Fibronectin, Laminin, and Collagen IV as Modulators of Cell Behavior ...

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The Journal of Clinical Endocrinology & Metabolism 87(4):1819 –1828 Copyright © 2002 by The Endocrine Society

Fibronectin, Laminin, and Collagen IV as Modulators of Cell Behavior during Adrenal Gland Development in the Human Fetus ` S NARCY, JEAN-GUY LEHOUX*, ESTELLE CHAMOUX, AGNE

AND

NICOLE GALLO-PAYET

Service of Endocrinology (E.C., N.G.-P.), Department of Biochemistry (A.N., J.-G.L.), Faculty of Medicine, Universite´ de Sherbrooke, Sherbrooke, Que´bec, Canada J1H 5N4 The specific development of the human fetal adrenal gland requires cell proliferation, migration, apoptosis, and zonespecific steroidogenic activity. The present work was designed to determine the physiological significance of the previously identified spatial distribution of extracellular matrix components in the fetal gland. Primary cultures of human fetal adrenal cells grown on collagen IV, laminin, or fibronectin revealed that cell morphology was affected by environmental cues. Matrices also modulated the profile of steroid secretion by the fetal cells. Collagen IV favored cortisol secretion after ACTH or angiotensin II stimulation and increased dehydroepiandrosterone production when the AT2 receptor of angiotensin II was specifically stimulated. These

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N THE HUMAN fetus, the adrenal gland plays a key role in feto-maternal communication as well as in preparation of extra-uterine life (1). As early as the sixth week of pregnancy, the fetal gland is capable of steroid synthesis (2). Due to strong expression of cytochrome P450C17, the fetal adrenal gland produces high amounts of dehydroepiandrosterone (DHEA)/DHEA sulfate (DHEAS) throughout the entire pregnancy. This type of enzymatic expression is mostly observed in cells of the major, inner part of the gland, referred to as the fetal zone. In contrast, definitive zone cells, found in the periphery, are nonsecreting cells, at least until the second trimester of pregnancy and may be devoted to proliferation. These cells rapidly move into the outer layers of the fetal zone and, by centripetal migration, become differentiated, e.g. steroidogenically active (for review, see Ref. 3). In the central part of the gland, highly differentiated fetal cells die by apoptosis (4, 5). At the end of the second trimester, a third transitional zone becomes distinguishable between the first two zones (6). Although it is generally assumed that the expression of the 3␤-hydroxysteroid dehydrogenase (3␤HSD) enzyme is initiated in this zone around the 24th wk of pregnancy (7), evidence suggests that the expression can be detected earlier (8). Indeed, cortisol has been measured in the 8th wk of gestation (for review, see Ref. 9). Whether the adrenal uses circulating progesterone at this time, or whether 3␤HSD has an early, but undetectable, level of expression has not yet been clearly established. Finally, little is known about the Abbreviations: Ang II, Angiotensin II; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; 3␤HSD, 3␤-hydroxysteroid dehydrogenase.

effects were correlated by changes in the mRNA levels of 3␤hydroxysteroid dehydrogenase and cytochrome P450C17. In contrast, fibronectin and laminin decreased cell responsiveness to ACTH in terms of cortisol secretion, but enhanced ACTH-stimulated androgen secretion. Finally, extracellular matrices were able to orchestrate cell behavior. Collagen IV and laminin enhanced cell proliferation, and fibronectin increased cell death. This study is the first to demonstrate that the nature of extracellular matrix coordinates specific steroidogenic pathways and cell turnover in the developing human fetal adrenal gland. (J Clin Endocrinol Metab 87: 1819 –1828, 2002)

initiation of aldosterone production by the fetus, but it may occur later during pregnancy. Among the factors that modulate the functional and morphological development of the gland, ACTH is the most documented. This hormone is known to regulate fetal adrenal activities. However, only the fetal zone has been proposed to be ACTH sensitive, and even if ACTH is thought to be involved in the onset of 3␤HSD expression and cortisol secretion, in vitro results do not correlate with in vivo observations. In primary cultures, ACTH is a potent stimulus of the induction of 3␤HSD expression and activity in fetal adrenal cells (10 –13). However, this enzyme is undetectable in the fetal gland between the 15th and 22nd wk of pregnancy despite the constant presence of circulating ACTH from the 15th wk (11, 14, 15). These conflicting data suggest that additional factors may interact with ACTH signals to downregulate 3␤HSD expression when the gland is intact. Our previous studies indicated a differential expression of angiotensin II (Ang II) receptor subtypes; the AT2 subtype was highly expressed in the fetal zone, whereas the AT1 subtype appeared in the definitive zone after 16 wk of pregnancy (16). We also identified a role for the AT2 receptor in regulation of apoptosis of fetal zone cells (5). Other studies reported zone-specific expression of ER (17), inhibin (4), or growth factors (for review, see Ref. 18), which could modulate either hormonal control of steroidogenesis (17) or specific cell behavior (4, 5). Nevertheless, little is known about the factors that direct zone-specific cell proliferation, migration, and apoptosis as well as localized expression of P450C17, 3␤HSD, ACTH sensitivity, or AT1/AT2 receptor expression. Components of extracellular matrix could imprint specific

