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Endocrinology Vol. 138, No. 3 1321-1327
Copyright © 1997 by The Endocrine Society

Articles

Expression of Laminin and Its Possible Role in Adrenal Cortex Homeostasis1

Sylvie Pellerin2, Michelle Keramidas, Edmond M. Chambaz and Jean-Jacques Feige

INSERM U-244, Biochemistry of Endocrine Cell Regulations, Department of Molecular and Structural Biology, Commissariat á l’Energie Atomique (Atomic Energy Committee) Grenoble, Grenoble, France.

Address all correspondence and requests for reprints to: INSERM U-244, Biochimie des Régulations Cellulaires Endocrines, Département de Biologie Moléculaire et Structurale, Commissariat á l’Energie Atomique (Atomic Energy Committee) Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France. E-mail: JJFeige{at}geant.ceng.cea.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adult mammalian adrenal cortex undergoes permanent regeneration. This process implies a cellular proliferation step restricted to the external zone of the tissue, and a subsequent centripetal cell migration during which phenotypic transition from glomerulosa into fasciculata and reticularis cells and elimination of senescent cells through apoptosis occur. As the molecular mechanisms implied in adrenocortical cell migration are still generally unknown, we addressed that question in the present study. Of several extracellular matrix proteins tested, laminin was the most potent chemotactic and haptotactic factor for bovine fasciculata adrenocortical cells. The maximal chemotactic effect (3-fold stimulation) was observed with 50–75 µg/ml laminin, whereas the haptotactic effect (3.5-fold stimulation) plateaued for laminin concentrations in the coating solution over 25 µg/ml. Using an anti-Engelbreth-Holm-Swarm laminin antibody, we could demonstrate that adrenocortical cells actively synthesize and secrete Engelbreth-Holm-Swarm-laminin, with the A chain produced in limiting quantities. ACTH treatment of adrenocortical cells specifically induced a 2.7- to 4.5-fold increase in A chain synthesis, resulting in a corresponding increase in the amount of secreted laminin. The distribution of laminin in the adrenal cortex tissue was then evaluated by standard immunohistochemistry. The protein appeared to be uniformly expressed in the three zones of the cortex. This observation does not favor the hypothesis that laminin acts as an attractant driving centripetal cell migration. Laminin, which is synthesized under the control of the systemic hormone ACTH, appears as a permissive factor that facilitates proper homeostasis of the adrenocortical tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADULT mammalian adrenal cortex is a differentiated endocrine tissue that undergoes permanent regeneration. The histology of the adrenal cortex reveals the presence of three distinct zones from the subcapsular area to the junction with the adrenal medulla, differing by the shape and the organization of their constituting cells. These three distinct cell types also differ in their steroidogenic capacities: glomerulosa cells synthesize mineralocorticoids, fasciculata cells synthesize glucocorticoids, and reticularis cells synthesize adrenal androgens in primates and glucocorticoids in other species. Following a long lasting controversy opposing the cell migration theory first proposed by Gottschau (1) and the zonal theory defended by Chester-Jones (2), it is now well established that these different cell types represent successive phenotypes of a single cell commonly named adrenocyte. Adrenocytes proliferate in the external zone, migrate inward, and die through apoptosis in the most inner zone of the tissue (3, 4, 5). In rats, a number of studies have characterized adrenocortical cell turnover (6, 7), showing that proliferation of the progenitor cells is restricted to the outer quarter of the cortex. After this initial proliferative step, rat adrenocytes displace centripetally, reaching the medulla after about 100 days (6). Half of the cells die on their way; the remaining ones are eliminated by apoptosis in the reticularis zone (5).

