Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5428-5432, June 1991 Medical Sciences
Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: A model for adrenal growth regulation SAM MESIANO, SYNTHIA H. MELLON, DENIS GOSPODAROWICZ, ANNA MARIA Di BLASIO, AND ROBERT B. JAFFE* Reproductive Endocrinology Center, University of California San Francisco, San Francisco, CA 94143
Communicated by Gerald D. Aurbach, February 28, 1991 (received for review November 19, 1990)
In the human, atrophy of the fetal adrenals and decreased adrenal steroid secretion have been reported after glucocorticoid treatment of the mother, presumably due to passage of glucocorticoids into the fetal compartment, where they exert a negative feedback effect on fetal pituitary ACTH secretion (9). Morphologic studies of adrenal gland development from anencephalic and apituitary human fetuses have shown normal development prior to 15 weeks of gestation but subsequent atrophy of the inner fetal zone thereafter (10). Adrenal atrophy in human anencephalic fetuses was prevented by ACTH treatment in utero (11-13). These data strongly suggest that human fetal adrenal growth, and in particular the rapid growth of the fetal zone, is dependent upon an intact pituitary and may be regulated by ACTH. Despite the many studies in vivo showing that adrenal growth can be regulated by ACTH, a stimulatory effect of ACTH on adrenal cell proliferation in vitro has not been demonstrated. This discrepancy raises the possibility that ACTH-stimulated adrenal growth may be mediated by a secondary growth factor. This hypothesis is not without precedent, as several classical growth-promoting hormones as well as some growth factors are now known to act through the stimulation of local growth factors which then function in an autocrine or paracrine manner to stimulate growth (1417). Growth factors have been shown to be capable of effecting the proliferation of steroid-secreting cells and, particularly relevant to the present study, human fetal adrenal cells in vitro (18). Previous studies from our laboratory have shown that both basic fibroblast growth factor (bFGF) and epidermal growth factor are mitogens for cultured human fetal adrenal cells (18), with bFGF being the more potent. In light of its potent mitogenic actions on human fetal adrenal cells and its presence in adult adrenal tissue in other species (19), we reasoned that bFGF may be a prime candidate as a local mediator of the stimulatory effects of ACTH on human fetal adrenal growth. Therefore, we examined whether bFGF is synthesized by the human fetal adrenal gland and whether its expression can be regulated by ACTH. Portions of these studies have been presented.t
ABSTRACT Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cells. Northern blot analysis of total RNA from whole human fetal adrenal glands revealed a characteristic 7-kilobase bFGF mRNA, indicating that the fetal adrenal bFGF bioactivity was most likely due to local synthesis. Slot blot and ribonuclease protection analysis showed that bFGF mRNA was present in very low amounts in total RNA d human fetal adrenal from primary cultures of u cells but was increased 2- to 3-fold in cells exposed to 10 nM ACTH-(1-24) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate for 24 hr. bFGF mRNA was localized to adrenocortical cells and not fibroblasts by in situ hybridization. bFGF mRNA was barely detectable in unstimulated cells, whereas it was markedly increased in cells exposed to either ACTH or 8-bromoadenosine 3',5'-cyclic monophosphate. These data support our hypothesis that the regulation of human fetal adrenal growth by ACTH at midgestation may be mediated by the stimulation of local growth factor production, and we suggest that bFGF may play a major role in this process.
The fetal adrenal glands are thought to play a pivotal regulatory and homeostatic role in intrauterine life and in preparation for extrauterine existence. In the human, the adrenals undergo a phase of rapid growth and development between 10 and 20 weeks of gestation, increasing their weight from 50 mg to 1 g and attaining a size equal to or greater than that of the fetal kidney by midpregnancy. The bulk of the gland (>80%o) consists of a large inner cortex referred to as the fetal zone, where much of the rapid growth after 15 weeks of gestation occurs (1-4). The factors responsible for the rapid growth of the human fetal adrenal are largely unknown. In adult mammals, adrenal growth and steroidogenesis are regulated by adrenocorticotropin (ACTH) in vivo (3, 5). Although similar studies in the fetus have been more difficult, a role for ACTH in fetal adrenal development is indicated (6, 7). Recent studies in the hypophysectomized fetal sheep have shown that adrenal cortical growth could be restored by in utero ACTH replacement therapy (8).
