0021-972X/90/7106-1678$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 6
Printed in U.S.A.
Human Recombinant Activin-A Inhibits Proliferation of Human Fetal Adrenal Cells In Vitro* SUSAN J. SPENCER, JARON RABINOVIC^, and ROBERT B. JAFFE Reproductive Endocrinology Center, Dept. of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143 ABSTRACT: Activins and inhibins are dimeric peptides which are structurally and functionally related to transforming growth factor-P (TGF-P). ThemRNAfor the activin and inhibin subunits is expressed in the adrenal gland. Because members of the TGF-P superfamily have effects on mitogenesis, we examined the effect of recombinant human activin-A(rh-activin-A) on proliferation of midgestation human fetal adrenal cells in vitro. Dose-dependent growth inhibition by rh-activin-A was obtained, with an EDso of 1 ng/ml. Rh-activin-A inhibited basal and epidermal growth factor (EGF)-stimulated fetal zone cell proliferation, but did not alter basic fibroblast growth factor (bFGF)stimulated growth. TGF-P combined with rh-activin-A demonstrated additive inhibition of fetal adrenal growth. These findings suggest a potential autocrine or paracrine role for activin-A in modulating the growth and/or subsequent involution of the human fetal adrenal gland. The human fetal adrenal gland undergoes striking growth during intrauterine life, reaching a size exceeding that of the kidney by midgestation (1, 2). Equally striking is the regression in size of the gland shortly afterbirth (2). Both the growth and, to an even greater degree, the regression of the gland occurs in the unique inner, fetal zone of the adrenal cortex. We have hypothesized that regulation of the growth of the gland is affected by locally produced growth factors and related peptides, and that the expression of these growth factors is governed by trophic hormones from the fetal pituitary and/or the placenta. In support of this thesis, we have shown that basic fibroblast growth factor (bFGF) and EGF are potert mitogens for the human fetal adrenal (3), that bFGF is synthesized by the fetal adrenal, and that expression of bFGF mRNA is enhanced by ACTH (4). Further, Voutilainen and Miller (5) as well as we (4) have shown that insulin-like growth factor-H (IGF-II) expression is enhanced by ACTH in the human fetal adrenal gland. Inhibition of fetal adrenal cell proliferation has been demonstrated by transforming growth factor-P (TGF-P) (6), which modulates growth of a variety of cell types (79). Activins and inhibins are members of the TGF-p peptide superfamily, based on their P-subunit sequence homology. They are dimeric proteins consisting of a, PA or PB subunits. Activins are P>p dimers whereas inhibins area-pheterodimers. These peptides were initially Submitted July 18,1990. Address all correspondence to: Robert B. JafTe, Reproductive Endocrinology Center, University of California, San Francisco, San Francisco, CA 94143. •Supported, in part, by NICHD Grant HD08478 and P30 Center Grant 11729. 'Recipient of Fogarty Fellowship TW04258-01.
characterized by their ability to modulate FSH secretion from the anterior pituitary (10, 11). Previous studies have shown that activin/inhibinsubunitmRNAis widely expressed in immature and adult tissues, including the adrenal gland (12-14). As activin and inhibin areTGFP-related peptides, and as activin/inhibin subunitmRNA is expressed in the adrenal gland, we asked whether activin might influence fetal adrenal gland growth. We hypothesized that, similar to TGF-P, activin exerts an effect on human fetal adrenal gland mitogenesis. Therefore, we assessed the effects of recombinanthuman activin-A (rh-activin-A) on human adrenal fetal zone proliferation. The proliferative effect of activin-A was also examined in combination with EGF, bFGF, and TGF-p. Our results indicate that activin-A inhibits fetal adrenal growth in vitro in a dose-dependent fashion. This inhibition is additive with TGF-p. Activin-A also inhibits EGF-induced proliferation, but does not alter bFGF-stimulated cell growth. Materials and Methods Materials: Medium 199 (M199) supplemented with Earle's balanced salt solution, Ham's F-12, glutamine, gentamicin, fetal bovine serum (FBS) and saline with 0.05% trypsin-versine were prepared by the Cell Culture Facility, University of California, San Francisco (UCSF). Collagenase-dispase was obtained from Boehringer-Mannheim (Mannheim, West Germany). Tissue culture dishes were from Falcon Plastic (Los Angeles, CA). Recombinanthuman activin-A andTGFP were generously provided by Dr. Ralph Schwall, Gen entech, South San Francisco. EGF and bFGF were gifts of Dr. Denis Gospodarowicz, UCSF. Methods: Human fetal adrenal glands of 17 to 20
1678
