A Novel Role of Follistatin, an Activin-binding Protein, in the Inhibition of Activin Action in Rat Pituitary Cells
ENDOCYTOTIC DEGRADATION OF ACTIVIN AND ITS ACCELERATION BY FOLLISTATIN ASSOCIATED WITH CELL-SURFACE HEPARAN SULFATE*

(Received for publication, September 18, 1996, and in revised form, December 30, 1996)

Osamu Hashimoto Dagger §, Takanori Nakamura Dagger , Hiroki Shoji Dagger , Shunichi Shimasaki , Yoshihiro Hayashi § and Hiromu Sugino Dagger par

From the Dagger  Institute for Enzyme Research, University of Tokushima, Kuramoto, Tokushima 770, Japan, the § Department of Veterinary Anatomy, Faculty of Agriculture, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan, and  Department of Reproductive Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093-0674

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

There are two types of the activin-binding protein follistatin (FS), FS-288 and FS-315. These result from alternative splicing of mRNA. FS-288 exhibits high affinity for cell-surface heparan sulfate proteoglycans, whereas FS-315 shows low affinity. To understand the physiological role of cell-associated FS, we investigated the binding of activin to cell-associated FS and its behavior on the cell surface using primary cultured rat pituitary cells. Affinity cross-linking experiments using 125I-activin A demonstrated that activin bound to rat pituitary cells via FS as well as to their receptors on the cell surface. FS-288 promoted the binding of activin A to the cell surface more markedly than FS-315. When the cells were incubated with 125I-activin A in the presence of FS-288, significant degradation of activin A was observed, and this was dependent on the FS-288 concentration. This activin degradation was abolished by heparan sulfate, chloroquine, and several lysosomal enzyme inhibitors. Moreover, FS-288 stimulated cellular uptake of activin A, whereas chloroquine suppressed lysosomal degradation following internalization, as demonstrated by microscopic autoradiography. These results suggest that cell-associated FS-288 accelerates the uptake of activin A into pituitary cells, leading to increased degradation by lysosomal enzymes, and thus plays a role in the activin clearance system.


INTRODUCTION

Three gonadal peptide factors, inhibin (1-4), activin (5, 6), and follistatin (FS)1 (7, 8) have been isolated and characterized by their ability to regulate follicle-stimulating hormone (FSH) synthesis and secretion by the pituitary. Inhibin and follistatin inhibit, and activin stimulates, FSH release from the pituitary both in vitro and in vivo. Inhibin and activin are structurally related and belong to the transforming growth factor beta  superfamily. Both are disulfide-linked dimers; inhibin is composed of alpha - and beta -subunits, whereas activin is a dimer of the inhibin beta -subunit. Two forms of inhibin (A and B) and three forms of activin (A, AB, and B) have been isolated from ovarian follicular fluid. These forms arise because of the existence of two homologous but distinct beta -subunits, called beta A and beta B (5, 6, 9-12).

In addition to stimulation of FSH synthesis and release, various biological roles of activins outside the reproductive system have been extensively investigated. Almost all functions associated with activins can now be interpreted by their proliferative, antiproliferative, and differentiative activities. In view of the widespread expression of activin subunits in both the embryo and the adult (13), as is also the case with activin receptors, it is not too surprising that activin effects have been noted in a multitude of diverse tissues and cell types. Activins exert their effects through specific binding to two different types of receptors, called types I (molecular mass ~53 kDa) and II (molecular mass ~70 kDa) that have recently been cloned. Types I and II receptors, which belong to the serine/threonine kinase receptor family, form heteromeric receptor complexes that are essential for signal transduction after ligand binding, but little is yet known about the events immediately following receptor activation (14).

The activity of FS resembles that of inhibin, but its structure is quite different because FS is a glycosylated single-chain protein (7). There are two forms of FS, resulting from alternative mRNA splicing and hence two different mRNAs that encode FS-315 and its carboxyl-terminal truncated homologue FS-288 (consisting of 315 and 288 amino acids, respectively) (15). Because of the widespread distribution of FS mRNA in extragonadal tissues, physiological roles of FS other than FSH suppression have been predicted (16). In line with these expectations, we previously demonstrated that FS is an activin-binding protein (17) and neutralizes the diverse activin bioactivities in various systems by stoichiometrically forming inactive complexes with activins (18). We have also shown that FS is capable of associating with cell-surface heparan sulfate proteoglycans and proposed that FS participates in the regulation of the multiple actions of activin (19). The affinities of the two FS proteins, purified from porcine ovaries, for activins were demonstrated to be essentially similar. However, the affinity of FS-288 for heparan sulfate side chains was found to be much higher than that of FS-315 (20). The widespread and similar tissue distributions of both FS and the beta -subunit mRNAs imply that FS and activin proteins are produced locally and that FS acts as a local modulator of activin activity. This assumption is supported by the findings that activin binding to FS inhibits the effect of activin on granulosa (21), embryonal carcinoma (22), Xenopus animal explant (23), erythroid (24), and pituitary (18) cells. To improve understanding of the role of FS in the local control of activin function, the association of FS with cell-surface proteoglycans could be of importance.

