and purified from cultured cells using the method of Chomczynski and Sacchi (24). For Northern blotting, total RNA was denatured in formaldehyde, subjected to electrophoresis through a 1.2% agarose gel, and transferred to nitrocellulose membranes (Nytran, Schleicher and Schuell, Keene, NH). For slot blots, total RNA was denatured in formaldehyde and applied to a nitrocellulose membrane using a slot blot manifold (Schleicher and Schuell). In both cases, the RNA was cross-linked to the membranes by exposure to UV radiation (Stratalinker, Stratagene, La Jolla, CA), then hybridized with [32P]deoxy-CTP-labeled cDNA probes (1–2 x 109 dpm/µg) synthesized by random primer extension of denatured full-length cDNAs. Prehybridization was performed in hybridization buffer (Quickhyb buffer, Stratagene, La Jolla, CA) at 68 C for 15 min. Denatured radiolabeled probe was then added to the membranes and incubated at 68 C for 90 min. To remove nonspecifically bound probe, membranes were washed in 2 x SSC (1 x SSC is 0.15 mol/L NaCl and 0.015 mol/L sodium citrate)-0.1% SDS at room temperature for 15 min and then in 0.1 x SSC-0.1% SDS at 60 C for 30 min. Membranes were subjected to autoradiography at -70 C. The relative abundance of mRNA transcripts was estimated by computer-assisted densitometry. All data were normalized to the abundance of transcripts encoding GAPDH that was constitutively expressed. Probes were removed by washing the membranes in distilled water at 100 C. Complete removal of probe was confirmed by autoradiography before reprobing.
Ribonuclease protection analyses were performed using a kit obtained from Ambion (HybSpeed RPA, Ambion, Austin, TX). Antisense 32P-labeled complementary RNA probes for P450scc (192 bases) and P450c17 (313 bases) were synthesized using cDNA templates subcloned into pBluescript SK (Stratagene, La Jolla, CA). The complementary RNA probes were gel-purified by electrophoresis and hybridized with 5 µg total RNA extracted from cultured fetal zone cells. Samples were then digested with ribonuclease A/T1, and the protected fragments were precipitated, resuspended, and subjected to electrophoresis under denaturing conditions (6 mol/L urea) in 6% polyacrylamide. The gel then was dried and subjected to autoradiography. The amount of radioactivity in the protected bands, which directly reflects the amount of target mRNA, was measured by scintillation counting of the excised bands.
RIAs
Cortisol and DHEA-S were measured in conditioned medium using specific RIAs, which we have described previously (23). Unconjugated cortisol was assayed using a kit purchased from Diagnostic Products Corp. (Los Angeles, CA). DHEA-S was assayed using a kit purchased from ICN Biomedicals (Costa Mesa, CA). All assays were validated for use in conditioned media from fetal adrenal cortical cell cultures, and each had inter- and intraassay coefficients of variation less than 10%.
Statistical analyses
All hormone data were normalized relative to cell number and expressed as picomoles per 105 cells/24 h and are presented as the mean ± SE. Data derived from Northern blot analyses were normalized relative to GAPDH and expressed in arbitrary units. All experiments were performed in triplicate wells and repeated at least three times. Statistical analyses were conducted by ANOVA followed by Newman-Keuls post-hoc test for significance between groups. Differences were considered statistically significant when P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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13 nmol/L)
significantly increased basal and agonist-stimulated [ACTH (1 nmol/L),
forskolin (1 µmol/L), or cAMP (0.1 mmol/L)] cortisol and DHEA-S
production by fetal zone cells (Fig. 1
). The effects of
IGF-II on ACTH-stimulated steroid production were dose dependent, with
an EC50 of 1.0–10 ng/mL (Fig. 2). In the
experiment shown in Fig. 2, the concentration of ACTH used (0.1 nmol/L)
was not sufficient to increase cortisol in the absence of IGF-II. In
the presence of increasing concentrations of IGF-II, cortisol increased
markedly in response to 0.1 nmol/L ACTH. IGF-II also increased DHEA-S
production in response to 0.1 nmol/L ACTH in a dose-responsive fashion.
Thus, with respect to cortisol and DHEA-S production, IGF-II augmented
the responsiveness of the fetal zone cells to ACTH.
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). With
respect to cortisol production, IGF-II shifted the ACTH dose-response
curve to the left by approximately 1 log10 order of
magnitude. The production of DHEA-S was more sensitive to ACTH
stimulation than was that of cortisol and was elicited with ACTH
concentrations as low as 0.1 pmol/L. In control cultures (no IGF-II),
DHEA-S production exhibited an apparent biphasic response to increasing
ACTH concentrations. Interestingly, the decrease in DHEA-S production
coincided with the increase in cortisol production. IGF-II eliminated
the biphasic pattern of DHEA-S production in response to increasing
concentrations of ACTH. In cells exposed to IGF-II, DHEA-S production
continued to increase with increasing concentrations of ACTH and
reached a plateau at 0.1 nmol/L. Thus, the net effect of IGF-II was to
augment DHEA-S synthesis.