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 21:11 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS weeks gestation were obtained after elective dilatation and evacuation. The study was approved by the UCSF Committee on Huiftan Research. Fetal age was estimated by measuring fetal foot length (15). Adrenal glands were decapsulated to remove the definitive zone, and the remaining fetal zone was then minced and incubated for 30 miti at 37C in M199 containing 1 mg/ml collagenase-dispase. Dispersed cells were filtered through nylon me$h and centrifuged at 200xg for 10 min. The pellet was resuspended in culture medium consisting of a 1:1 (V:v)mixtureofM199 and Ham's F-12 with 10% FBS, 2 mM glutamine and 50 mg/ml gentamicin. Cells were plated on plastic dishes (24 multiwell plates, Falcon 3047, B&D Labware, Lincoln Park, NJ) at a density of 20,000 cells per well and were cultured at 37C in a 95% air, 5% CO2 humidified environment. After 48h incubation, medium was changed and test substances were added. In addition to rh-activin-A, growth factors tested included EGF (50 ng/ml), bFGF (1 ng/ml), and TGF-|$ (1 ng/ml). Medium and test substances were renewed every 48h. Cell number in each well was determined using a Coulter counter (Coulter Electronics, Hialeah, FL). Cells were trypsinized for counting after 2, 4, or 6d incubation in the presence or absence of test substances. Statistical Analysis: Experiments were performed at least 3 times, with each sample in duplicate or triplicate. Statistical comparisons between treatment groups were made by one-way analysis of variance (ANOVA) and Fisher PLSD or Schefle F-test as post-tests. Results are expressed asmean±SE. Significance was assumed at P ^ 0.05.
1679
inhibition relative to control was achieved with doses equal to or greater than 0.1 ng/ml of activin-A (4 pmol/ L activin-A). This inhibition occurred after 2d incubation with activin-A, reaching a plateau at 6d. Maximal inhibition, to 50% of control at 6d, was achieved with doses of activin-A ^3 ng/ml. Half maximal inhibition was achieved at a dose of 1 ng/ml (40 pmol/L) of rhactivin-A.
100.
. 90 .
09
S
i
FGF
N
s
*
0)
2
- I
i\
*
—i
TGF-p
80 EGF
70 60
0.1 1 10 Activln-A (ng/ml)
100
Fig. 2: EflectofActivin-Aongrowthfactortreatedcultures(day 4) expressed as percent ofcell number in the presence of growth factor alone. Dosesof growth factors: FGF-lng/ml;TGF-f}-lng/ ml; EGF-50ng/ml. (*p
Vol. 71, No. 6
Printed in U.S.A.
Human Recombinant Activin-A Inhibits Proliferation of Human Fetal Adrenal Cells In Vitro* SUSAN J. SPENCER, JARON RABINOVIC^, and ROBERT B. JAFFE Reproductive Endocrinology Center, Dept. of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143 ABSTRACT: Activins and inhibins are dimeric peptides which are structurally and functionally related to transforming growth factor-P (TGF-P). ThemRNAfor the activin and inhibin subunits is expressed in the adrenal gland. Because members of the TGF-P superfamily have effects on mitogenesis, we examined the effect of recombinant human activin-A(rh-activin-A) on proliferation of midgestation human fetal adrenal cells in vitro. Dose-dependent growth inhibition by rh-activin-A was obtained, with an EDso of 1 ng/ml. Rh-activin-A inhibited basal and epidermal growth factor (EGF)-stimulated fetal zone cell proliferation, but did not alter basic fibroblast growth factor (bFGF)stimulated growth. TGF-P combined with rh-activin-A demonstrated additive inhibition of fetal adrenal growth. These findings suggest a potential autocrine or paracrine role for activin-A in modulating the growth and/or subsequent involution of the human fetal adrenal gland. The human fetal adrenal gland undergoes striking growth during intrauterine life, reaching a size exceeding that of the kidney by midgestation (1, 2). Equally striking is the regression in size of the gland shortly afterbirth (2). Both the growth and, to an even greater degree, the regression of the gland occurs in the unique inner, fetal zone of the adrenal cortex. We have hypothesized that regulation of the growth of the gland is affected by locally produced growth factors and related peptides, and that the expression of these growth factors is governed by trophic hormones from the fetal pituitary and/or the placenta. In support of this thesis, we have shown that basic fibroblast growth factor (bFGF) and EGF are potert mitogens for the human fetal adrenal (3), that bFGF is synthesized by the fetal adrenal, and that expression of bFGF mRNA is enhanced by ACTH (4). Further, Voutilainen and Miller (5) as well as we (4) have shown that insulin-like growth factor-H (IGF-II) expression is enhanced by ACTH in the human fetal adrenal gland. Inhibition of fetal adrenal cell proliferation has been demonstrated by transforming growth factor-P (TGF-P) (6), which modulates growth of a variety of cell types (79). Activins and inhibins are members of the TGF-p peptide superfamily, based on their P-subunit sequence homology. They are dimeric proteins consisting of a, PA or PB subunits. Activins are P>p dimers whereas inhibins area-pheterodimers. These peptides were initially Submitted July 18,1990. Address all correspondence to: Robert B. JafTe, Reproductive Endocrinology Center, University of California, San Francisco, San Francisco, CA 94143. •Supported, in part, by NICHD Grant HD08478 and P30 Center Grant 11729. 'Recipient of Fogarty Fellowship TW04258-01.