The pituitary has been identified as the production site of a number of cell growth factors and peptide hormones, including activins and FS. Some of these have been shown to locally modulate pituitary function. Therefore, a study was undertaken to investigate the physiological significance of the activin-FS-glycosaminoglycan interaction in cultured pituitary cells. The results demonstrate that cell-associated FS-288 (carboxyl-terminal truncated FS) accelerates the endocytotic internalization of activin into pituitary cells leading to its degradation by lysosomal enzymes. Cell-associated FS, therefore, plays a role in clearance of the activin signal from the cell surface.


EXPERIMENTAL PROCEDURES

Materials

Activin A was purified from porcine follicular fluid as described previously (25). Recombinant human follistatins with 315 (FS-315) and 288 (FS-288) amino acids were prepared as described previously (26). Heparan sulfate from bovine kidney was purchased from Seikagaku Kogyo (Tokyo, Japan). A rat FSH radioimmunoassay kit was obtained from the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. Chloroquine and trans-epoxy succinyl-L-leucylamido-3-methylbutane (E-64c) were purchased from Sigma. Chymostatin, leupeptin, and pepstatin A were obtained from Peptide Institute, Inc. (Osaka, Japan). Glycosaminoglycan-degrading enzymes (chondroitinase ABC and heparitinase) were purchased from Seikagaku Co. (Tokyo, Japan). All other chemicals were of analytical grade or the highest quality commercially available.

Cell Culture

COS-7 cells were obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin in a humidified CO2 (5%) air incubator at 37 °C. Rat anterior pituitary cells were obtained from 10-week-old female Wistar rats (Japan SLC, Inc., Hamamatsu, Japan). The rats were decapitated then their pituitaries were minced and enzymatically dispersed as described previously (27). The cells were suspended in DMEM supplemented with 2.5% FBS, 10% horse serum, 40 µg/ml gentamicin sulfate, 1 µg/ml fungizone, 0.5 mg/ml glutamine, and 0.1 mg/ml NaHCO3. For affinity cross-linking experiments, the cells were seeded into 24-well tissue plates at a density of 5 × 105 cells/0.5 ml/well. For endocytotic degradation experiments, the cells were seeded into 96-well plates at a density of 8 × 104 cells/0.1 ml/well. Pituitary cells were cultured in a humidified CO2 (5%) air incubator at 37 °C.

Radiolabeling of Ligands

Activin A, FS-288, and FS-315 were iodinated with Na125I using the chloramine-T method as described previously (27). The specific activity of 15,000-20,000 cpm/ng protein was then ascertained. In brief, 10 µg of protein was dissolved in 25 µl of 0.3 M phosphate buffer (pH 7.2). Na125I (400 µCi) in 8 µl of distilled water and 8 µl of chloramine-T solution (250 µg/ml) were then added and vortexed for 1 min. To dilute and terminate the reaction, 150 µl of phosphate-buffered saline (PBS) containing 10 mM sodium pyrosulfite and 0.05% CHAPS was added. The solution was chromatographed on a Bio-Gel P-10 (Bio-Rad) column (0.8 × 6 cm) equilibrated with PBS containing 10 mM sodium pyrosulfite and 0.05% CHAPS. The column was eluted with the same buffer, and the labeled protein was collected. The iodinated preparations were bioactive; the affinity of labeled activin A for receptors and its apoptotic activity were not affected by labeling, and the activin-binding activity of labeled FS was unchanged after labeling.

Transient Transfection of cDNAs

A cDNA clone of the type I activin receptor was isolated by screening a mouse embryonal carcinoma cell line (P19) cDNA library using a polymerase chain reaction-amplified fragment (nucleotides 601-837 (28)) as a probe. A cDNA fragment that included the entire coding region for the type IIA activin receptor (nucleotides 59-1824 (29)) was obtained by amplification of the reverse transcriptase products of total RNA from P19 cells using polymerase chain reaction. The cDNAs for type I or type II activin receptors were subcloned into a pcDLSRalpha expression vector (30) and used for transient transfection. The plasmid DNA (2 µg) was transfected into COS-7 cells plated into 6-well plates at a density of 2 × 105 cells/1 ml/well by a DEAE-dextran method (31). After 2 days of culture, the cells were used for affinity cross-linking.