MATERIALS AND METHODS Cell Culture. Human fetal adrenal glands were obtained from second-trimester aborted fetuses. Abortions were performed by dilatation and evacuation, and gestational age was estimated by foot length. An adult adrenal was obtained from a kidney donor from the Kidney Transplant Unit, University of California San Francisco. Abbreviations: ACTH, adrenocorticotropin; bFGF, basic fibroblast growth factor; aFGF, acidic fibroblast growth factor; IGF, insulinlike growth factor. *To whom reprint requests should be addressed. tMesiano, S., Mellon, S. H., DiBlasio, A. M., Gospodarowicz, D. & Jaffe, R. B., 72nd Annual Meeting of the Endocrine Society, June 20-23, 1990, Atlanta, GA, abstr. 21.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
5428
Proc. Natl. Acad. Sci. USA 88 (1991)
Medical Sciences: Mesiano et al. For cell culture experiments, human fetal adrenal glands were decapsulated to remove most of the definitive zone, minced into 1-mm3 pieces, and incubated in 0.1% collagenase (Sigma) at 37TC for 30 min (20). The dispersed cells were filtered through a nylon mesh, centrifuged, and resuspended in culture medium, a 1:1 (vol/vol) mixture of medium 199 and Ham's nutrient mixture F-12, supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 gg of gentamicin per ml. Cells were plated on Falcon culture plates at -10,000 cells per cm2 and incubated in a humidified environment at 370C in 95% air/5% CO2. Medium was changed every 48 hr. In some plates, ACTH-(1-24) (10 nM; Organon) or 8-bromocAMP (8-Br-cAMP; 1 mM; Sigma) was added and the cells were incubated for a further 24 hr. bFGF Bioassay. Extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cortical cells were subjected to heparin-Sepharose affinity chromatography and the eluted fractions were assayed for mitogenic activity (21, 22). For the whole glands, three human fetal adrenals (16-23 weeks) were homogenized in 10 mM Tris Cl, pH 7.0/0.5% Triton X-100. The homogenate was adjusted to 0.6 M NaCl and centrifuged (15 min; 40C; 20,000 x g), and the supernatant was applied to a heparin-Sepharose affinity column (0.5-ml bed volume) preequilibrated with 10 mM Tris Cl, pH 7.0/0.6 M NaCl. The column was sequentially eluted with 10 mM Tris Cl, pH 7.0, containing 0.6, 1.0, and 3 M NaCl. An aliquot of each fraction was assayed for mitogenic activity in a cell proliferation bioassay using bovine adrenal capillary endothelial cells as target cells (19). For cultured cells, fetal adrenal cortical cells were dispersed and cultured for 5 days on 10-cm plastic culture dishes, harvested, lysed in 0.8 ml of 0.5% Triton X-100/10 mM dithiothreitol/10 mM Tris Cl, pH 7.0, and centrifuged (30 min at 20,000 x g). The NaCl concentration was adjusted to 0.6 M and the lysate was applied to a heparin-Sepharose column (0.1-ml bed volume) and eluted with 10 mM Tris Cl buffer containing increasing concentrations of NaCl as described above. Each fraction was then bioassayed (19). Effect of bFGF on Human Fetal Adrenal Cell Proliferation. Fetal adrenal cells were plated in 24-well culture dishes at 20,000 cells per well. The culture medium was changed every 48 hr. After 1 week, bFGF (21) was added in various concentrations (0.01-100 ng/ml) in triplicate wells and the cells were incubated 48 hr. Cells were then harvested and counted in a particle counter (Coulter) (20). Extraction of RNA. Total RNA was isolated from human fetal adrenal glands and an adult adrenal gland. The tissue was homogenized in 5 M guanidinium thiocyanate, and RNA was purified on a CsCI gradient (23, 24). Total RNA was also isolated from cultured human fetal adrenal cells (25). Northern and Slot Blot Analysis. A 32P-labeled bovine bFGF cDNA probe