Recently, a stratum of cells devoid of both aldosterone synthase cytochrome P-450 and cytochrome P-45011ß was identified at the junction between the glomerulosa and fasciculata zones and was proposed to represent the adrenocyte stem cells (8). Although adrenocortical cell proliferation and phenotypic differentiation have been extensively studied both in vitro and in vivo (9, 10, 11, 12, 13, 14), the mechanisms and the molecules implied in adrenocortical cell migration are still mostly unknown. Several studies have established that adrenocytes move en masse toward the adrenal medulla (3, 6, 7). During this process, the cells advance together with their microenvironment and keep their same neighbors. Therefore, the term migration may be inappropriate to define the displacement of adrenocortical cells in the gland, because this process is clearly distinct from the migration observed during embryonic organogenesis when cells pass one another and modify their relative positions. In this article, we will use this term as a synonym of oriented cell displacement.

Concerning the driving force of adrenocortical cell migration, two hypotheses can be formulated. The mitotic pressure occurring in the subcapsular region may drive a streaming of adrenocortical cells toward the zone of cell deletion (zona reticularis). The second possibility is that adrenocortical cell migration is driven by a gradient of chemoattractant secreted by the most inner zones of the cortex. The two hypotheses are not exclusive. As it has long been known that these different processes (proliferation, phenotypic differentiation, apoptosis, and migration) are tightly controlled by the trophic hormone ACTH (3, 4), we investigated in the present study the effects of several extracellular matrix proteins on adrenocortical cell migration. Laminin was the most potent chemotactic and hapotactic molecule in vitro. This prompted us to analyze the regulation of its synthesis by adrenocortical cells and its expression and distribution in the adrenal gland. The presence and function of laminin in the adrenal cortex were uncharacterized before this study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Mouse laminin purified from the extracellular matrix of the Engelbreth-Holm-Swarm (EHS) tumor cell line was purchased from Collaborative Biomedical Products (Bedford, MA). Bovine plasma fibronectin was obtained from Sigma Chemical Co. (St. Louis, MO). Thrombospondin-1 was purified from human platelets, and corticotropin-induced secreted protein/thrombospondin-2 was purified from the conditioned medium of ACTH-treated primary cultures of bovine adrenocortical cells according to previously described protocols (15, 16). ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) (Synacthene) was purchased from Ciba-Geigy (Basel, Switzerland). Recombinant human basic fibroblast growth factor (bFGF) was a generous gift from Dr. A. Baird (Whittier Institute, La Jolla, CA). Recombinant human transforming growth factor-ß1 (TGFß1) and recombinant human insulin-like growth factor I (IGF-I) were purchased from R&D Systems (Oxon, UK) and Boehringer Mannheim (Meylan, France), respectively. Tetradecanoyl phorbol acetate (TPA), angiotensin II, and other chemicals (highest purity grade available) were purchased from Sigma.

Rabbit polyclonal IgGs raised against EHS tumor laminin were generously provided by Dr. J. F. Riou and Dr. D. L. Shi (Laboratoire de Biologie Expérimentale, CNRS URA 1135, Paris, France). Protein A-Sepharose was purchased from Pharmacia (Uppsala, Sweden). The mixture of radiolabeled amino acids (73% [35S]methionine and 22% [35S]cysteine) used for cell metabolic labeling (SA of the mixture, 1175 Ci/mmol) was obtained from DuPont-New England Nuclear (Wilmington, DE).

Cell culture
Bovine adrenocortical (BAC) fasciculata-reticularis cell suspensions were prepared from freshly collected adrenals, and the primary cultures were initiated in Ham’s F-12 medium (Life Technologies, Grand Island, NY) supplemented with 10% horse serum and 2.5% FCS as previously described (17). At the onset of the culture, more than 99% of the cells were immunoreactive for cholesterol side-chain cleavage cytochrome P-450 (P-450scc), and 80–95% were immunoreactive for P-45017{alpha}, indicating that the cell population was constituted of pure adrenocytes essentially of fasciculata-reticularis origin (18). Cells were seeded in six-well plates or in petri dishes (Falcon, Oxnard, CA), at a density of 12.5 x 104 cells/cm2 and grown at 37 C in a humidified atmosphere (5% CO2-95% air).