Affinity Cross-linking

The cells were washed once with binding buffer (DMEM containing 25 mM HEPES (pH 7.4) and 0.2% bovine serum albumin) and incubated on ice for 2 h with 40 ng/ml 125I-activin A in the presence or absence of unlabeled activin A in the binding buffer. After incubation, the cells were washed three times with ice-cold PBS and incubated in PBS containing 1 mM disuccinimidyl suberate (DSS) for 20 min on ice. The reaction was quenched with PBS. The cells were scraped off, rinsed with 20 mM Tris-HCl (pH 7.2) containing 2 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide, and 2 mM diisopropyl fluorophosphate, centrifuged, and resuspended in solubilization buffer (50 mM Tris-HCl (pH 7.2) containing 150 mM NaCl, 2 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide, 2 mM diisopropyl fluorophosphate, 1% Triton X-100, and 10% glycerol), followed by gentle stirring for 1 h on ice. The cell lysates were introduced into 2% SDS and boiled at 100 °C for 10 min. The resulting affinity-labeled samples were subjected to SDS-PAGE (7.5 or 8% gels). Thereafter, the gels were fixed, stained with 0.25% Coomassie Brilliant Blue R-250, destained, and air-dried and then autoradiographed using a Fuji BAS1500 Bio-Imaging analyzer (Fuji Photo Film, Tokyo, Japan) and Hyperfilm (Amersham Corp.).

Assay for Binding of FS and the Activin·FS Complex to Cultured Rat Pituitary Cells

Rat pituitary cells grown in 96-well plates (8 × 104 cells/well) were washed once with culture medium and incubated with various concentrations of 125I-FS-288 or 125I-FS-315 in 100 µl of culture medium for 2 h at 37 °C. After washing three times with fresh medium, 100 µl of 10% SDS was added to each well to solubilize the cells, and the radioactivity (cell-bound FS) was quantified by gamma -spectrometry. To examine the association of the activin·FS complex with the cell surface, 125I-activin A (40 ng/ml) and FS-288 or FS-315 (200 ng/ml) were preincubated in DMEM containing 10% horse serum and 2.5% FBS for 1 h at 37 °C to ensure complete formation of the complex. The resulting complex fraction (10 µl) was added to the pituitary cell culture (8 × 104 cells/well). The amount of the cell-bound complex was determined as described above. The effect of heparan sulfate on binding was investigated by co-incubation with 10 µg/ml heparan sulfate.

Treatment of Rat Pituitary Cells with Glycosaminoglycan-degrading Enzymes (GDE)

Rat pituitary cells (8 × 104 cells) grown in 96-well plates were washed twice with DMEM containing 0.2% bovine serum albumin and 20 mM HEPES (pH 7.3), and treated with GDE (0.02 units/ml) at 37 °C for 90 min. The cells were washed twice with the same buffer, and FS binding and activin degradation in treated cells were determined using 125I-activin A.

Assay of Endocytotic Degradation of Activin and FS

Rat pituitary cells grown in 96-well plates (8 × 104 cells/well) were incubated in 100 µl of culture medium with 40 ng/ml 125I-activin A in the presence of increasing concentrations of FS (0-400 ng/ml). The incubation medium was collected at various incubation times. An equal volume of 30% trichloroacetic acid solution was added to the medium. After centrifugation, radioactivity in the supernatant (trichloroacetic acid-soluble fraction), in which degraded activin was recovered, was quantified by gamma -spectrometry. Degradation was expressed as the percentage of radioactivity in the trichloroacetic acid-soluble fraction relative to the total radioactivity added. To examine the effects of various chemical inhibitors on the endocytotic degradation of activin by pituitary cells, the cells (8 × 104 cells/well) were incubated with 125I-activin A (40 ng/ml), FS-288 or FS-315 (200 µg/ml), and various concentrations of inhibitors for 24 h at 37 °C in 100 µl of DMEM containing 10% horse serum and 2.5% FBS.

Determination of the FSH Release-inhibiting Activity of FS

FSH release-inhibiting activity was determined by the cultured rat pituitary cell assay (27). Cells plated into 96-well plates (8 × 104 cells in 0.2 ml/well) were cultured in the presence of FS (0-100 ng/ml) for 60 h at 37 °C. The culture medium was then removed and tested for FSH using the radioimmunoassay kit.

Autoradiography

Pituitary cells were plated into a Lab-Tek tissue culture chamber slide (8 chambers, Nunc Inc., IL) at a density of 4 × 104 cells/0.3 ml/chamber and incubated with 40 ng/ml 125I-activin A (0.3 ml/well) in the presence or absence of FS (200 ng/ml) for 12 h at 37 °C. The effect of heparan sulfate (10 µg/ml) or chloroquine (0.1 mM) on the endocytotic degradation of activin A was also examined. After incubation, the cells were washed three times with ice-cold PBS. To remove iodinated activin A bound to the cell surface, the cells were rinsed twice with 20 mM HEPES buffer (pH 7.4) containing 2 M NaCl and twice with 20 mM sodium acetate buffer (pH 4.0) containing 2 M NaCl (acid/salt buffer). The cells were then fixed with Carnoy's solution (ethanol/chloroform/acetic acid, = 6:3:1 (v/v)) for 10 min at room temperature after which the slides were coated with a layer of K.5 emulsion (Ilford, UK), exposed for 5 days at 4 °C, and developed. The internalized activin A was observed microscopically.