corresponding to the protein coding region and exhibiting 99% sequence identity with human bFGF was used (26). Total RNA (20 ,ug) was denatured by glyoxal treatment, electrophoresed through a 1.2% agarose gel, and blotted onto a nylon membrane (Nytran; Schleicher & Schuell). The filter was baked and then prehybridized overnight in 50% (vol/vol) formamide/5x standard saline citrate (SSC)/5x Denhardt's solution/1.0o SDS/10 mM sodium phosphate buffer, pH 8.0, with salmon sperm DNA at 100 ,ug/ml. A 32P-labeled bFGF cDNA probe corresponding to nucleotides 103-605 (26) was synthesized by random primer extension and hybridized to the membrane at 42°C overnight in hybridization solution. The membrane was then washed at 65°C in 0.1x SSC/0.1% SDS and autoradiographed for 2 weeks at -70°C. For slot blot analysis, RNA (20 ,ug) was immobilized on a Nytran membrane under vacuum. The membrane was then hybridized with a 32p_ labeled bFGF cDNA probe, washed under high stringency, and autoradiographed for 1 week at -70'C. Integrity and
5429
amount of RNA loaded were confirmed by hybridization of Northern blots with a human y-actin probe (27) and slot blot filters with a rat cyclophilin probe (28). Ribonuclease Protection Analysis. A 205-base 32P-labeled bFGF cRNA probe was synthesized from a bovine bFGF cDNA fragment corresponding to nucleotides 103-255. The cRNA was purified by gel electrophoresis and hybridized at 550C overnight in 200 mM Pipes/2 M NaCl/5 mM EDTA with 10 Ag of total RNA from control and ACTH-treated human fetal adrenal cells. The hybridization solution was then treated with RNase A (10 Ag/ml; Sigma) in 10 mM Tris Cl, pH 7.5/300 mM NaCl/5 mM EDTA at 370C for 30 min and electrophoresed in a 6 M urea/6% polyacrylamide gel (29). The gel was then dried and subjected to autoradiography for 1 week at -70'C. In Situ Hybridization. Fetal adrenal cortical cells (18-week fetus) were plated onto eight-chamber glass microscope slides (Lab-Tek chamber slides; Nunc) at 10,000 cells per chamber. After 5 days the cells were stimulated for 24 hr with 10 nM ACTH-(1-24) or 1 mM 8-Br-cAMP. The cells were then fixed in 4% paraformaldehyde and subjected to in situ hybridization with a digoxigenin-labeled bFGF cDNA probe (Genius 2 DNA labeling kit; Boehringer Mannheim) at 55°C overnight in 50%o formamide/4x SSC/1 x Denhardt's solution containing salmon sperm DNA (100 lg/ml) and yeast tRNA (100 lg/ml). The cells then were washed for 15 min in 2x SSC at room temperature and four times for 15 min in 0.5x SSC at 55°C. Specifically hybridized cDNA probe was detected with a Genius nucleic acid detection kit (Boehringer Mannheim).
RESULTS Effect of bFGF on Human Fetal Adrenal Cell Proliferation. bFGF was found to be a potent mitogen for cultured human fetal adrenal cells. Addition of bFGF caused a dosedependent increase in cell number with an ED50 of around 0.01 ng/ml (0.6 pM; Fig. 1). Detection of bFGF Bioactivity in the Human Fetal Adrenal Gland. The bioassay is based on the heparin-Sepharose affinity chromatography elution profile of bFGF, which is in the 3.0 M NaCl fractions. In both whole adrenal glands and extracts of cultured cells, a major peak of mitogenic activity was detected in the 3.0 M NaCl fractions (Fig. 2). A minor peak of mitogenic activity was also detected in the 1 M NaCl eluates. This peak of mitogenic activity probably represents acidic FGF (aFGF). The material eluted at 3.0 M NaCl showed bFGF immunoreactivity in a specific radioimmunoassay (data not shown). These data demonstrate that bFGF 4000, 3600 0
x
C)
3200 2800
2400 2000
-
-
-
-I
I
-T
0
0.01
0.1
I
1.0
10
loo
FGF (ng/ml) FIG. 1. Effect of bFGF on human fetal adrenal cortical cell proliferation. (Mean + SD, n = 3.)