Metabolic labeling and immunoprecipitation of radiolabeled laminin
BAC cells were grown in six-well plates and metabolically labeled with a [35S]methionine/[35S]cysteine mixture (50 µCi/ml) either for 3 h in methionine-free DMEM (for analysis of cellular proteins) or for 24 h in standard DMEM (for analysis of secreted proteins). At the end of the incubation, the media were collected, and the cells were washed with PBS and lysed for 30 min at 4 C in 0.25 ml RIPA medium [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS] containing a mixture of protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). Cell lysates were centrifuged for 10 min at 10,000 x g to eliminate insoluble debris. Radioactive media and cell lysates were incubated for 1 h at 4 C in the presence of 10 µg/ml antilaminin IgGs. The immune complexes were adsorbed onto protein A-Sepharose beads for 30 min at 4 C, which were then washed three times with RIPA and once with 0.1% SDS. After boiling the beads in Laemmli’s sample buffer, the immunoadsorbed proteins were analyzed by 0.1% SDS-6% PAGE under reducing conditions. The radioactive bands were visualized using a ß-imager (PhosphorImager, Molecular Dynamics, Sunnyvale, CA). The intensity of the bands was quantified from the digitalized pictures of the autoradiograms using ImageQuant software (Molecular Dynamics). The background signal was subtracted from the specific signals in each individual lane. As the PhosphorImager generates a signal proportional to the radioactive content over a dynamic range of 5 orders of magnitude, no saturation problem was encountered during the quantitative analysis.

Immunohistochemical detection of laminin in the adrenal cortex
Fresh bovine adrenal glands were obtained from the local slaughterhouse, sagittally cut, and immediately fixed in Bouin-Hollande solution. After overnight fixation, the pieces were rinsed, dehydrated through graded ethanol, and embedded in paraffin. Eight-micron sections were deparaffinized and hydrated for standard indirect peroxidase immunohistochemistry.

Briefly, the preparations were microwaved for 5 min at 900 watts in 0.1 M citrate buffer, pH 6.0. They were then incubated for 1 h at room temperature with 3% donkey serum in 50 mM Tris-HCl buffer (pH 7.4), 0.9% NaCl, and 0.3% Tween-20. This was followed by overnight incubation at 4 C with 20 or 50 µg/ml antilaminin IgGs. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide for 30 min at room temperature. The preparations were incubated for 45 min with biotinylated donkey antirabbit Igs (Amersham Corp., Arlington Heights, IL) diluted 1:250, washed, and incubated for 30 min with horseradish peroxidase-labeled streptavidin (Amersham) diluted 1:250. Peroxidase was revealed using the metal enhanced diaminobenzidine substrate kit (Pierce Chemical Co., Rockford, IL). Brief nuclear counterstaining was performed using Harris’ hematoxylin, and sections were directly mounted in merckoglass (Merck, Rahway, NJ). For controls, nonimmune IgGs were used instead of the first antibody.

Haptotaxis and chemotaxis assays
Migration of the bovine adrenocortical cells was assayed using Boyden blind well microchambers (NeuroProbe, Cabin John, MD). The assays used 8-µm pore size polyvinylpyrrolidone-free polycarbonate membranes from Poretics Corp. (Livermore, CA). To make the assay quantitative, BAC cells were prelabeled with [35S]methionine (50 µCi/ml) for 5–15 h in standard culture medium. The cells for the assay were obtained by brief exposure of subconfluent cultures to 0.05% trypsin-0.02% EDTA. Trypsinization was stopped by the addition of F-12 medium containing 10% FCS. After two PBS washes, the cells were resuspended in Ham’s F-12 medium supplemented with 0.1% BSA and counted in a Neubauer hematimeter, and their radioactive content was determined. Proteins to be tested in the chemotaxis assay were diluted to the appropriate concentrations in Ham’s F-12 medium supplemented with 0.1% BSA. Twenty-five microliters of these samples were distributed into blind well portions of the microchambers. Filters were overlayed onto the wells, the chambers were assembled, and 50 µl cell suspension (~50,000 cells) were added to each top well. Chambers were incubated overnight at 37 C in a humidified incubator flushed with 5% CO2-95% air. Cells that had not migrated were removed from the upper surface with cotton swabs. The wiped filters were then counted in a liquid scintillation ß-counter. Each sample was tested in triplicate, and data represent the mean ± SEM. As a control, some filters were fixed, stained with Diff-Quick (Poretics Corp., Livermore, CA), mounted, and observed under a light microscope (Axiovert 35, Zeiss, Oberkochen, Germany). A good correlation was observed between the radioactive counts of the wiped filters and the number of cells observed under the microscope.