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J Clin Endocrinol Metab, April 2002, 87(4):1819 –1828

adrenocortical cell behavior. Cheng and Hornsby (19) were the first to demonstrate that extracellular matrix contributed to the maintenance of P450C11 and P450C21 enzymatic activities in cultured bovine adrenal cells. Thereafter, Feige’s group described the extracellular matrix distribution in bovine and human adrenal glands and determined a possible role for these factors in adrenocortical cell migration (20, 21). Also, it was proposed that ACTH responsiveness could be dependent on the architectural integrity, at least in the rat adrenal cortex (22). Finally, we recently showed a particular spatial distribution of matrix components and integrin receptors in the developing fetal adrenal gland (23). It is now well accepted that extracellular matrix can induce intracellular cell signals or interact with hormonal or growth factor transduction pathways, leading to specific cell behaviors, such as proliferation, migration, apoptosis, and gene expression (24, 25). In a previous work we have shown that type IV collagen was uniformly expressed throughout the human fetal adrenal gland. In contrast, laminin was mostly present in the periphery, whereas fibronectin had a gradient exhibiting a higher concentration in the central part of the gland. We also demonstrated that these matrix components may find specific receptors in their respective localizations. These results led to the hypothesis that extracellular matrix components present in the human fetal adrenal gland may provide environmental information modulating cell behavior or hormonal responsiveness. The present study was designed to gain insight into the individual and coordinate effects of extracellular matrix components interacting with ACTH and Ang II signals leading to fetal adrenal development. We therefore cultured human fetal adrenal cells on collagen IV, laminin, or fibronectin and analyzed their hormonal responses. We used phase contrast microscopy and fluorescence to determine effects on cell morphology, cytoskeletal architecture, and apoptosis. The rate of cell proliferation was assessed by spectrophotometry. Finally, we determined the combined effect of matrix, ACTH, and Ang II on P450C17 and 3␤HSD mRNA expression as well as on DHEA, DHEAS, and cortisol secretion. Materials and Mehtods Chemicals The chemicals used in the present study were obtained from the following sources: Tissue-Tek embedding medium for frozen tissue specimens from Miles (Elkhart, IN) and gelatin from Fisher Scientific (Nepean, Canada). The in situ apoptosis detection kit (Apoptag) was obtained from Intergen (Purchase, NY); Vectashield mounting medium was purchased from Vector Laboratories, Inc. (Burlingame, CA). Crystal violet and deoxyribonuclease were purchased from Sigma (St. Louis, MO); collagenase, Eagle’s MEM, and OPTI-MEM were obtained from Life Technologies, Inc.(Burlington, Canada). Matrix-coated dishes (collagen IV, fibronectin, and laminin) were obtained from BD-VWR Canlab (Ville Mont-Royal, Canada). Anticortisol antibody for RIA was purchased from ICN Biochemicals, Inc. (Costa Mesa, CA), anti-DHEA and anti-DHEAS antibodies were provided by Dr. Alain Be´ langer (Laboratoire d’Endocrinologie Mole´ culaire, Center de Recherche du CHUL, Ste-Foy, Canada), tritiated cortisol was obtained from Amersham Pharmacia Biotech (Oakville, Canada), and tritiated DHEA and DHEAS were purchased from NEN Life Science Products-DuPont (Missauga, Canada). ACTH-(1–24) peptide (Cortrosyn) was obtained from Organon (Toronto, Canada), and angiotensin II from Bachem (Marina Delphen, CA). CGP 42112, PD 123319, and DUP753 were synthesized at Ciba-Geigy (Basel, Switzerland). All other chemicals were of A grade purity.

Chamoux et al. • Extracellular Matrix and Human Fetal Adrenal Cell Behavior

Retrieval and preparation of glands Fetal adrenal glands were obtained from fetuses aged 14 –21 wk (postfertilization) at the time of therapeutic abortion. Fetal ages were estimated by foot length and time after last menstruation according to the method of Streeter et al. (26). The project was approved by the human subject review committee of our institution. After retrieval, glands were cleansed of fat in MEM and immediately processed for cellular preparation.

Cell culture As described previously (5, 16), whole tissues were used for cell preparation, without separation of fetal zone, neocortex, or chromaffin cells. Briefly, small pieces of tissue (1–2 mm) (3) were dissociated with collagenase (2 mg/ml) and deoxyribonuclease (25 ␮g/ml) in Eagle’s MEM containing 2% antibiotics. After three incubation periods of 20 min each, cells were dissociated, filtered, and centrifuged for 10 min at 100 ⫻ g. The cell pellet was suspended in Opti-MEM medium containing 2% FCS and antibiotics. Cells were plated at a density of about 1 ⫻ 105 in 35 mm either on plastic petri dishes or in dishes coated with various matrices, including collagen IV, fibronectin, or laminin. Cells were grown for 3 d in a humidified atmosphere of 95% air/5% CO2 at 37 C in the absence or presence of ACTH (10⫺9 m), Ang (10⫺7 m), or CGP42112 (10⫺8 m) after an initial resting period of 24 h. The culture medium was harvested every 24 h and stored at ⫺20 C until assayed for steroid secretion. DHEA, DHEA/S, and cortisol were determined by RIA using specific antisera and tritiated steroid as tracer. Cells were examined daily, and phase contrast photographs were taken using a Leica Corp. microscope (Deerfield, IL) equipped with a ⫻32 objective.

Actin labeling After 3 d of culture on defined matrices, cells were fixed for 20 min in 3% paraformaldehyde in PBS buffer, permeabilized for 10 min in 1% Triton X-100, and incubated for 20 min at room temperature with 1 U rhodamine/phalloidin solution as described previously (5). After three washes, coverslips were mounted on petri dishes with Vectashield mounting medium and examined on a Nikon Eclipse 300 microscope (Melville, NY) equipped with a G2A-rhodamin filter and a Coolsnap digital camera.

