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.

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.

Three gonadal peptide factors, inhibin (1)(2)(3)(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 ␤ superfamily. Both are disulfide-linked dimers; inhibin is composed of ␣and ␤-subunits, whereas activin is a dimer of the inhibin ␤-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 ␤-subunits, called ␤A and ␤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 ϳ53 kDa) and II (molecular mass ϳ70 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 activinbinding 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 ␤-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 CO 2 (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 g/ml gentamicin sulfate, 1 g/ml fungizone, 0.5 mg/ml glutamine, and 0.1 mg/ml NaHCO 3 . For affinity cross-linking experiments, the cells were seeded into 24-well tissue plates at a density of 5 ϫ 10 5 cells/0.5 ml/well. For endocytotic degradation experiments, the cells were seeded into 96-well plates at a density of 8 ϫ 10 4 cells/0.1 ml/well. Pituitary cells were cultured in a humidified CO 2 (5%) air incubator at 37°C.
Radiolabeling of Ligands-Activin A, FS-288, and FS-315 were iodinated with Na 125 I 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 g of protein was dissolved in 25 l of 0.3 M phosphate buffer (pH 7.2). Na 125 I (400 Ci) in 8 l of distilled water and 8 l of chloramine-T solution (250 g/ml) were then added and vortexed for 1 min. To dilute and terminate the reaction, 150 l of phosphatebuffered 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 ϫ 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 pcDLSR␣ expression vector (30) and used for transient transfection. The plasmid DNA (2 g) was transfected into COS-7 cells plated into 6-well plates at a density of 2 ϫ 10 5 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 125 I-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 ϫ 10 4 cells/well) were washed once with culture medium and incubated with various concentrations of 125 I-FS-288 or 125 I-FS-315 in 100 l of culture medium for 2 h at 37°C. After washing three times with fresh medium, 100 l of 10% SDS was added to each well to solubilize the cells, and the radioactivity (cell-bound FS) was quantified by ␥-spectrometry. To examine the association of the activin•FS complex with the cell surface, 125 I-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 l) was added to the pituitary cell culture (8 ϫ 10 4 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 g/ml heparan sulfate.

Treatment of Rat Pituitary Cells with Glycosaminoglycan-degrading Enzymes (GDE)-Rat pituitary cells (8 ϫ 10 4 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 125 I-activin A.
Assay of Endocytotic Degradation of Activin and FS-Rat pituitary cells grown in 96-well plates (8 ϫ 10 4 cells/well) were incubated in 100 l of culture medium with 40 ng/ml 125 I-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 ␥-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 ϫ 10 4 cells/well) were incubated with 125 I-activin A (40 ng/ml), FS-288 or FS-315 (200 g/ml), and various concentrations of inhibitors for 24 h at 37°C in 100 l 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 ϫ 10 4 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 ϫ 10 4 cells/0.3 ml/chamber and incubated with 40 ng/ml 125 I-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 g/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, ϭ 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.

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 125 I-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 g/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 125 I-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.
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 g/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 cellassociated FS-288 is more positively involved in controlling activin activity on cell surfaces than FS-315.
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 125 I-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.
Endocytotic Degradation of the Activin A⅐FS 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 ␥-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 A⅐FS-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 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 125 I-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 125 Iactivin 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.
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 endo- cytosis, activin A is hydrolyzed, probably together with FS-288, in the lysosome. 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 crosslinking experiments also showed this inhibition of activin bind-ing 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, Sugahara 3 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 cellassociated 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 heparitinaseand 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 break-2 O. Hashimoto, unpublished data. 3 K. Sugahara, unpublished data.  down 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 125 I-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 125 I-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 125 I-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), granulocytemacrophage 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 A⅐FS-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 125 I-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 receptormediated endocytosis, there must be a scavenger mechanism for clearing signaling molecules away from their target cell surfaces.