5430
Medical Sciences: Mesiano et al. 0.6M NaCI
5000-
Proc. Natl. Acad. Sci. USA 88 (1991)
adrenal (19). The amount of total RNA analyzed, the long
3.OM NaCI
1.0M NaCI
A
exposure time (2 weeks), and the intensity of the signal reflect
4000-
C4
0
x
3000 -j -j
w
a
2000-
_____________________________________ 1000 0o 10 20 30 40 I-OmNaCI lM NaC
3000 00.6M .6MNaCI 3000 aCI
2500-
Cl
3.OM NaCI 3.OMNaCI
B
observed on three separate occasions.
0
2000-
\
2=
V, -i
\
or \ B \
1500
w
the low abundance of bFGF mRNA in the human fetal adrenal gland, possibly due to the mRNA being highly unstable and/or regulated stringently. The intensity of the 7-kb band was reduced in RNA from an adult human adrenal and barely detectable in RNA from 18- and 22.5-week human fetal adrenal glands. Similar results were obtained by slot blot. Effect of ACTH on Human Fetal Adrenal bFGF Expression. Glucocorticoid concentrations increased 10-fold in the conditioned medium from ACTH- and 8-Br-cAMP-treated cells, indicating that steroidogenic capacity was maintained. bFGF mRNA accumulation increased 3-fold in cells treated with ACTH or 8-Br-cAMP (Fig. 4). This increase was evident from 24 to 96 hr of incubation with either ACTH or 8-Br-cAMP. This effect of ACTH on bFGF mRNA accumulation was
a 1000
-
500
10
0
20 30 FRACTION NUMBER
40
Fet 3. 2. Heparin-Sepharose elution profile of bFGF bioactivity in human fetal adrenal glands (A) and primary cultures of extra cts of A__1A ---I
I- __^ _ DX Ai URAL __:_ mean oi human &ttl tetal adrenal cortical cells (B). Each point Is: the duplicate determinations (SD c5%). Similar chromatographic profiles were seen in three separate experiments. _
--
In the cultured fetal adrenal cells, bFGF gene expression was identified in cortical cells by in situ hybridization using a nonradioactive alkaline phosphatase-linked detection system (Fig. 5). Cytoplasmic staining was markedly increased in ACTH- and 8-Br-cAMP-treated cells relative to control. In the ACTH- and 8-Br-cAMP-treated cells, dark cytoplasmic staining was visible after 1-2 hr of exposure to the color reagent. No staining was detected when ACTH-treated cells were treated with RNase A (10 pzg/ml; Sigma) for 30 min at 370C prior to hybridization or in cells probed with a rat growth hormone cDNA probe (30) (Fig. 5). Thus, ACTH can stimulate bFGF mRNA accumulation specifically in cultured human fetal adrenal cortical cells. We obtained similar results in identical cells probed with human insulin-like growth factor II (IGF-II) cDNA,t confirming that IGF-II expression in human fetal adrenal cells is regulated by ACTH (31).
_
is present in the intact human fetal adrenal gland as well as in primary cultures of human fetal adrenal cells. Expression of bFGF by the Human Fetal Adrenal Gland. A bFGF transcript of -7 kilobases (kb) was detected in RNA prepared from 19-, 16.5-, and 16-week fetal adrenals (Fig. 3). The 7-kb bFGF transcript was similar in size to one of the bFGF gene transcripts reported previously in the adult bovine
DISCUSSION Several studies in the human have provided strong evidence that the growth of the fetal adrenal, particularly the rapid proliferation of the fetal zone, is dependent on ACTH (10-13, 32). The present study was part of an ongoing project designed to determine the factors that regulate the rapid growth of the human fetal adrenal gland during midgestation.