RESULTS

Association of FS with Rat Pituitary Cell Surfaces

Activin receptor mRNAs have been reported to be distributed in the rat pituitary (32), but the receptor protein has yet to be identified. We attempted to analyze the pituitary activin receptor by affinity cross-linking 125I-activin A to rat pituitary cells using the bifunctional chemical cross-linker DSS. Faint but definite cross-linked bands of 80 and 100 kDa were observed (Fig. 1A), and these corresponded well with those of the activin receptors transiently coexpressed in COS-7 cells (Fig. 1B). These bands were displaced by the addition of excess unlabeled activin, demonstrating the specificity of the activin·receptor complex. The binding of labeled activin to both types I and II activin receptors was completely abolished in the presence of excess FS-288 or FS-315. These findings indicate that the activin·FS complex cannot bind to activin receptors and would account for the inhibitory effect of FS on activin-induced stimulation of FSH secretion by pituitary cells. Although formation of activin·receptor complexes was prevented by the addition of FS, broad bands with molecular masses ranging from 45 to 65 kDa and from 70 to 100 kDa were visible after treatment with either FS-288 or FS-315. These bands were not related to activin receptors, because they were also yielded by COS-7 cells that were not transfected with activin receptor DNAs. Based upon their molecular sizes, the lower band (45-65 kDa) was assumed to be a 1:1 molar complex between activin and FS and the higher band (70-100 kDa) to be a 1:2 molar complex. Labeling of these bands was completely inhibited by incubation with heparan sulfate and with excess unlabeled activin, suggesting that labeled activin is held on the cell surface by FS bound to the heparan sulfate side chains of proteoglycans. It was confirmed by binding experiments using an FS-coated microplate that heparan sulfate (10 µg/ml) had no inhibitory effect on formation of activin and FS (data not shown). It should be noted that FS-288 yielded a much more intense band than FS-315, which was consistent with our previous finding that FS-288 showed a much higher affinity than FS-315 for heparan sulfate proteoglycans on rat ovarian granulosa cells (20). To confirm that this also applied to pituitary cells, rat pituitary cells were incubated with various concentrations of radioiodinated FS, and the results are shown in Fig. 2. As expected, FS-288 showed quite high affinity for the pituitary cells, whereas the affinity of FS-315 was low. Bound 125I-FS-288 was displaced by the addition of excess unlabeled FS-288 but not by unlabeled FS-315 (data not shown). The association of FS-288 with the cells was completely suppressed by excess heparan sulfate or heparin but not by keratan sulfate, chondroitin sulfate A, or dermatan sulfate (data not shown), indicating that FS-288 binds to heparan sulfate on pituitary cell surfaces. The binding site of FS on the pituitary cell surface was examined by determining the binding of FS to the cells after treatment with GDE such as chondroitinase ABC and heparitinase. Heparitinase-treated cells showed a significantly reduced binding capacity for FS-288, whereas chondroitinase ABC treatment was found to have no significant effect on FS-288 binding (Fig. 3A). The affinity of FS-315 for the cell surface remained low even after GDE treatment (Fig. 3B). These data support the hypothesis that FS-288 recognizes and attaches to the heparan sulfate side chains of proteoglycans on the pituitary cell surface.