In the haptotaxis assay, membranes were coated with 50 µg/ml (in PBS) or otherwise indicated concentrations of the tested proteins for 3 h at 37 C, rinsed with PBS, and air-dried before use. The lower compartment of the chamber was filled with Ham’s F-12 medium containing 0.1% BSA. The experimental procedure was identical to that used in the chemotaxis assay.

Statistical analysis
Statistical analyses were performed using paired Student’s t test for comparison of two groups. Values were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Laminin is chemotactic and haptotactic to adrenocortical cells
To determine which extracellular matrix proteins may have the potency to promote adrenocortical cell migration, we developed a migration assay using modified Boyden chambers. [35S]Methionine-labeled fasciculata cells were induced to migrate through 8-µm pore size polycarbonate membranes either toward a solution of attractant protein (chemotaxis assay) or toward an attractant protein immobilized on the underface of the membrane (haptotaxis assay). As shown in Fig. 1Go, fibronectin and laminin (coated at a concentration of 50 µg/ml) were equally potent in the haptotaxis assay; they stimulated adrenocortical cell migration by a factor of 3–3.5. In the chemotaxis assay, adrenocortical cells migrated more efficiently toward a 50 µg/ml solution of laminin (migration, x3) than fibronectin (migration, x1.6). Thrombospondin-1 and thrombospondin-2, tested in parallel in these two assays, were unable to stimulate cell migration. The dramatic effects of laminin on cell migration were dose dependent as shown in Fig. 2Go. The maximal chemotactic effect was observed with 50–75 µg/ml laminin, whereas the haptotactic effect plateaued for laminin concentrations over 25 µg/ml in the coating solution. The migration of adrenocortical cells toward 100 µg/ml laminin was also quantified by staining the lower face of the filter at the end of the experiment and counting the cells that had migrated through the membrane. A very good correlation with the radioactive counts was observed (r = 0.91 and r = 0.93 between both methods tested in parallel on two distinct cell preparations), ruling out the possibility of differences in radioactive counts being due to variations in the wiping procedure.



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  		Figure 1. Effects of various extracellular matrix components on adrenocortical cell migration. Migration of BAC cells was assayed using Boyden blind well microchambers as described in Materials and Methods. Each protein was tested at a concentration of 50 µg/ml in both the haptotaxis and chemotaxis assays. Each value is the mean ± SD of triplicate determinations in one experiment. The results are representative of those obtained in two additional experiments. **, P < 0.01; *, P < 0.05 (vs. respective controls).

 


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  		Figure 2. Dose-dependent stimulation of adrenocortical cell migration by laminin. The migration of BAC cells toward the indicated concentrations of a laminin solution (chemotaxis; A) or toward filters coated on their lower face with the indicated concentrations of laminin (haptotaxis; B) was measured as described in Materials and Methods. Each value is the mean ± SD of triplicate determinations.