RNA isolation and cDNA synthesis Total RNA was isolated from human adrenal cells cultured on various matrices using RNAqueous-4PCR (Ambion, Inc., Austin, TX), according to the manufacturer’s recommendations. RNA content and quality were determined photometrically. Two micrograms of total RNA were denatured (70 C, 10 min) in the presence of 200 ␮m p(deoxythymidine)12–18 (Promega Corp., Madison, WI) and reverse transcribed at 42 C for 50 min in 20 ␮l of 1 ⫻ RT buffer [25 mm Tris-HCl (pH 8.3), 37.5 mm KCl, and 1.5 mm MgCl2] containing 15 mm dithiothreitol (Promega Corp.), 200 ␮m deoxy-NTPs (Amersham Pharmacia Biotech, Piscataway, NJ), 25 U rRNasin ribonuclease inhibitor (Promega Corp.), and 200 U Moloney murine leukemia virus reverse transcriptase (Promega Corp.). Inactivation of the enzyme (70 C, 10 min) was followed by glyceraldehyde3-phosphate dehydrogenase PCR to asses the quality of the template cDNA.

PCR experiments cDNA samples (4 ␮l) were used for subsequent PCR amplifications in 50 ␮l of 1 ⫻ PCR buffer [10 mm Tris (pH 8.3), 50 mm KCl, and 1.5 mm MgCl2] containing 200 ␮m deoxy-NTPs (Amersham Pharmacia Biotech, Piscataway, NJ), 10 pmol each of the sense and antisense primers, and 2.5 U Taq DNA polymerase (Amersham Pharmacia Biotech). Primers used for the amplification of 3␤HSD cDNA were chosen according to a previous report (27): P1, 5⬘-TGGAGCTGCCTTGTGACAGGA-3⬘; and P2, 5⬘-TATCATAGCTTTGGTGAGGCG-3⬘; for P450C17 cDNA (28) the primers were: P1, 5⬘-CCCATCTATTCTGTTCGTATGGGCAC-3⬘; and P2, 5⬘-GCCCCAAAGATGTCCCCTATGGTGGT-3⬘. PCR was carried out in a Perkin-Elmer Corp. (Norwalk, CT) thermocycler at 95 C for 2 min, followed by 35 cycles at 95 C for 2 min, 55 C for 1 min, 74 C for 1.5 min, and a final extension at 74 C for 5 min. In


PCR experiments, amplifications in the absence of cDNA and in the presence of 2 ␮g RNA were performed as controls. The PCR products (10 ␮l) were analyzed on 1.5% (wt/vol) agarose gel (Life Technologies, Inc., Burlington, Canada) and visualized by ethidium bromide staining. The lengths of the PCR products were estimated with a 100-bp DNA ladder (Promega Corp.).

In situ apoptosis detection Cells were processed according to the protocol provided by the manufacturer of the Apoptag kit (Intergen). In brief, adherent cells were fixed in 1% paraformaldehyde in HBSS for 10 min, washed briefly, and postfixed in ethanol/acetic acid (2:1) for 5 min at ⫺20 C. Cells were then progressively rehydrated with successive HBSS (130 mm NaCl, 3.5 mm KCl, 1.8 mm MgCl2 2.5 mm NaHCO3, and 5 mm HEPES) washings. After 10 min in equilibration buffer at room temperature, cells were incubated for 1 h and 30 min at 37 C in a solution containing the terminal deoxynucleotidyl transferase enzyme and the reaction buffer containing digoxygenin-labeled nucleotides. Cells were then washed twice in HBSS and incubated with an anti-digoxygenin fluorescein isothiocyanate-coupled antibody. After two washes, sections were counterstained with 0.1% Evan’s Blue solution to visualize normal cells. Plastic slides were then mounted onto dishes with Vectashield mounting medium and observed under a Nikon Eclipse 300 fluorescent microscope equipped with G2A-rhodamine and B1A-FITC filters. Photographs were made with a CoolSnap color digital camera. To better quantify apoptotic cells present in dishes, apoptotic nuclei were counted in 10 fields for each condition and plotted against the total number of cells present in each field.

Proliferation assays The method used to evaluate cell proliferation was adapted from that described by Basora et al. (29). In brief, cells were plated onto matrixcoated 96-well plates and treated with hormones after a 24-h resting period. Three days after treatment, cells were fixed with 1% paraformaldehyde in PBS for 10 min at room temperature. After PBS washing, cells were incubated for 10 min at room temperature with a solution containing 1% crystal violet. After extensive water washings, cells were lysed by incubation for 10 min in 100 ␮l 1% SDS solution. OD at 595 nm was then read with a ␮Quant spectrophotometer. At the time of experimentation, a standard curve was performed with serial dilutions of a cell solution in which cell number was evaluated under a classical hemocytometer.