characterized by their ability to modulate FSH secretion from the anterior pituitary (10, 11). Previous studies have shown that activin/inhibinsubunitmRNAis widely expressed in immature and adult tissues, including the adrenal gland (12-14). As activin and inhibin areTGFP-related peptides, and as activin/inhibin subunitmRNA is expressed in the adrenal gland, we asked whether activin might influence fetal adrenal gland growth. We hypothesized that, similar to TGF-P, activin exerts an effect on human fetal adrenal gland mitogenesis. Therefore, we assessed the effects of recombinanthuman activin-A (rh-activin-A) on human adrenal fetal zone proliferation. The proliferative effect of activin-A was also examined in combination with EGF, bFGF, and TGF-p. Our results indicate that activin-A inhibits fetal adrenal growth in vitro in a dose-dependent fashion. This inhibition is additive with TGF-p. Activin-A also inhibits EGF-induced proliferation, but does not alter bFGF-stimulated cell growth. Materials and Methods Materials: Medium 199 (M199) supplemented with Earle's balanced salt solution, Ham's F-12, glutamine, gentamicin, fetal bovine serum (FBS) and saline with 0.05% trypsin-versine were prepared by the Cell Culture Facility, University of California, San Francisco (UCSF). Collagenase-dispase was obtained from Boehringer-Mannheim (Mannheim, West Germany). Tissue culture dishes were from Falcon Plastic (Los Angeles, CA). Recombinanthuman activin-A andTGFP were generously provided by Dr. Ralph Schwall, Gen entech, South San Francisco. EGF and bFGF were gifts of Dr. Denis Gospodarowicz, UCSF. Methods: Human fetal adrenal glands of 17 to 20
1678
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 21:11 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS weeks gestation were obtained after elective dilatation and evacuation. The study was approved by the UCSF Committee on Huiftan Research. Fetal age was estimated by measuring fetal foot length (15). Adrenal glands were decapsulated to remove the definitive zone, and the remaining fetal zone was then minced and incubated for 30 miti at 37C in M199 containing 1 mg/ml collagenase-dispase. Dispersed cells were filtered through nylon me$h and centrifuged at 200xg for 10 min. The pellet was resuspended in culture medium consisting of a 1:1 (V:v)mixtureofM199 and Ham's F-12 with 10% FBS, 2 mM glutamine and 50 mg/ml gentamicin. Cells were plated on plastic dishes (24 multiwell plates, Falcon 3047, B&D Labware, Lincoln Park, NJ) at a density of 20,000 cells per well and were cultured at 37C in a 95% air, 5% CO2 humidified environment. After 48h incubation, medium was changed and test substances were added. In addition to rh-activin-A, growth factors tested included EGF (50 ng/ml), bFGF (1 ng/ml), and TGF-|$ (1 ng/ml). Medium and test substances were renewed every 48h. Cell number in each well was determined using a Coulter counter (Coulter Electronics, Hialeah, FL). Cells were trypsinized for counting after 2, 4, or 6d incubation in the presence or absence of test substances. Statistical Analysis: Experiments were performed at least 3 times, with each sample in duplicate or triplicate. Statistical comparisons between treatment groups were made by one-way analysis of variance (ANOVA) and Fisher PLSD or Schefle F-test as post-tests. Results are expressed asmean±SE. Significance was assumed at P ^ 0.05.
1679
inhibition relative to control was achieved with doses equal to or greater than 0.1 ng/ml of activin-A (4 pmol/ L activin-A). This inhibition occurred after 2d incubation with activin-A, reaching a plateau at 6d. Maximal inhibition, to 50% of control at 6d, was achieved with doses of activin-A ^3 ng/ml. Half maximal inhibition was achieved at a dose of 1 ng/ml (40 pmol/L) of rhactivin-A.
100.
. 90 .
09
S
i
FGF
N
s
*
0)
2
- I
i\
*
—i
TGF-p
80 EGF
70 60
0.1 1 10 Activln-A (ng/ml)
100
Fig. 2: EflectofActivin-Aongrowthfactortreatedcultures(day 4) expressed as percent ofcell number in the presence of growth factor alone. Dosesof growth factors: FGF-lng/ml;TGF-f}-lng/ ml; EGF-50ng/ml. (*p