 
ACTH stimulates laminin synthesis by adrenocortical cells
We then wondered whether laminin was expressed by adrenocortical cells. BAC cells were metabolically labeled with [35S]methionine, and radiolabeled laminin was immunoprecipitated from the cell extracts and from the medium using a specific antibody. As shown in Fig. 3Go, two distinct immunoreactive laminin subunits of approximately 400 and 200 kDa Mr were detected, corresponding to the A chain (400 kDa) and the comigrating B1 and B2 chains (200 kDa). The ratio of B to A chains in the cell extracts (~13) was very different from that in the medium (~2). This suggested that B chains are synthesized in excess, but that laminin is secreted as a stoichiometric A(B)2 complex. We then investigated the regulation of laminin expression in this model. BAC cells were treated for 24 h with different hormones (0.1 µM ACTH and 0.1 µM angiotensin II), growth factors (2 ng/ml TGFß, 10 ng/ml bFGF, and 50 ng/ml IGF-I), or 1 µM TPA, an activator of protein kinase C. They were metabolically labeled with [35S]methionine during the last 3 h of treatment, and radiolabeled laminin was immunoprecipitated from the cell lysates and visualized with a ß-imager (Fig. 4Go) and quantified (Table 1Go). As observed in Table 1Go, the level of synthesis of B chains was not affected by more than 20% under the various treatments. However, the abundance of A chain was strongly increased by ACTH (x3.2), was slightly increased by angiotensin II (x1.4), and was decreased about 2-fold by TGFß, bFGF, and IGF-I. TPA induced the synthesis of an additional chain of about 300 kDa. To characterize in more detail the effects of ACTH, we analyzed the time (Fig. 5Go) and dose dependence (Fig. 6Go) of laminin synthesis during ACTH treatment. The results indicate that laminin A chain synthesis was stimulated within 8 h of ACTH treatment and reached a plateau between 16–24 h (Fig. 5Go). The dose-response study indicated that the stimulation was maximal for ACTH concentrations equal to or larger than 10-10 M (Fig. 6Go). The maximal level of induction of laminin A chain synthesis varied between 2.7- and 4.5-fold in five independent experiments. Immunoprecipitation of laminin from the medium of metabolically labeled cells revealed that the amounts of A and B chains were stimulated 3- to 5-fold after ACTH treatment (data not shown). This confirms that A chain is the limiting factor in laminin synthesis.



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  		Figure 3. Biosynthesis of laminin by adrenocortical cells in primary culture. BAC cells were metabolically labeled for 24 h with 50 µCi/ml [35S]methionine. Radiolabeled laminin was then immunoprecipitated from the conditioned medium (M) and the cell extract (C) and analyzed by 6% PAGE-SDS, as described in Materials and Methods. Radiolabeled subunits of laminin were visualized using a ß-imager, and their position is shown by arrows. B1 and B2 chains comigrated under these experimental conditions. The positions and sizes (in kilodaltons) of mol wt markers are indicated in the left lane.

 


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  		Figure 4. Effects of various cellular effectors on BAC cell laminin biosynthesis. BAC cells were serum deprived for 6 h and subsequently treated for 24 h in serum-free F-12 medium containing 1 mg/ml BSA and either no addition (CTL), 10-7 M ACTH, 10 ng/ml TGFß1, 10-7 M angiotensin II (AII), 10 ng/ml bFGF, 50 ng/ml IGF-I, or 1 µM TPA. Cells were rinsed and incubated for 3 additional h in methionine-free medium containing 50 µCi/mol of a [35S]methionine/[35S]cysteine mixture, then lysed in RIPA buffer. Radiolabeled laminin was immunoprecipitated from the cell extracts as described in Materials and Methods and visualized using a ß-imager. The positions and sizes of mol wt standards are indicated in the left lane.

 

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  		Table 1. Quantitative analysis of the regulation of laminin biosynthesis by adrenocortical cell effectors

 


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  		Figure 5. Time-dependent induction of laminin A chain biosynthesis by ACTH. BAC cells were serum deprived for 6 h and subsequently treated for the indicated periods of time in serum-free F-12 medium supplemented with 1 mg/ml BSA and either no (-ACTH) or 10-7 M ACTH (+ACTH). Cells were rinsed and incubated for 3 additional h in methionine-free medium containing 50 µCi/ml of a [35S]methionine/[35S]cysteine mixture, then lysed in RIPA buffer. Radiolabeled laminin was immunoprecipitated from the cell extracts and analyzed by PAGE as described in Materials and Methods. It was then visualized using a ß-imager. The positions and sizes of mol wt markers are indicated in the left lane.