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).
IGF-II had no effect on basal expression of P450scc. However, basal
expression of P450c17 was increased by IGF-II 2.2-fold. We also
examined the effects of IGF-I and -II on P450c17 expression by Northern
and slot blot analysis and found that IGF-I increased basal and
ACTH-stimulated expression of P450c17 in a dose-responsive fashion
(Fig. 5). Consistent with its augmentation of ACTH
responsiveness, IGF-II also increased responsiveness to ACTH with
respect to P450c17 expression and shifted the ACTH dose-response curve
to the left (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5
C19 steroids. In adult bovine (26) and fetal ovine (27)
adrenal cortical cells, IGF-I also has been shown to enhance
steroidogenic responsiveness to ACTH and proliferation of adrenal
cortical cells. Although we have shown that IGF-I is not expressed in
the human fetal adrenal cortex (13), IGF-II appears to exert its
actions by interacting with the IGF-I receptor (type I IGF receptor).
Thus, the actions of IGF-II in the human fetal adrenal may be analogous
to those of IGF-I in the postnatal adrenal. IGF-II markedly increased the responsiveness of fetal zone cells to agonist (ACTH, forskolin, and cAMP) stimulation. These effects were closely mimicked by IGF-I, and based on their relative potencies, this suggests that the actions of these growth factors were mediated via the type I IGF receptor. This is consistent with previous studies in which we showed that the mitogenic actions of IGF-II on fetal zone cells were probably mediated via the type I IGF receptor (13). This receptor shares structural and functional homology with the insulin receptor, binds both IGF-I and IGF-II with similarly high affinities and insulin with low affinity, and is thought to mediate most of the biological actions of IGF-II (28). Ligand binding autoradiographic studies have shown that fetal zone cells express the type I IGF receptor (19). Although a specific high affinity IGF-II receptor has been identified (i.e. the type II IGF/mannose-6-phosphate receptor), the biological actions that it mediates are unknown, and its expression by fetal zone cells has not been determined.
IGF-II increased ACTH-stimulated production of cortisol and DHEA-S by
primary cultures of fetal zone cells in a dose-responsive fashion. With
respect to cortisol production, IGF-II shifted the ACTH dose-response
curve to the left by 1 log10 order of magnitude, reflecting
its augmentation of ACTH responsiveness. In contrast, IGF-II did not
shift the ACTH dose-response curve with respect to DHEA-S production.
Instead, IGF-II eliminated the decrease in DHEA-S production that
occurred at higher ACTH concentrations. The decrease in DHEA-S
production in control cultures coincided with the increase in cortisol
production, and, therefore, was probably due to the diversion of
substrate away from the 5 pathway to the
4–3 ketosteroid pathway involved in cortisol synthesis.
In the presence of IGF-II, DHEA-S production continued to rise in
response to increasing concentrations of ACTH, despite the initiation
of cortisol synthesis, and reached a plateau at 0.01–0.1 nmol/L ACTH.
This continued increase in DHEA-S production may have been due to
increased 17-hydroxylase activity induced by IGF-II (see below). It
should be noted that the fetal zone does not express
3ß-hydroxysteroid dehydrogenase in vivo and, therefore,
cannot synthesize cortisol de novo (4). Thus, in
vivo this effect of IGF-II on augmenting DHEA-S production by
fetal zone cells may be more pronounced as steroidogenesis would be
committed to the 5 pathway.
To examine the mechanism by which IGF-II augmented fetal zone cell steroidogenesis, we assessed its effects on basal and ACTH-stimulated expression of the key steroidogenic enzymes P450scc and P450c17. We found that IGF-II (and IGF-I) markedly increased ACTH-stimulated expression of P450scc and P450c17. This suggests that IGF-II increases ACTH-stimulated steroid production by increasing the expression, and therefore activity, of these steroidogenic enzymes. These data are consistent with our previous findings (23), and in terms of steroidogenic enzyme expression, further demonstrate IGF-induced increased responsiveness to ACTH. Interestingly, we found that IGF-II also increased basal P450c17 expression, but did not affect basal expression of P450scc. These effects also were mimicked by IGF-I and are consistent with previous studies in which IGF-I was shown to increase P450c17 activity (17) and expression (18) in human adult adrenal cortical cells. In some experiments, we found that the magnitude by which the IGFs (both 13 nmol/L) increased basal P450c17 expression in fetal zone cells was comparable to that caused by 1 nmol/L ACTH. Thus, either IGF-I or IGF-II activation of the type I IGF receptor in fetal zone cells not only increases ACTH-stimulated expression of P450scc and P450c17, but also augments basal P450c17 expression. The increased basal P450c17 expression may account for the increase in basal cortisol and DHEA-S production induced by IGF-I and IGF-II. The consequence of such an increase in basal P450c17 expression may be to enhance the capacity of fetal zone cells for androgen production upon ACTH stimulation. In vivo, P450c17 is highly expressed by fetal zone cells. Our data suggest that IGF-II plays a role in maintaining this elevated level of P450c17 expression and, therefore, can serve as a pivotal factor in the regulation of fetal zone androgen production.