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Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: A model for adrenal growth regulation SAM MESIANO, SYNTHIA H. MELLON, DENIS GOSPODAROWICZ, ANNA MARIA Di BLASIO, AND ROBERT B. JAFFE* Reproductive Endocrinology Center, University of California San Francisco, San Francisco, CA 94143
Communicated by Gerald D. Aurbach, February 28, 1991 (received for review November 19, 1990)
In the human, atrophy of the fetal adrenals and decreased adrenal steroid secretion have been reported after glucocorticoid treatment of the mother, presumably due to passage of glucocorticoids into the fetal compartment, where they exert a negative feedback effect on fetal pituitary ACTH secretion (9). Morphologic studies of adrenal gland development from anencephalic and apituitary human fetuses have shown normal development prior to 15 weeks of gestation but subsequent atrophy of the inner fetal zone thereafter (10). Adrenal atrophy in human anencephalic fetuses was prevented by ACTH treatment in utero (11-13). These data strongly suggest that human fetal adrenal growth, and in particular the rapid growth of the fetal zone, is dependent upon an intact pituitary and may be regulated by ACTH. Despite the many studies in vivo showing that adrenal growth can be regulated by ACTH, a stimulatory effect of ACTH on adrenal cell proliferation in vitro has not been demonstrated. This discrepancy raises the possibility that ACTH-stimulated adrenal growth may be mediated by a secondary growth factor. This hypothesis is not without precedent, as several classical growth-promoting hormones as well as some growth factors are now known to act through the stimulation of local growth factors which then function in an autocrine or paracrine manner to stimulate growth (1417). Growth factors have been shown to be capable of effecting the proliferation of steroid-secreting cells and, particularly relevant to the present study, human fetal adrenal cells in vitro (18). Previous studies from our laboratory have shown that both basic fibroblast growth factor (bFGF) and epidermal growth factor are mitogens for cultured human fetal adrenal cells (18), with bFGF being the more potent. In light of its potent mitogenic actions on human fetal adrenal cells and its presence in adult adrenal tissue in other species (19), we reasoned that bFGF may be a prime candidate as a local mediator of the stimulatory effects of ACTH on human fetal adrenal growth. Therefore, we examined whether bFGF is synthesized by the human fetal adrenal gland and whether its expression can be regulated by ACTH. Portions of these studies have been presented.t
ABSTRACT Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cells. Northern blot analysis of total RNA from whole human fetal adrenal glands revealed a characteristic 7-kilobase bFGF mRNA, indicating that the fetal adrenal bFGF bioactivity was most likely due to local synthesis. Slot blot and ribonuclease protection analysis showed that bFGF mRNA was present in very low amounts in total RNA d human fetal adrenal from primary cultures of u cells but was increased 2- to 3-fold in cells exposed to 10 nM ACTH-(1-24) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate for 24 hr. bFGF mRNA was localized to adrenocortical cells and not fibroblasts by in situ hybridization. bFGF mRNA was barely detectable in unstimulated cells, whereas it was markedly increased in cells exposed to either ACTH or 8-bromoadenosine 3',5'-cyclic monophosphate. These data support our hypothesis that the regulation of human fetal adrenal growth by ACTH at midgestation may be mediated by the stimulation of local growth factor production, and we suggest that bFGF may play a major role in this process.
The fetal adrenal glands are thought to play a pivotal regulatory and homeostatic role in intrauterine life and in preparation for extrauterine existence. In the human, the adrenals undergo a phase of rapid growth and development between 10 and 20 weeks of gestation, increasing their weight from 50 mg to 1 g and attaining a size equal to or greater than that of the fetal kidney by midpregnancy. The bulk of the gland (>80%o) consists of a large inner cortex referred to as the fetal zone, where much of the rapid growth after 15 weeks of gestation occurs (1-4). The factors responsible for the rapid growth of the human fetal adrenal are largely unknown. In adult mammals, adrenal growth and steroidogenesis are regulated by adrenocorticotropin (ACTH) in vivo (3, 5). Although similar studies in the fetus have been more difficult, a role for ACTH in fetal adrenal development is indicated (6, 7). Recent studies in the hypophysectomized fetal sheep have shown that adrenal cortical growth could be restored by in utero ACTH replacement therapy (8).