 


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  		Figure 6. Dose-dependent stimulation of laminin A chain biosynthesis by ACTH. BAC cells were serum deprived for 6 h and subsequently treated for 21 h in serum-free F-12 medium containing 1 mg/ml BSA and either no (C) or the indicated concentrations (from 10-12-10-7 M) of ACTH. They were then incubated for 3 additional h in methionine-free, cysteine-free culture medium containing 50 µCi/ml of a [35S]methionine/[35S]cysteine mix and the same concentrations of ACTH and lysed in RIPA buffer. Radiolabeled laminin was immunoprecipitated from the cell extracts, analyzed by PAGE, and visualized as described in Materials and Methods. The positions and sizes of the mol wt markers are indicated in the left lane.

 
Laminin is uniformly distributed in the adult adrenal cortex
The synthesis of certain proteins, e.g. {alpha}2-macroglobulin, appears to be induced when adrenocortical cells are placed in primary culture, although it is repressed in vivo (18). To check whether laminin is present in the adrenal gland in vivo and to determine its distribution, we localized it in sagittal sections of bovine adrenal glands using standard immunohistochemical techniques. Figure 7Go shows that laminin is uniformly distributed in the glomerulosa, fasciculata, and reticularis zones of the cortex, whereas it is not detected in the capsule or medulla. No concentration gradient in the protein distribution could be detected among the different zones of the adrenal cortex.



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  		Figure 7. Immunohistochemical localization of laminin in bovine adrenal cortex section. Sections of bovine adrenal glands were immunostained either by antilaminin IgG or by nonimmune IgG, as a control, as described in Materials and Methods. Strong laminin immunoreactivity was observed in the glomerulosa, fasciculata, and reticularis zones of the cortex. A weak signal was observed in the capsule, and few individual cells were labeled in the adrenal medulla. Peroxidase staining with hematoxylin counterstain. Magnification, x20.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here that laminin, the major glycoprotein component of basement membranes, is actively synthesized and secreted by adrenocortical cells in primary culture. Initially thought to be a single molecule, laminin has been redefined as a multigene family of related proteins (19, 20, 21). All of them are large heterotrimeric glycoproteins composed of one heavy chain (200–400 kDa) and two distinct lighter chains (150–200 kDa), arranged altogether in a cruciform structure (22, 23). In this study, laminin was immunoprecipitated from adrenocortical cell extracts with a polyclonal antibody raised against the classical form of laminin (EHS laminin) composed of A, B1, and B2 subunits. The two radiolabeled bands (400 and 200 kDa) corresponded to A and comigrating B1/B2 chains, respectively. It is remarkable that the A chain appears to be synthesized in limiting quantities compared to the B chains. As the antibody we used was raised against a form of laminin composed of A, B1, and B2 chains, this observation could reflect the existence of other forms of laminin containing B1 and B2 chains but a distinct heavy chain, such as M-laminin or K-laminin (20). However, two observations argue against this hypothesis. First, the same antibody immunoprecipitates a distinct ratio of A and B chains (close to 1:2) from the conditioned medium of BAC cells. Second, in TPA-treated cells, the same antibody immunoprecipitates a 300-kDa protein, corresponding probably to the M heavy chain of merosin (M-laminin). We thus favor the hypothesis that BAC cells synthesize B1/B2 chains in excess over A chains, but secrete only the stoichiometric trimeric assemblies of A-B1-B2 chains. The steady state level of the A chain messenger RNA (mRNA) has been reported to be the lowest of the three mRNAs in several tissues, and its noncanonical polyadenylation signal has been shown to be responsible for its rapid degradation (24). Among the different effectors that we tested, ACTH selectively stimulated biosynthesis of the A chain. This effect was time and dose dependent, and the mean stimulation factor reached 3.6-fold. ACTH has been shown previously to profoundly modify the composition of BAC cell extracellular matrix. It stimulates the synthesis of thrombospondin-2 and heparan sulfate proteoglycans (15, 25), whereas it inhibits that of thrombospondin-1 (26). Several researchers have described the induction of laminin B1 and B2 mRNAs by cAMP (27, 28), but to our knowledge this is the first report of a positive regulation of laminin A chain by a cAMP-inducing hormone. We presently do not know by which mechanism(s) ACTH regulates laminin A chain synthesis. The possibility exists that the regulation occurs at the level of gene transcription, mRNA stability, translation, protein stability, or a combination of these processes. This is under current investigation.