Results Effect of matrices on cell morphology

The architectural organization of the actin cytoskeleton has previously been reported to be distinct for each cell population of the human fetal adrenal gland and can be used as a marker to discriminate fetal and definitive zone cells (30). Therefore, we compared the actin network on fetal adrenal cells cultured for 3 d on each substrate with phase contrast microphotographs. As shown in Fig. 1, when cultured on plastic, the large cells of the fetal zone had a polygonal morphology, and exhibited an extensive cytoplasmic actin network with well-developed stress fibers (Fig. 1, Aa and Ab, arrows). In contrast, the small cells of the definitive zone were grouped in clusters and retained a rounded shape with actin fibers distributed at the membrane level. This distribution was distinguishable even though the labeling was saturated due to the small size of these cells (Fig. 1, Aa and Ab, arrowheads). On collagen IV-coated dishes, fetal zone cells exhibited a more elongated shape (Fig. 1Ba) with strong actin labeling at specific focal adhesion points (Fig. 1Bb). On fibronectin, fetal zone cells appeared to be less adherent, with a lot of cytoplasmic blebs (Fig. 1Ca), suggesting apoptotic

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cells (5). Moreover, the appearance of the actin cytoskeleton was quite variable; cells adherent to this matrix have a preserved, typical network of stress fibers (Fig. 1Cb, arrows), although the overlapping rounded cells have a more diffuse actin network. On both collagen IV and fibronectin, cells of the definitive zone were almost absent, and when present, groups were of smaller size compared with those observed on laminin (Fig. 1, Bb and Ca, arrowhead). Indeed, laminincoated dishes drastically altered the morphology of adrenocortical cells; most of them appeared rounded, clustered, and reduced in size compared with the fetal cells in other culture conditions (Fig. 1Da, arrowheads). Such a morphology has usually been described for proliferative cells of the definitive zone (31). In fact, actin labeling indicated that most of the cells cultured on this matrix were of the definitive zone type, grouped in large clusters (Fig. 1Db, arrowheads). Due to the thickness of these clusters, the focus was difficult to obtain. However, a few characteristic cells from the fetal zone were distinguishable and constituted a layer under the groups of rounded cells (Fig. 1Db, arrows). Effect of matrices on steroid secretion

We next investigated whether the matrices could influence basal and hormone-stimulated production of fetal adrenal steroids. DHEA, DHEAS, and cortisol were measured in the culture medium after 3 d with or without ACTH (10⫺9 m) or Ang II (10⫺7 m) treatment. CGP42112 (10⫺8 m) was also used to evaluate the AT2 receptor-specific response of Ang II. Because of the diversity inherent to human material, it was almost impossible to perform statistical analyses of the basal level of steroid secretion, which ranged, as indicated in Table 1, from 5.5 ⫾ 0.2 to 107 ⫾ 22 ng/ml for DHEA, from 1.1⫾ 0.1 to 1707 ⫾ 278 ng/ml for DHEAS, and from 0.7 ⫾ 0.1 to 6.1 ⫾ 0.1 ng/ml for cortisol. The results presented in Fig. 2 were expressed as a stimulation ratio observed in four different experiments. As shown in Fig. 2, A and B, the different environmental conditions strongly modified the corticosteroid secretion profiles. Indeed, fibronectin and laminin culture conditions significantly enhanced human fetal adrenal cell responsiveness to ACTH in terms of DHEA and DHEAS production (P ⬍ 0.001; n ⫽ 4 in all conditions). Collagen IV had little effect on DHEA and DHEAS secretion in response to ACTH. In contrast, as illustrated in Fig. 2C, collagen IV conditions strongly enhanced cortisol secretion after ACTH stimulation in comparison to that observed on plastic. Fibronectin culture conditions reduced ACTH-stimulated cortisol production by 45.5% compared with that observed on plastic and by 73% to that observed on collagen IV (P ⬍ 0.001; n ⫽ 4). Under the same conditions, laminin also had a slight, but not significant, inhibitory effect on cortisol secretion. As illustrated in Fig. 3, A and B, Ang II responsiveness was strongly dependent on the nature of matrices. Ang II did not significantly modify DHEA or DHEAS secretion regardless of the matrix used (Fig. 3, A and B). However, a selective AT2 receptor stimulation with the agonist CGP42112 strongly decreased DHEA secretion on plastic (P ⬍ 0.01), but increased DHEA secretion on collagen IV (Fig. 3A; P ⬍ 0.02; n ⫽ 4). DHEAS production remained unaffected under these

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FIG. 1. Phase contrast morphology and fluorescent labeling of actin cytoskeleton of human fetal adrenal cells grown on various extracellular matrices. A, Human fetal adrenal cells cultured on plastic. B, Cells grown on collagen IV-coated dishes. C, Cells grown on laminin-coated dishes. D, Cells grown on fibronectin-coated dishes. Phase contrast microphotographs (Aa, Ba, Ca, and Da, respectively) and actin labeling (Ab, Bb, Cb, and Db, respectively) were carried out after 3 d of culture for each condition. Arrows indicate cells of the fetal zone, and arrowheads indicate cells of the definitive zone. Images were taken with a phase contrast Leica Corp. microscope with a ⫻32 objective for phase contrast observations, and actin labeling was observed under a fluorescent Nikon Eclipse 300 microscope equipped with a G2-A rhodamine filter and a Coolsnap digital camera. Scale bars represent 10 ␮m in phase contrast microphotographs and 25 ␮m in fluorescent microphotographs.

same conditions (Fig. 3B). Fibronectin increased both DHEA and DHEAS secretion, whereas laminin increased DHEA secretion only (Fig. 3, A and B). As shown in Fig. 3C, plastic, laminin, or fibronectin did not influence basal or Ang IIstimulated cortisol secretion. Only the collagen IV matrix favored Ang II stimulation on cortisol secretion, presumably through its AT1 receptor subtype, because CGP42112 had no effect. These data suggest that extracellular matrices imprint

particular steroid production patterns, either by influencing basal levels of steroidogenic enzyme activity or by modulating cell responsiveness to ACTH or Ang II. To determine whether the combined effects of hormones and matrices could affect the expression of steroidogenic enzymes, semiquantitative RT-PCR amplification of 3␤HSD and P450C17 mRNA was conducted. When nonstimulated cells were cultured on collagen IV, fibronectin, or plastic petri