MATERIALS AND METHODS Cell Culture. Human fetal adrenal glands were obtained from second-trimester aborted fetuses. Abortions were performed by dilatation and evacuation, and gestational age was estimated by foot length. An adult adrenal was obtained from a kidney donor from the Kidney Transplant Unit, University of California San Francisco. Abbreviations: ACTH, adrenocorticotropin; bFGF, basic fibroblast growth factor; aFGF, acidic fibroblast growth factor; IGF, insulinlike growth factor. *To whom reprint requests should be addressed. tMesiano, S., Mellon, S. H., DiBlasio, A. M., Gospodarowicz, D. & Jaffe, R. B., 72nd Annual Meeting of the Endocrine Society, June 20-23, 1990, Atlanta, GA, abstr. 21.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
5428
Proc. Natl. Acad. Sci. USA 88 (1991)
Medical Sciences: Mesiano et al. For cell culture experiments, human fetal adrenal glands were decapsulated to remove most of the definitive zone, minced into 1-mm3 pieces, and incubated in 0.1% collagenase (Sigma) at 37TC for 30 min (20). The dispersed cells were filtered through a nylon mesh, centrifuged, and resuspended in culture medium, a 1:1 (vol/vol) mixture of medium 199 and Ham's nutrient mixture F-12, supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 gg of gentamicin per ml. Cells were plated on Falcon culture plates at -10,000 cells per cm2 and incubated in a humidified environment at 370C in 95% air/5% CO2. Medium was changed every 48 hr. In some plates, ACTH-(1-24) (10 nM; Organon) or 8-bromocAMP (8-Br-cAMP; 1 mM; Sigma) was added and the cells were incubated for a further 24 hr. bFGF Bioassay. Extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cortical cells were subjected to heparin-Sepharose affinity chromatography and the eluted fractions were assayed for mitogenic activity (21, 22). For the whole glands, three human fetal adrenals (16-23 weeks) were homogenized in 10 mM Tris Cl, pH 7.0/0.5% Triton X-100. The homogenate was adjusted to 0.6 M NaCl and centrifuged (15 min; 40C; 20,000 x g), and the supernatant was applied to a heparin-Sepharose affinity column (0.5-ml bed volume) preequilibrated with 10 mM Tris Cl, pH 7.0/0.6 M NaCl. The column was sequentially eluted with 10 mM Tris Cl, pH 7.0, containing 0.6, 1.0, and 3 M NaCl. An aliquot of each fraction was assayed for mitogenic activity in a cell proliferation bioassay using bovine adrenal capillary endothelial cells as target cells (19). For cultured cells, fetal adrenal cortical cells were dispersed and cultured for 5 days on 10-cm plastic culture dishes, harvested, lysed in 0.8 ml of 0.5% Triton X-100/10 mM dithiothreitol/10 mM Tris Cl, pH 7.0, and centrifuged (30 min at 20,000 x g). The NaCl concentration was adjusted to 0.6 M and the lysate was applied to a heparin-Sepharose column (0.1-ml bed volume) and eluted with 10 mM Tris Cl buffer containing increasing concentrations of NaCl as described above. Each fraction was then bioassayed (19). Effect of bFGF on Human Fetal Adrenal Cell Proliferation. Fetal adrenal cells were plated in 24-well culture dishes at 20,000 cells per well. The culture medium was changed every 48 hr. After 1 week, bFGF (21) was added in various concentrations (0.01-100 ng/ml) in triplicate wells and the cells were incubated 48 hr. Cells were then harvested and counted in a particle counter (Coulter) (20). Extraction of RNA. Total RNA was isolated from human fetal adrenal glands and an adult adrenal gland. The tissue was homogenized in 5 M guanidinium thiocyanate, and RNA was purified on a CsCI gradient (23, 24). Total RNA was also isolated from cultured human fetal adrenal cells (25). Northern and Slot Blot Analysis. A 32P-labeled bovine bFGF cDNA probe