Another novel piece of information brought up by this work, is that laminin, and to a lesser extent fibronectin, are potent chemotactic and haptotactic agents for adrenocortical cells. One cannot exclude the possibility that these effects are not induced by laminin per se, but rather are due to an associated chemotactic peptide such as, for example, FGF-2. Although FGF-2 is chemotactic for adrenal capillary endothelial cells in the nanograms per ml concentration range, we did not observe any effect of the recombinant factor on the migration of steroidogenic adrenocortical cells, ruling out the possible mediation of the laminin effect by this specific factor.

Whereas it is now well established that adrenocytes migrate centripetally from the capsule toward the medulla during the permanent process of adult adrenal cortex regeneration, the nature of the molecules that drive this migration is still unknown. ACTH is essential to this process, as demonstrated by experiments with hypophysectomized animals that exhibit reversible adrenal atrophy. It was thus tempting to speculate that the ACTH-induced chemotactic protein laminin was participating in the process of adrenocortical cell centripetal migration. However, the in vivo distribution of laminin in the adrenal cortex appears very uniform across the three zones. No gradient of protein distribution is observed. Therefore, we think that laminin must be considered as a favorable environment facilitating adrenocortical cell migration, rather than as the attractant driving the cells inward. It is more likely that the mitotic pressure resulting from cell division in the subcapsular glomerulosa zone is the real driving force for adrenocortical cell displacement and that laminin is a permissive factor for this process. Time-lapse microscopic observation of adrenocortical cells grown on laminin-coated surfaces should allow determination of whether laminin stimulates chemokinesis, i.e. random motility, in addition to chemotaxis. It could also be informative to examine how laminin expression and distribution are modified during pathophysiological situations that increase the trophicity of the adrenal cortex, such as those induced by ACTH perfusion, adrenal enucleation, or unilateral adrenalectomy. This will require conversion of the assays described here to the rat species.

A second possible function for laminin in the adrenal cortex is the maintenance of a differentiated phenotype. Cheng and Hornsby reported previously that expression of steroid 11ß-hydroxylase and 21-hydroxylase by BAC in culture (at a population doubling level of 10) was undetectable in cultures grown on plastic, but was greatly enhanced in cultures grown on Matrigel (29). Matrigel is a commercial preparation of extracellular matrix from the EHS tumor, of which laminin AB1B2 is the major component. Although purified laminin did not substitute for Matrigel in these effects, it may require additional factors to regulate the expression of these steroidogenic enzymes. The uniform distribution of laminin in the adrenal cortex, as observed in this study, could participate in the maintenance of its differentiated steroid-producing phenotype.

Taken together, the present observations define laminin as an important permissive factor synthesized under the control of the systemic hormone ACTH that allows proper differentiation and regeneration of the adrenal cortex.


   Acknowledgments
 
We are indebted to Drs. V. Richoux and J.-F. Riou (Laboratoire de Biologie Expérimentale, CNRS URA 1135, Paris, France), for the generous gift of antilaminin antibodies, and to Dr. M. Aumailley (CNRS UPR 412, Lyon, France) for helpful discussions. We thank Claude Blanc-Brude and Isabelle Gaillard for their skillful assistance in the preparation of primary cultures of BAC cells, and Sonia Lidy for her help in preparing the manuscript.


   Footnotes
 
1 This work was supported by INSERM (U-244), the CEA (Division des Sciences du Vivant/Biochimie des Régulations Cellulaires Endocrines, Département de Bisur Moléculaire et Structurale), the Association pour la Recherche sur le Cancer, the Fédération Nationale des Centres de Lutte contre le Cancer, the Fondation pour la Recherche Médicale, and the Ligue Nationale contre le Cancer. Back

2 Recipient of a postdoctoral fellowship from the Ligue Nationale contre le Cancer. Back

Received July 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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