Fig. 1. Inhibitory effects of FS on 125I-activin A binding to activin receptors on pituitary cells and COS-7 cells transfected with activin receptor cDNAs. A, rat pituitary cells (5 × 105 cells) cultured in 24-well plates were incubated with 125I-activin A (40 ng/ml) in the absence (a) or presence (b) of unlabeled activin A (400 ng/ml), and in the presence of FS-288 (200 ng/ml) (c) FS-315 (200 ng/ml) (d), FS-288 (200 ng/ml) and heparan sulfate (10 µg/ml) (e), or FS-315 (200 ng/ml) and heparan sulfate (10 µg/ml) (f), cross-linked with DSS, and analyzed by SDS-PAGE (8% gels) as described under "Experimental Procedures." B, COS-7 cells cultured in 6-well plates (2 × 105 cells) and transfected with type IA and type IIA activin receptor cDNAs were incubated with 125I-activin A (40 ng/ml) A in the absence (a) or presence (b) of activin A (400 ng/ml), or porcine FS (200 ng/ml) (c), cross-linked with DSS, and analyzed by SDS-PAGE (7.5% gels) as described under "Experimental Procedures." Untransfected COS-7 cells were also analyzed under the same conditions in the absence (d) or presence (e) of porcine FS (200 ng/ml) or porcine FS (200 ng/ml) and heparan sulfate (10 µg/ml) (f).
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Fig. 2. Binding of 125I-FS to rat pituitary cells. Pituitary cells cultured in 96-well plates (8 × 104 cells/well) were incubated with increasing concentrations of 125I-FS-288 (bullet ) or FS-315 (open circle ) for 2 h at 37 °C. The amount of cell-bound FS was determined as described under "Experimental Procedures." The results are expressed as means ± S.D. from triplicate experiments.
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Fig. 3. Inhibition of the binding of FS to pituitary cells by GDE treatment. Rat pituitary cells (8 × 104 cells/well) were treated with 0.02 units/ml GDE at 37 °C for 90 min and incubated with 40 ng/ml 125I-activin A and 200 ng/ml FS-288 (A) or FS-315 (B) in the presence of GDE for 2 h at 37 °C. After washing the cells, the amount of cell-bound FS was determined as described under "Experimental Procedures." The relative FS binding in the absence of GDE was designated as 100%. The results are expressed as means ± S.D. from triplicate experiments.
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Inhibitory Effect of Heparan Sulfate on the FSH-suppressing Activity of FS

FS was identified as an inhibitor of FSH secretion by cultured pituitary cells, but its potency was shown to be only 10-30% that of inhibin (7, 18). The mechanism by which FS acts is unclear, but it has been suggested that it binds endogenous activin and neutralizes activin-stimulated FSH secretion. To understand the role of the interaction of FS and proteoglycans in the FSH-suppressing effect of FS, we examined the effect of heparan sulfate on this inhibitory action of FS in rat pituitary cells. FS-288 and FS-315 dose-response curves for the inhibition of basal FSH secretion into the culture medium were prepared in the presence or absence of heparan sulfate (10 µg/ml) (Fig. 4). As reported previously, FS-288 was about 6-7 times more potent than FS-315. Heparan sulfate reduced the inhibitory activity of FS-288 by about 50%, whereas the potency of FS-315 remained unchanged regardless of the presence of heparan sulfate. This suggests that cell-associated FS-288 is more positively involved in controlling activin activity on cell surfaces than FS-315.


Fig. 4. Effect of heparan sulfate on suppression of pituitary FSH release by FS. Rat pituitary cells cultured in 96-well plates (8 × 104 cells/well) were incubated with various concentrations of FS-288 (A) or FS-315 (B) in the presence (bullet ) or absence (open circle ) of heparan sulfate (10 µg/ml) for 60 h at 37 °C. The amount of FSH released was determined by a radioimmunoassay kit. The results are expressed as means ± S.D. from triplicate experiments.
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Binding of Activin A to Cell-Associated FS

We then examined the binding of activin to FS associated with pituitary cell surfaces. In the presence of various concentrations of FS-288 or FS-315, rat pituitary cells were incubated with increasing amounts of 125I-activin A (0-100 ng/ml), and the cell-bound radioactivities (activin·FS complex) were determined (Fig. 5). The binding activity of activin A alone was difficult to detect, probably due to the very small number of activin receptors on pituitary cells. However, FS-288 markedly increased the affinity of activin A for cell surfaces in a concentration-dependent manner, whereas FS-315 did not enhance activin A binding to cell surfaces. These results suggest that activin A can adhere strongly to cells by forming a complex with FS-288 on the cell surface.


Fig. 5. Effect of FS on binding of 125I-activin A to pituitary cells. Rat pituitary cells cultured in 96-well plates (8 × 104 cells/well) were incubated with increasing concentrations of 125I-activin A (0-100 ng/ml) in the presence of various concentrations of FS-288 (A) or FS-315 (B) at 37 °C. After a 2-h incubation, specific binding was determined as described under "Experimental Procedures." Concentrations of FS added were 0 (bullet ), 25 (black-diamond ), 100 (black-square), and 400 (black-triangle) ng/ml. The results are expressed as means ± S.D. from triplicate experiments.
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Endocytotic Degradation of the Activin A·FS Complex