TABLE 1. Basal level of steroid secretion by human fetal adrenal cells after 3 d in culture on plastic petri dishes Steroid measurements

Exp 1

Exp 2

Exp 3

Exp 4

DHEA (ng/ml) DHEAS (ng/ml) Cortisol (ng/ml)

5.5 ⫾ 0.2 1.1 ⫾ 0.1 1.2 ⫾ 0.1

77.9 ⫾ 16.2 1707 ⫾ 278 1.3 ⫾ 0.5

28.3 ⫾ 8.6 180 ⫾ 8 6.0 ⫾ 0.1

107 ⫾ 22 168 ⫾ 35 0.7 ⫾ 0.1

Human fetal adrenal cells obtained from 16- to 18-wk-old fetuses were cultured for 3 d on various matrixes, and media were collected every 24 h. RIA measurement of corticosteroids present in culture media after 3 d are presented in this table. Four independent experiments were performed; each point is the mean of triplicate determinations.

FIG. 3. Steroid secretion in human fetal adrenal cells cultured on various extracellular matrices. Stimulation of DHEA (A), DHEAS (B), or cortisol (C) secretion after 3 d of culture on defined matrices in the absence (䡺) or presence (o) of 10⫺7 M Ang II or 10⫺8 M CGP42112 (f). Results are expressed as a stimulation ratio. Matrix conditions were normalized against plastic conditions, as described in Materials and Methods. Statistical significance is indicated as follows: #, P ⬍ 0.05; *, 0.05 ⬍ P ⬍ 0.02; **, P ⬍ 0.01; ***, P ⬍ 0.001.

FIG. 2. Steroid secretion in human fetal adrenal cells cultured on extracellular matrices. Stimulation of DHEA (A), DHEAS (B), or cortisol (C) secretion after 3 d of culture on defined matrices in the absence (䡺) or presence (u) of 10⫺9 M ACTH. Results are expressed as a stimulation ratio. Matrix conditions were normalized against plastic conditions, as described in Materials and Methods. Statistical significance is indicated as follows: #, P ⬍ 0.05; *, 0.05 ⬍ P ⬍ 0.02; **, P ⬍ 0.01; ***, P ⬍ 0.001.

dishes, 3␤HSD mRNA was virtually absent (Fig. 4A), whereas laminin culture conditions induced a basal level of expression. However, 24-h stimulation with ACTH increased the expression of 3␤HSD mRNA, mainly on collagen IV or plastic. In contrast, stimulation with ACTH did not increase

3␤HSD mRNA expression when cells were cultured on fibronectin. On all of the matrices tested, Ang II alone had poor effects on 3␤HSD mRNA production, but, when used together with ACTH, clearly inhibited the stimulation observed with ACTH, except for plastic culture conditions. As shown in Fig. 4B, the expression of P450C17 mRNA was subject to hormonal control as well as to environmental conditions. First, the basal level of P450C17 expression in cells grown on plastic or fibronectin was high, corroborating with the secretion pattern observed in vivo. Under these conditions, Ang II and ACTH had poorly discernable effects; the expression level was similar to that of nonstimulated cells. However, a combination of the two hormones used on plastic abrogated this expression, which remained unchanged on fibronectin. On the contrary, cells grown on laminin or collagen IV had a lesser level of P450C17 mRNA, but stimulation with Ang II enhanced this expression, an effect reversed when both ACTH and Ang II were used together. ACTH used alone had opposite effects on collagen IV and laminin, acting as a stimulus on laminin and as an inhibitor on col-

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lagen IV. These data suggest that environmental conditions can influence steroid secretion by modulating the mRNA production of the key enzymes in steroidogenesis, P450C17 and 3␤HSD. Effect of matrices on cell behavior

The development of the human adrenal gland involves proliferation at the periphery of the gland and apoptosis in the central portion. To determine whether the distinct spatial


distribution of extracellular matrix components we identified previously could modulate cell behavior, we examined apoptosis and cell proliferation in defined matrices-culture conditions. Apoptosis was induced with the AT2 superagonist CGP42112, as previously reported (5). TUNEL fluorescence photographs as well as their quantifications are illustrated in Fig. 5. On plastic, few apoptotic cells were present (Fig. 5Aa), but cell death was clearly enhanced when CGP42112 was

FIG. 4. RT-PCR amplification of steroidogenic enzymes in human fetal adrenal cells. Cells were cultured for 24 h in the absence or presence of the following treatments: Ang II (10⫺7 M), ACTH (10⫺9 M), or a combination of both hormones. A, Amplified cDNA fragment corresponding to 3␤HSD mRNA (743 bp); B, amplification of P450C17 cDNA fragment (751 bp); C, amplification of glyceraldehyde-3-phosphate dehydrogenase (192 bp), which was used to ensure RNA quality and amounts.