corresponding to the protein coding region and exhibiting 99% sequence identity with human bFGF was used (26). Total RNA (20 ,ug) was denatured by glyoxal treatment, electrophoresed through a 1.2% agarose gel, and blotted onto a nylon membrane (Nytran; Schleicher & Schuell). The filter was baked and then prehybridized overnight in 50% (vol/vol) formamide/5x standard saline citrate (SSC)/5x Denhardt's solution/1.0o SDS/10 mM sodium phosphate buffer, pH 8.0, with salmon sperm DNA at 100 ,ug/ml. A 32P-labeled bFGF cDNA probe corresponding to nucleotides 103-605 (26) was synthesized by random primer extension and hybridized to the membrane at 42°C overnight in hybridization solution. The membrane was then washed at 65°C in 0.1x SSC/0.1% SDS and autoradiographed for 2 weeks at -70°C. For slot blot analysis, RNA (20 ,ug) was immobilized on a Nytran membrane under vacuum. The membrane was then hybridized with a 32p_ labeled bFGF cDNA probe, washed under high stringency, and autoradiographed for 1 week at -70'C. Integrity and
5429
amount of RNA loaded were confirmed by hybridization of Northern blots with a human y-actin probe (27) and slot blot filters with a rat cyclophilin probe (28). Ribonuclease Protection Analysis. A 205-base 32P-labeled bFGF cRNA probe was synthesized from a bovine bFGF cDNA fragment corresponding to nucleotides 103-255. The cRNA was purified by gel electrophoresis and hybridized at 550C overnight in 200 mM Pipes/2 M NaCl/5 mM EDTA with 10 Ag of total RNA from control and ACTH-treated human fetal adrenal cells. The hybridization solution was then treated with RNase A (10 Ag/ml; Sigma) in 10 mM Tris Cl, pH 7.5/300 mM NaCl/5 mM EDTA at 370C for 30 min and electrophoresed in a 6 M urea/6% polyacrylamide gel (29). The gel was then dried and subjected to autoradiography for 1 week at -70'C. In Situ Hybridization. Fetal adrenal cortical cells (18-week fetus) were plated onto eight-chamber glass microscope slides (Lab-Tek chamber slides; Nunc) at 10,000 cells per chamber. After 5 days the cells were stimulated for 24 hr with 10 nM ACTH-(1-24) or 1 mM 8-Br-cAMP. The cells were then fixed in 4% paraformaldehyde and subjected to in situ hybridization with a digoxigenin-labeled bFGF cDNA probe (Genius 2 DNA labeling kit; Boehringer Mannheim) at 55°C overnight in 50%o formamide/4x SSC/1 x Denhardt's solution containing salmon sperm DNA (100 lg/ml) and yeast tRNA (100 lg/ml). The cells then were washed for 15 min in 2x SSC at room temperature and four times for 15 min in 0.5x SSC at 55°C. Specifically hybridized cDNA probe was detected with a Genius nucleic acid detection kit (Boehringer Mannheim).
RESULTS Effect of bFGF on Human Fetal Adrenal Cell Proliferation. bFGF was found to be a potent mitogen for cultured human fetal adrenal cells. Addition of bFGF caused a dosedependent increase in cell number with an ED50 of around 0.01 ng/ml (0.6 pM; Fig. 1). Detection of bFGF Bioactivity in the Human Fetal Adrenal Gland. The bioassay is based on the heparin-Sepharose affinity chromatography elution profile of bFGF, which is in the 3.0 M NaCl fractions. In both whole adrenal glands and extracts of cultured cells, a major peak of mitogenic activity was detected in the 3.0 M NaCl fractions (Fig. 2). A minor peak of mitogenic activity was also detected in the 1 M NaCl eluates. This peak of mitogenic activity probably represents acidic FGF (aFGF). The material eluted at 3.0 M NaCl showed bFGF immunoreactivity in a specific radioimmunoassay (data not shown). These data demonstrate that bFGF 4000, 3600 0
x
C)
3200 2800
2400 2000
-
-
-
-I
I
-T
0
0.01
0.1
I
1.0
10
loo
FGF (ng/ml) FIG. 1. Effect of bFGF on human fetal adrenal cortical cell proliferation. (Mean + SD, n = 3.)