We followed the behavior of the cell-associated activin·FS complex and found that it was degraded endocytotically. Rat pituitary cells were incubated with radioiodinated activin A (40 ng/ml) in the presence of increasing concentrations of FS-288 or FS-315 for various incubation periods, after which the radioactivities recovered from the trichloroacetic acid-soluble fractions (degraded activin) of the incubation media were determined using a gamma -spectrometer. As shown in Fig. 6, FS-288 stimulated activin A degradation significantly in a time- and concentration-dependent manner and to a greater extent than FS-315. This stimulatory effect of FS-288 was abolished by adding heparin or heparan sulfate to the culture medium (data not shown). SDS-PAGE of the trichloroacetic acid-soluble fractions showed that activin A was degraded into small peptides and/or amino acids (data not shown). This reflects the cell-surface adhesiveness of the complex; the more strongly the activin A·FS-288 complex binds to cell-surface heparan sulfate, the more easily it is degraded. This was also supported by our finding that FS degradation was markedly reduced in heparitinase-treated cells (data not shown). Moreover, degradation was dependent on the number of pituitary cells as shown in Fig. 7; increasing their number stimulated degradation of activin A bound to the cell surfaces via FS. These degradation data were obtained by monitoring the degradation of 125I-activin A. SDS-PAGE and gel filtration of samples of the complex demonstrated that the FS component of the activin·FS complex was also degraded (data not shown).


Fig. 6. Effect of FS on endocytotic degradation of 125I-activin A by pituitary cells. Rat pituitary cells cultured in 96-well plates (8 × 104 cells/well) were incubated with 40 ng/ml 125I-activin A in the presence of various concentrations of FS-288 (A) or FS-315 (B) at 37 °C. Medium was collected at the indicated time points and subjected to trichloroacetic acid treatment. The radioactivity of the trichloroacetic acid-soluble fraction (degraded 125I-activin A) was determined in a gamma -counter. Concentrations of FS added were 0 (bullet ), 40 (black-diamond ), 120 (black-square), and 400 (black-triangle) ng/ml. The results are expressed as means ± S.D. from triplicate experiments.
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Fig. 7. Effect of cell density on endocytotic degradation of 125I-activin A by pituitary cells. Various numbers of rat pituitary cells cultured in 96-well plates were incubated with 125I-activin A (40 ng/ml) in the absence or presence of FS-288 or FS-315 (200 ng/ml) for 24 h at 37 °C. Degradation of 125I-activin A was determined as indicated under "Experimental Procedures." Cell densities used were 2.4 × 104 (square ), 8 × 104 () and 2.4 × 105 (black-square). The results are expressed as means ± S.D. from triplicate experiments.
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Endocytotic Internalization of Activin A

The degradation of cell-bound activin and/or FS by pituitary cells led us to hypothesize that endocytotic internalization occurs in the cells and that the resulting endocytotic vesicles ultimately fuse with lysosomes, after which most of the vesicle contents are rapidly broken down. To explore this idea, autoradiographic experiments using radioiodinated activin A were performed. Pituitary cells were incubated with 125I-activin A at 37 °C in the presence or absence of FS, heparin, and chloroquine for 12 h, and the cells were washed with acid/salt buffer to strip 125I-activin A from their surfaces and then autoradiographed. As shown in Fig. 8, it is obvious that FS-288 markedly accelerated the uptake of activin A by pituitary cells and had a greater effect than FS-315. Heparan sulfate significantly suppressed uptake, which agreed well with the degradation data described above. Co-incubation with chloroquine, which increases the pH inside lysosomes, inhibited the degradation of activin A taken up by the cells, probably in the lysosomes, resulting in activin A accumulation within the cells. Microscopic observations supported our hypothesis that activin A bound to pituitary cell surfaces via FS-288 is taken up and packaged into endocytic vesicles, which fuse with lysosomes. This is followed by proteolytic degradation of their contents.


Fig. 8. Uptake of 125I-activin A by rat pituitary cells. Rat pituitary cells cultured in chamber slides were incubated with 125I-activin A (40 ng/ml) in the absence (A) or presence (B) of FS-288 (200 ng/ml), FS-315 (200 ng/ml) (C), FS-288 (200 ng/ml) and heparan sulfate (10 µg/ml) (D) or FS-288 (200 ng/ml) and chloroquine (0.1 mM) (E) for 12 h at 37 °C. After extensive washing, the cells were fixed and subjected to autoradiography as described under "Experimental Procedures." Scale = 50 µm.
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Inhibition of Endocytotic Degradation of Cell-associated Activin A by Lysosomal Enzyme Inhibitors

To demonstrate the participation of lysosomes in the degradation of activin A after endocytosis, we examined the effects of various types of lysosomal enzyme inhibitor on activin A degradation in rat pituitary cells. The results are summarized in Table I. Lysosomal enzyme inhibitors reduced degradation significantly, but the serine protease inhibitor aprotinin had no effect. As expected from the results shown in Fig. 8, chloroquine markedly inhibited activin A breakdown. Both heparin and heparan sulfate suppressed activin degradation significantly, strongly suggesting that degradation does not occur until FS binds to the pituitary cells. These results clearly indicate that after endocytosis, activin A is hydrolyzed, probably together with FS-288, in the lysosome.