FIG. 5. In situ apoptosis detection in cells grown on various extracellular matrices. Human fetal adrenal cells were cultured for 3 d in the absence (Aa, Ba, Ca, and Da) or presence of the apoptosis-inducing AT2 receptor agonist, CGP42112 (10⫺8 M; Ab, Bb, Cb, and Db). Matrix conditions are as follows: A, plastic; B, collagen IV; C, fibronectin; D, laminin. E, Variations in the number of apoptotic cells, measured on a total of 10 fields. White boxes represent unstimulated cells, and black boxes represent CGP-stimulated cells. Results are reported as the mean ⫾ SE of the total number of cells vs. apoptotic cells. Statistical significance is indicated as follows: #, P ⬍ 0.05; *, 0.05 ⬍ P ⬍ 0.02; **, P ⬍ 0.01.


added to the culture medium (Fig. 5, Ab and E). The results observed in cells grown on collagen IV were quite similar to those from cells grown on plastic (Fig. 5, Ba and Bb). In contrast, fibronectin alone strongly induced apoptosis of fetal adrenal cells (Fig. 5, Ca, Cb, and E), an effect not statistically changed after CGP42112 addition. This observation corroborates the phase contrast illustrations shown in Fig. 1, where cytoplasmic blebs were seen on fibronectin, and is consistent with the central localization of both apoptotic cells and fibronectin in the intact gland. Interestingly, laminin prevented cell death not only in control conditions (Fig. 5Da), but also in AT2 receptor-stimulated cells (Fig. 5, Db and E). We next examined the ability of cells to proliferate under the different culture conditions (Fig. 6). Grown on plastic, cells had a proliferation ratio of 1.4 ⫾ 0.1, a level not significantly affected by hormones. Fibronectin did not favor proliferation, either alone or in the presence of hormones; the ratio remained stable at 1.1 ⫾ 0.1. In contrast, on collagen IV this ratio attained a value of 1.9 ⫾ 0.2 and was enhanced to 2.6 ⫾ 0.1 when cells were stimulated with ACTH. Cell proliferation on laminin was also high (1.9 ⫾ 0.2 after 3 d), but stimulation with ACTH reduced the level of proliferation to 1.5 ⫾ 0.2. In all of the matrix culture conditions tested, Ang II alone reduced the rate of cell proliferation, but had minor effects when used in combination with ACTH. Discussion

The present study is the first to demonstrate that extracellular matrix components can modulate cell behavior and hormonal responsiveness during human fetal adrenal gland development. Our results show that primary cultures grown on collagen IV and, to a lesser extent, on laminin favor cell proliferation, whereas fibronectin enhances apoptosis. We also show that collagen IV increases cell responsiveness to ACTH and mostly favors known AT1 receptor effects of Ang II. Fibronectin clearly reduces cell sensitivity to ACTH, but enhances the effects of Ang II on AT2 receptor. Finally, laminin appears to reduce cell responses to ACTH as well as AT2

FIG. 6. Proliferation of human fetal adrenal cells cultured on various extracellular matrices. After 3 d of culture on plastic, fibronectin, collagen IV, or laminin, cells were counted using a colorimetric method described in Materials and Methods. The figure illustrates results from one representative experiment of 3; each experimental condition is mean ⫾ SE from 12 individual samples. Cells were cultured in the absence (䡺) or presence of 10⫺9 M ACTH ( ), 10⫺7 M Ang II ( ), or a combination of both hormones ( ). Statistical significance is indicated as follows: **, P ⬍ 0.01; ***, P ⬍ 0.001.

;

; ;


receptor responsiveness. In line with our previous study, the present data may explain some of the regulation of zonespecific cellular behavior observed in the developing human adrenal gland. Environmental cues provided by extracellular matrix are known to regulate cell behavior and functions, although the interactions between hormonal responses and matrix elements are less well understood. The recognition of matrix components by their specific integrin receptors present at the plasma membrane leads to the recruitment of multiple cytoskeleton-associated proteins and the subsequent activation of intracellular pathways, ultimately leading to cell growth, survival, differentiation, and motility (24, 25). In the present work we show that the composition of extracellular matrix modulates cell morphology and function. Indeed, on collagen IV, cells resemble the large fetal/transitional steroidogenic cells, whereas cells grown on laminin have the proliferative appearance of cells from the definitive zone. On fibronectin, cells exhibit two types of morphology, i.e. polygonal steroidogenically active cells or rounded picnotic cells. These results in culture are in concordance with the observation that in situ, collagen IV is present in the whole fetal cortex, laminin is concentrated at the periphery, and fibronectin is in the center (23). Moreover, we have previously shown the presence of integrins ␣1␤1 in periphery and ␣2␤1 in the transitional zone, which would allow the recognition of both collagen and laminin matrices (32). Whether cells adopt a defined functional morphology on each matrix or whether they are selected due to the integrins expressed at their membrane surface remains unclear. Previous studies have shown that laminin is expressed throughout the adult human adrenal cortex, whereas fibronectin exhibits a gradient and is more concentrated at the center than in the periphery (20, 21). The presence of collagen IV was not examined in these studies. Taken together, these results suggest that there is a developmental regulation of extracellular matrix, particularly laminin, in the adrenal gland, that could influence cell steroidogenesis and behavior (proliferation vs. apoptosis). ACTH and Ang II responsiveness are also strongly affected by environmental signals. We show that cortisol secretion stimulated by ACTH is enhanced by collagen IV and decreased by laminin and fibronectin. This result is correlated by the expression patterns of 3␤HSD mRNA, which is strongly stimulated by ACTH on collagen IV, but is not affected on fibronectin. The mechanisms that initiate the expression of 3␤HSD, the enzyme allowing fetal glucocorticoid synthesis, are still poorly understood. Indeed, confusion concerning 3␤HSD expression and cortisol secretion results from the observations that cortisol is detected early in gestation at a time when 3␤HSD is not detected (for review, see Ref. 9). Even if the consensus is that this enzyme is first expressed in the definitive or transitional zone (7, 15, 33), the ontogenic expression of 3␤HSD has not been clearly established. From previous publications in the field, it is reasonable to assume that 3␤HSD has a biphasic level of expression (8), which may be under intraadrenal regulation (present data). In vitro studies clearly indicate that ACTH increases 3␤HSD mRNA expression levels (10, 11, 13, 18, 34). From these studies it was assumed that ACTH provides the initi-