It is possible that our data reflect a general effect of type I IGF receptor activation on human adrenal cortical cell function. These findings have important implications regarding the role of the IGFs in the regulation of adrenal androgen production and suggest that activation of the type I IGF receptor by IGF-I, IGF-II, and/or insulin may increase ACTH responsiveness and P450c17 activity and, therefore, the capacity for adrenal androgen synthesis. A role for the IGFs and activation of the type I IGF receptor also has been suggested in pathological conditions of androgen excess, particularly hyperandrogenic amenorrhea (polycystic ovary syndrome) (29, 30). This syndrome is associated with insulin resistance and hyperinsulinemia. It has been hypothesized that the increased insulin concentrations may be sufficient to activate the type I IGF receptor, leading to increased adrenal androgen production (29). Our present data support this idea and indicate that activation of the type I IGF receptor on human adrenal cortical cells may have profound effects not only on fetal adrenal development and function, but also on adrenal cortical function postnatally.
The mechanism by which the IGFs increase ACTH responsiveness is not known. Previous studies have suggested that the IGFs augment the responsiveness of adrenal cortical cells to ACTH by increasing ACTH receptor expression and ACTH-binding capacity (18, 31). We addressed this issue in fetal zone cells by determining whether the IGFs modulate ACTH receptor expression. Neither IGF-I nor IGF-II altered ACTH receptor expression by fetal zone cells. Although ACTH binding was not assessed, these findings indicate that the IGFs do not increase ACTH responsiveness in human fetal zone cells by increasing the number of ACTH-binding sites. Instead, our data indicate that the IGFs modulate the ACTH signal transduction pathway at some point distal to ACTH receptor binding, as IGF-I and -II increased responsiveness not only to ACTH, but also to forskolin and cAMP. The nature of this mechanism is unknown, and studies are underway to explore further the interaction between the growth factor/IGF and ACTH signal transduction pathways that may lead to increased tropic hormone responsiveness.
In summary, we have shown that IGF-II is a major modulator of fetal zone function and responsiveness to ACTH. Our present data indicate that IGF-II, via activation of the type I IGF receptor, may contribute to the increased responsiveness of the fetal zone during late gestation. In addition, our data suggest that IGF-II also increases the capacity of fetal zone cells for androgen synthesis by directly augmenting the expression of P450c17. Thus, IGF-II may play a pivotal role in establishing and maintaining the differentiated function of the fetal zone. This effect of IGF-II may reflect a general action of the IGFs on adrenal cortical responsiveness to ACTH and androgen production throughout life.
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Acknowledgments |
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Footnotes |
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Received November 21, 1996.
Revised January 24, 1997.
Accepted February 7, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Lacroix, N. N'Diaye, J. Tremblay, and P. Hamet Ectopic and Abnormal Hormone Receptors in Adrenal Cushing's Syndrome Endocr. Rev., February 1, 2001; 22(1): 75 - 110. [Abstract] [Full Text]
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F. Wilkin, N. Gagné, J. Paquette, L. L. Oligny, and C. Deal Pediatric Adrenocortical Tumors: Molecular Events Leading to Insulin-Like Growth Factor II Gene Overexpression J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 2048 - 2056. [Abstract] [Full Text]
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A. Biason-Lauber, B. Kempken, E. Werder, M. G. Forest, S. Einaudi, M. B. Ranke, N. Matsuo, V. Brunelli, E. J. Schönle, and M. Zachmann 17{alpha}-Hydroxylase/17,20-Lyase Deficiency as a Model to Study Enzymatic Activity Regulation: Role of Phosphorylation J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1226 - 1231. [Abstract] [Full Text]
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H. J. H. M. T. van Dessel, P. D. K. Lee, G. Faessen, B. C. J. M. Fauser, and L. C. Giudice Elevated Serum Levels of Free Insulin-Like Growth Factor I in Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., September 1, 1999; 84(9): 3030 - 3035. [Abstract] [Full Text]
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