Table I. Effects of various lysosomal enzyme inhibitors on FS-288-induced endocytotic degradation of activin A by rat pituitary cells

Rat pituitary cells (8 × 104 cells/well) cultured in 96-well plates were incubated with 125I-activin A (40 ng/ml) and FS (200 ng/ml) in the presence of various inhibitors for 24 h at 37 °C, after which the media were collected and 125I-activin A degradation was measured as described under "Experimental Procedures." Activin degradation in the absence of inhibitors was designated 100%. The results are expressed as means ± S.D. from triplicate experiments. Rat pituitary cells (8 × 104 cells/well) cultured in 96-well plates were incubated with 125I-activin A (40 ng/ml) and FS (200 ng/ml) in the presence of various inhibitors for 24 h at 37 °C, after which the media were collected and 125I-activin A degradation was measured as described under "Experimental Procedures." Activin degradation in the absence of inhibitors was designated 100%. The results are expressed as means ± S.D. from triplicate experiments.

Inhibitors (concentration) Degradation

%
None 100  ± 7.5
Lysosomal protease inhibitors
  E-64 (1 mM) 64  ± 6.8
  Leupeptin (1 mM) 58  ± 4.7
  1,10-Phenanthroline (0.05 mM) 68  ± 5.1
  Pepstatin A (1 mM) 89  ± 5.8
Serine protease inhibitor
  Aprotinin (200 µg/ml) 103  ± 7.2
Lysosome function inhibitor
  Chloroquine (0.1 mM) 14  ± 4.8
FS binding inhibitor
  Heparin (10 µg/ml) 26  ± 3.8
  Heparan sulfate (10 µg/ml) 40  ± 4.3


DISCUSSION

FS binds stoichiometrically to activin to form an inactive complex, which results in blockade of various activin bioactivities. However, the physiological significance of this complex formation is not fully understood. Recently, de Winter et al. (33) demonstrated that the preincubation of radioiodinated activin A with FS completely abolished binding to type II activin receptors and consequently binding to type I receptors and proposed that FS can neutralize activin bioactivity by interfering with activin binding to type II receptors. Our affinity cross-linking experiments also showed this inhibition of activin binding to its receptors on rat pituitary cells (Fig. 1). This may be explained by assuming that FS masks the as yet unidentified receptor binding domain of the activin molecule, thus preventing activin from transducing its signal in responsive cells. Furthermore, in the present study, we investigated the importance of FS adhesiveness to the cell surface in its role in controlling activin bioavailability. The two FS isoforms studied showed different degrees of adhesion to rat pituitary cell surfaces in the cross-linking experiment; the intensity of the FS-288 band (the COOH-terminal truncated form) was much stronger than that of FS-315 (full-length form) (Fig. 1). More direct evidence was obtained as shown in Fig. 2; FS-288 showed a higher affinity for the rat pituitary cell surface than FS-315. As previously observed in rat granulosa cells (19), heparitinase treatment of pituitary cells resulted in significant suppression of FS-288 binding to the cell, whereas treatment with chondroitinase ABC had no effect. Furthermore, the binding of FS-288 to pituitary cells was significantly reduced after cells had been cultured in the presence of sodium chlorate, which is a potent inhibitor of protein and carbohydrate sulfation (34).2 These findings clearly support our idea that FS-288 binds mainly to the heparan sulfate side chains of proteoglycans on the cell surface.

Significant binding of radioiodinated activin A to pituitary cell surfaces was observed only in the presence of FS (Fig. 5). As expected, FS-288 markedly promoted this binding and had a greater effect than FS-315. Recently, Sugahara3 found that the smallest heparin oligosaccharides that could be recognized by FS-288 was a dodecasaccharide, suggesting that FS-288 distinguishes certain glycosaminoglycan configurations.

When incubated with pituitary cells in the presence of FS-288, activin A in the medium appeared to be trapped by cell-associated FS-288. Binding to heparan sulfate side chains via FS-288 on the cell surfaces thus appears necessary for the first step of activin A degradation. This idea was further supported by our finding that activin A is not degraded in heparitinase- and sodium chlorate-treated pituitary cells, to which it cannot bind.2 After being captured on the cell surface, activin A may, together with FS-288 and proteoglycans, be ingested by endocytotic vesicles that fuse with primary lysosomes. Most of the vesicle contents were found to be hydrolyzed into small breakdown products and secreted to the exterior. There is little doubt that activin A is broken down by such an endocytotic degradation process, because various inhibitors of each stage of this process blocked activin A degradation; the inhibitors tested included chloroquine and several lysosomal protease inhibitors. Monensin, an endosome lysosome fusion inhibitor, also almost completely inhibited degradation (data not shown). As this process was visualized (Fig. 8), activin A was collected within vesicles, which were probably lysosomes because chloroquine prevented the latter half of the degradation process. As is the case with radioiodinated activin A, the proteolytic degradation of 125I-labeled FS-288 was also observed in pituitary cells (data not shown). On the other hand, the complex between FS-315 and activin A showed low affinity for cell surfaces, resulting in the avoidance of endocytotic degradation by these complexes. Indeed, these complexes were found to be relatively stable under our incubation conditions for at least 48 h (data not shown). Activin might select FS, which adheres with difficulty to cell surfaces, as its binding partner and could therefore defend itself against endocytotic internalization followed by proteolytic attack. The anterior pituitary gland consists of many different cell types, classified on the basis of size, shape, and the hormone secreted. Therefore, it is also important to identify the type of pituitary cells that undertake the degradation process.