5430
Medical Sciences: Mesiano et al. 0.6M NaCI
5000-
Proc. Natl. Acad. Sci. USA 88 (1991)
adrenal (19). The amount of total RNA analyzed, the long
3.OM NaCI
1.0M NaCI
A
exposure time (2 weeks), and the intensity of the signal reflect
4000-
C4
0
x
3000 -j -j
w
a
2000-
_____________________________________ 1000 0o 10 20 30 40 I-OmNaCI lM NaC
3000 00.6M .6MNaCI 3000 aCI
2500-
Cl
3.OM NaCI 3.OMNaCI
B
observed on three separate occasions.
0
2000-
\
2=
V, -i
\
or \ B \
1500
w
the low abundance of bFGF mRNA in the human fetal adrenal gland, possibly due to the mRNA being highly unstable and/or regulated stringently. The intensity of the 7-kb band was reduced in RNA from an adult human adrenal and barely detectable in RNA from 18- and 22.5-week human fetal adrenal glands. Similar results were obtained by slot blot. Effect of ACTH on Human Fetal Adrenal bFGF Expression. Glucocorticoid concentrations increased 10-fold in the conditioned medium from ACTH- and 8-Br-cAMP-treated cells, indicating that steroidogenic capacity was maintained. bFGF mRNA accumulation increased 3-fold in cells treated with ACTH or 8-Br-cAMP (Fig. 4). This increase was evident from 24 to 96 hr of incubation with either ACTH or 8-Br-cAMP. This effect of ACTH on bFGF mRNA accumulation was
a 1000
-
500
10
0
20 30 FRACTION NUMBER
40
Fet 3. 2. Heparin-Sepharose elution profile of bFGF bioactivity in human fetal adrenal glands (A) and primary cultures of extra cts of A__1A ---I
I- __^ _ DX Ai URAL __:_ mean oi human &ttl tetal adrenal cortical cells (B). Each point Is: the duplicate determinations (SD c5%). Similar chromatographic profiles were seen in three separate experiments. _
--
In the cultured fetal adrenal cells, bFGF gene expression was identified in cortical cells by in situ hybridization using a nonradioactive alkaline phosphatase-linked detection system (Fig. 5). Cytoplasmic staining was markedly increased in ACTH- and 8-Br-cAMP-treated cells relative to control. In the ACTH- and 8-Br-cAMP-treated cells, dark cytoplasmic staining was visible after 1-2 hr of exposure to the color reagent. No staining was detected when ACTH-treated cells were treated with RNase A (10 pzg/ml; Sigma) for 30 min at 370C prior to hybridization or in cells probed with a rat growth hormone cDNA probe (30) (Fig. 5). Thus, ACTH can stimulate bFGF mRNA accumulation specifically in cultured human fetal adrenal cortical cells. We obtained similar results in identical cells probed with human insulin-like growth factor II (IGF-II) cDNA,t confirming that IGF-II expression in human fetal adrenal cells is regulated by ACTH (31).
_
is present in the intact human fetal adrenal gland as well as in primary cultures of human fetal adrenal cells. Expression of bFGF by the Human Fetal Adrenal Gland. A bFGF transcript of -7 kilobases (kb) was detected in RNA prepared from 19-, 16.5-, and 16-week fetal adrenals (Fig. 3). The 7-kb bFGF transcript was similar in size to one of the bFGF gene transcripts reported previously in the adult bovine
DISCUSSION Several studies in the human have provided strong evidence that the growth of the fetal adrenal, particularly the rapid proliferation of the fetal zone, is dependent on ACTH (10-13, 32). The present study was part of an ongoing project designed to determine the factors that regulate the rapid growth of the human fetal adrenal gland during midgestation.
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