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ating signal for 3␤HSD expression and/or cortisol secretion in cultured fetal cells. Even if it is well established that, after the 15th wk of pregnancy, adrenal development depends on the presence of ACTH (35, 36), 3␣HSD was never described between the 15th and 22nd wk of fetal life. These differences observed between the in vivo and in vitro regulation of 3␤HSD led to the hypothesis that other signals, dependent on gland integrity, modulate cell responsiveness to ACTH. Two observations contributed to such a hypothesis. First, fibronectin favored the expression of P450C17, rather than that of 3␤HSD. This correlates with the presence of fibronectin and its potential receptor ␣3␤1 in the fetal zone. In contrast, the presence of collagen and its binding sites on ␣1␤1 and ␣2␤1 in the transitional zone may explain why 3␤HSD is first expressed in this zone. Nevertheless, it remains difficult to explain why collagen (which is present in vivo between the 15th and 22nd wk) favors the in vitro expression of 3␤HSD mRNA and cortisol secretion, whereas this is not the case in vivo between these stages of maturation. Second, when added together, ACTH and Ang II abrogate 3␤HSD expression, which will be discussed below. On laminin, 3␤HSD mRNA levels appear higher than on other matrices and remain unaffected by hormone stimulation, correlating with a poor effect of ACTH or Ang II on cortisol secretion on this matrix. These observations may reflect the fact that cells grown on laminin are mostly of the definitive zone type, a cell population reported to be nonsensitive to ACTH (1, 3). This may also explain why despite the presence of 3␤HSD in both zones, cortisol is secreted in the transitional zone (collagen IV and fibronectin) rather than in the definitive zone (laminin and collagen IV). Matrices may thereby imprint the future pattern of secretion in the zona fasciculata and zona glomerulosa, respectively (6). On fibronectin, ACTH stimulation increases DHEA and DHEAS secretion, a result compatible with the strong basal level of P450C17 expression on this matrix. Such an enzymatic expression pattern is consistent with what is observed in vivo, where P450C17 is expressed in the fetal adrenal cortex as early as the 6th wk of pregnancy (2) and maintains high levels throughout pregnancy (7, 15) and into the neonate stage (37). Ang II alone has no effect on DHEA/DHEAS secretion regardless of the matrix used. Only specific stimulation of the AT2 receptor enhances DHEA on collagen IV. These results may be compatible with the in vivo situation, because at this stage only AT2 receptors are observed in the fetal zone (16), where fibronectin is the major matrix. In contrast, Ang II increases cortisol secretion on collagen, a matrix that favors 3␤HSD expression. These results are thus in accordance with previous studies showing that the AT1 receptor is localized at the periphery of the gland during the second trimester of pregnancy (16). The effect of Ang II on cortisol secretion seen in the human fetus is then compatible with the stimulation observed in the adult (38 – 41). Finally, it should be noted that the expression of AT1 receptor and corresponding cortisol responses are species specific. Indeed, Ang II/cortisol responsiveness is observed in bovine, but not in sheep. In the latter model, a high level of AT1 receptor coincides with a low level of P450C17, whereas later in gestation a rise in P450C17 is preceded by a fall in AT1 receptor (42, 43). Our data suggest that collagen IV favors Ang II effects via