As shown in Figs. 5 and 6, FS-288 was approximately twice as effective as FS-315 in assisting degradation of 125I-activin A, and binding of FS-288 to the cells was more than 10 times higher than that of FS-315 (Fig. 2). There is no direct evidence to explain these phenomena clearly, but there are several possible explanations. As described in our previous paper (20), the majority of FS isolated from porcine ovaries is FS-303, which is thought to be derived from FS-315 by proteolytic cleavage of the 12 COOH-terminal amino acids and shows moderate affinity for cell surfaces. During incubation of FS-315 with pituitary cells, proteolytic degradation of the COOH-terminal portion of FS-315 may occur, and the resulting COOH-terminal truncated FS becomes attached to cells so that it can be degraded by endocytosis. Another possibility is that 125I-activin A may be more effectively degraded when it binds to FS-315.

The number of growth factors and cytokines discovered to bind to heparin and heparan sulfate is increasing steadily, and the list includes fibroblast growth factors (FGFs), granulocyte-macrophage colony stimulating factor, interleukin-3, pleiotrophin, hepatocyte growth factor, vascular endothelial growth factor, and midkine, among others. On the basis of our present results, we speculate that, like the activin A·FS-288 complex, these growth factors bind to cell-surface heparan sulfate proteoglycans, become internalized, and are eventually degraded in lysosomes. In fact, we found that 125I-basic FGF bound to cultured rat pituitary cells in a concentration-dependent manner and that its intracellular degradation was time-dependent.4 Therefore, it is conceivable that endocytotic degradation is a common mechanism for eliminating signaling molecules from cell surfaces.

It is well documented that the interaction between FGF and heparin-like molecules in the extracellular matrix is important for various biological functions, such as protection of this factor against proteolytic degradation and regulating its concentration on cell surfaces. The role of heparin-like molecules in the signal transduction of FGF is noteworthy; binding of basic FGF to its receptor requires prior binding either to the heparan sulfate side chains of cell-surface proteoglycans or to free heparin to present the ligand to the receptor (35). De Winter et al. (33) attempted to determine whether cell surface-bound FS-288 presents activin A to the activin receptors on human erythroleukemic K562 cells and found that FS-288 and the activin A·type IIA receptor complex were not co-precipitated by an anti-type IIA activin receptor antibody, suggesting that, unlike basic FGF, cell surface-associated FS cannot present ligands to signaling receptors. Judging from these results, FS appears to be nothing more than a negative regulator for activin, its function being to form an inactive complex with activin and thereby neutralize its activity. There are some situations in which FS traps activin on the cell-surface heparan sulfate and leads to endocytotic degradation. However, taking these findings together, we hypothesize that endocytotic degradation of growth factors via cell-surface heparan sulfate is necessary to erase their signals from cell surfaces when they become excessive and thus useless. It has been established that the binding of a signaling ligand to its receptor stimulates a biological response and triggers a sequence of events leading to cellular desensitization to the ligand to regulate the responsiveness of the target cell to the ligand. We propose that, in addition to such receptor-mediated endocytosis, there must be a scavenger mechanism for clearing signaling molecules away from their target cell surfaces.


FOOTNOTES

*   This work was supported in part by grants for scientific research from the Ministry of Education, Science, and Culture, Japan (to H. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed.
1   The abbreviations used are: FS, follistatin; FSH, follicle-stimulating hormone; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis; DSS, disuccinimidyl suberate; PBS, phosphate-buffered saline; GDE, glycosaminoglycan-degrading enzyme; FGFs, fibroblast growth factors.
2   O. Hashimoto, unpublished data.
3   K. Sugahara, unpublished data.
4   T. Nakamura, unpublished data.

ACKNOWLEDGEMENTS

We thank Dr. K. Sugahara of Kobe Pharmaceutical University for permission to use his unpublished observation in this paper and the National Hormone and Pituitary Program, National Institute of Diabetes and Digestion and Kidney Diseases for supplying the rat FSH assay kit. We are also grateful to Dr. K. Sugino for useful discussions and reading of the manuscript.


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