its AT1 receptor in cells of the transitional zone and potentates the effects on AT2 receptor in cells of the fetal zone, the former designated to become the zona fasciculata and the latter to become more similar to the zona reticularis (3). The synergistic effect of the AT2 receptor/collagen IV seems to be specific to P450C17 activity, because no increase was observed for DHEAS production. This observation is of interest, as DHEA-sulfotransferase activity usually parallels that of P450C17 (44). However, the effects of collagen IV on Ang II responsiveness are poorly reflected in enzymatic mRNA expression, suggesting a specific hormonal/environmental regulation of the sulfotransferase and/or lyase activity. The relationship between Ang II and collagens has been extensively studied in heart diseases (for review, see Ref. 45). In fact, the AT1 receptor subtype favors collagen deposition (46), whereas the AT2 receptor may play a significant role in cardiac remodeling by antagonizing AT1 receptor effects and inducing the death of damaged cells (47). Finally, when added together, ACTH and Ang II inhibit the mRNA expression of P450C17 and 3␤HSD in cells grown on collagen IV or laminin. In contrast, no inhibition of P450C17 mRNA levels is observed on fibronectin, although 3␤HSD mRNA is abrogated under the same conditions. These results may be compared with the in vivo physiology: P450C17 is largely expressed in the fetal zone, but is absent in the periphery, where collagen and laminin are both present. In contrast, 3␤HSD is present at the periphery, where fibronectin is less present. This observation may explain why 3␤HSD expression is repressed in vivo; both hormones are present in the fetal adrenal. In fact, this combined stimulation represents a more physiological context for fetal cells, which express both the ACTH receptor and the AT2 receptor for Ang II. Taken together, these data highlight the intricate regulation of 3␤HSD onset and P450C17 maintenance in the human fetal adrenal gland and some of the differences observed between in vitro and in vivo data. We have previously shown that the AT2 receptor and fibronectin were mainly localized in the central part of the gland, as are apoptotic cells (5, 16, 23). The present work indicates that fibronectin enhanced the inhibitory effect of the AT2 receptor on cortisol secretion. This matrix is known to favor proliferation when recognized by its high affinity receptor ␣5␤1. No typical receptor for fibronectin is present in the human fetal adrenal gland, but ␣3␤1 integrin, which can serve as a moderate affinity receptor (32), is specifically expressed in the fetal zone (23). Recent studies have shown that ␣3␤1 integrin is not essential for cell survival and disrupts cell-cell adhesion (48, 49), which is concordant with the migration associated with the in vivo differentiation of cells of the fetal zone. Thus, it is possible that cells retained on fibronectin-coated dishes are cells from the fetal zone (bearing ␣3␤1) rather than the others. The absence of ␣5␤1 may also explain why these cells are nonproliferative and are subject to apoptosis. Indeed, we show that this matrix promotes a high level of cell death, again enhanced by AT2 receptor stimulation. Cells grown on this matrix proliferate poorly compared with cells grown on the other matrices studied. Morphological data together with metabolic observations strongly suggest that fibronectin favors the maintenance of cells from the fetal zone in culture, cells known to be non-


FIG. 7. Summary of the profile of steroid secretion in correlation with the differential distribution of extracellular matrix components (ECM) in the human fetal adrenal gland in the second trimester of gestation. The definitive zone (DZ) contains mainly laminin and collagen IV as well as the AT1 receptor for Ang II, all elements that favor proliferation; the transitional zone (TZ) contains mainly collagen IV and expresses both AT1 and AT2 receptors for Ang II as well as ACTH receptor. All of these elements favor cortisol secretion. However, the fetal zone (FZ) expresses mainly fibronectin and both ACTH and AT2 receptors of Ang II. On this matrix, stimulation of the AT2 receptor induces apoptosis, whereas ACTH stimulation induces steroid secretion.

proliferative (3) and subject to cell death (4, 5). Due to the critical role of Ang II in heart and vessel fibrosis, interactions between fibronectin and Ang II have been well studied (50 – 52). Fibronectin and RGD-containing peptides (the ligandbinding domain of some integrins) have been reported to inhibit AT1 receptor contractile effects of Ang II in rat aortic rings (53). Moreover, in a recent study Fisher et al. (54) indicated that activation of the AT2 receptor enhanced the expression of fibronectin mRNA, thus corroborating with our finding that fibronectin favors AT2 receptor-mediated effects of Ang II in terms of inhibition of proliferation, apoptosis, and steroid secretion. Based on the literature on fibronectin and Ang II, we can hypothesize that fibronectin does not induce proliferative activity of definitive zone cells, inhibits the AT1 receptor effects, and favors AT2 receptorsensitive cells of the fetal zone. In contrast to fibronectin, both laminin and collagen IV favored cell proliferation, an effect described in other endocrine models (55). Interestingly, the present results together with the previous in situ data indicate that cellular function is dependent on the nature of the culture matrix. Indeed, the morphological appearance as well as the steroid secretion pattern and the proliferating/apoptotic behavior of cells attached to laminin were hallmarks of cells from the definitive zone. Cells grown on laminin were not only less sensitive to cell death, but were, in fact, protected against AT2 receptorinduced apoptosis. Accordingly, the protective effect of laminin against cell death has been shown in other models (56, 57). In situ, laminin was reported to be mainly present in the periphery, as was its ␣1␤1 integrin receptor (23). In contrast, collagen IV did not protect cells from AT2 receptor-induced apoptosis despite a potent stimulation of cell proliferation on


this matrix. In agreement with the in vivo expression pattern of this matrix component and its receptors throughout the fetal cortex, collagen appears to enhance typical cell responses associated with the definitive/transitional zone (cortisol secretion, 3␤HSD expression, and AT1 receptor responsiveness) as well as typical cell responses of the fetal zone (ACTH responsiveness and AT2 receptor sensitivity). In summary, the results of the present study highlight that laminin favored the attachment of cells exhibiting morphological and metabolic characteristics of proliferative cells from the definitive zone, whereas fibronectin contributed to cell responses typical of cells from the fetal zone (steroid secretion and apoptosis). Collagen IV, expressed throughout the fetal cortex, greatly enhanced some characteristics of the definitive/transitional zone as well as those of the fetal zone (Fig. 7). In line with our previous study the present data may explain some of the zone-specific cellular behavior observed in the developing human adrenal gland. Acknowledgments The authors deeply thank Lucie Chouinard for experimental assistance, and Dr. Nuria Basora for critical review of the manuscript. Received September 7, 2001. Accepted December 18, 2001. Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, Universite´ de Sherbrooke, 3001 12th Avenue North, Sherbrooke, Que´ bec, Canada J1H 5N4. E-mail: [email protected]. This work was supported by grants from the Medical Research Council of Canada (to N.G.-P. and J.-G.L.; MOP-37891). * Chercheur-boursier de carrie`re of Fonds pour la Recherche en Sante´ du Que´ bec.

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