| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Reproductive Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Alan Schneyer, Ph.D., Reproductive Endocrine Unit BHX-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail Schneyer.alan{at}mgh.harvard.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
FST has a number of putative physiological roles, including regulating pituitary FSH production (4), ovarian follicle maturation (5), spermatogenesis (6), liver homeostasis (7), wound repair (8), and response to inflammatory stimuli (9). Moreover, disruption of the mouse Fst gene resulted in developmental abnormalities including loss of hair, weakened musculature, XX sex reversal, and early neonatal death, demonstrating that FST is required for normal mammalian development (10, 11). However, the identification of precise activities and mechanisms of action for FST in both embryos and adults has been hampered by the fact that multiple isoforms of FST are produced from the FST gene, each with potentially distinct activities. The primary FST transcript undergoes alternative splicing to produce mRNAs that code for two FST proteins, termed FST288 and FST315 (12). The FST315 isoform contains all six exons, whereas the FST288 splice variant is missing exon 6, which codes for the acidic C-terminal tail. A third isoform, FST303, appears to arise from proteolytic cleavage of the FST315 C-terminal tail between residues 300 and 303 (13). All three isoforms contain a region of basic residues known as the heparin-binding sequence (HBS), which is essential for binding to cell-surface heparin-sulfated proteoglycans (14, 15, 16, 17). However, it has been proposed that the acidic tail in FST315 interacts with the basic residues within the HBS, thereby suppressing the cell-surface binding activity of FST315. FST303, with its shortened tail, has cell-surface binding activity intermediate between FST315 and FST288 (13). These biochemical distinctions suggest that each isoform may be responsible for different subsets of biological activities depending on their degree of cell-surface localization and subsequent compartmentalization within the body, a concept supported by the finding that FST315 is the predominant circulating FST isoform in human serum (18), whereas ovarian follicular fluid contains primarily FST303 (14). Nevertheless, differential biological activity as well as the underlying mechanisms among the FST isoforms remain to be fully elucidated.
FST-like-3 (FSTL3), also known as FST-related gene (FLRG) (19) and FST-related protein (FSRP) (20), shares substantial structural and functional homology with FST (21), including inhibition of activin bioactivity in vivo (22). Importantly, FSTL3 does not have an HBS, cannot bind to cell-surface proteoglycans (23), and is a weak antagonist of endogenous (autocrine) activin despite being only slightly less potent in neutralizing exogenous (endocrine/paracrine) activin (23). These distinctions between FSTL3 and FST support the concept that the presence of a functional HBS and resultant cell-surface binding is a critical biochemical determinant for endogenous activin inhibition.
In addition to activin, FST and FSTL3 bind other members of the TGFß superfamily, including myostatin (24, 25) and some bone morphogenetic proteins (BMPs) (26, 27). FSTL3 was recently identified as the circulating binding protein for myostatin in mice and humans (24), suggesting that FSTL3 may have an important role in regulating muscle development and/or adult muscle mass. These studies indicate that both FST and FSTL3 likely play important roles in regulating the physiological and homeostatic activities of activin and related TGFß superfamily ligands and that the physiological significance of these regulatory interactions, as well as quantitative differences in specificity among the binding proteins, remains to be determined systematically.
Given the different biochemical properties and compartmentalization of the FST isoforms and FSTL3, we hypothesized that these proteins will also have distinct mechanisms of action and ligand selectivity that will ultimately determine their range of physiological actions in vivo. To explore molecular mechanisms responsible for this differential bioactivity, we produced recombinant FST isoforms and investigated their differential biochemical and biological activities compared with FSTL3. Our results indicate that the biological activities of the FST isoforms and FSTL3 are determined primarily by their differential cell-surface binding and ligand specificity and further support the concept that FST isoforms may have distinct physiological roles in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparation of recombinant tagged FST and FSTL3 proteins
Human FST288, FST315, and FSTL3 (the latter a gift from Millennium Pharmaceuticals, Cambridge, MA) coding sequences were subcloned into pCDNA3.1Myc-His (Invitrogen, Carlsbad CA). The FST303 expression construct was prepared from the FST315 cDNA by deleting all sequence 3' to codon 303 up to the start of the Myc-His tag using standard PCR methods. The resulting C-terminal Myc-His-tagged cDNA constructs were transfected into HEK-293-F cell suspension cultures in Freestyle serum-free medium (Invitrogen) as described (28). Recombinant proteins were purified by nickel-Sepharose affinity chromatography (QIAGEN, Valencia, CA) and concentrated by centrifugal dialysis into Dulbecco’s PBS.
Quantitation of secreted proteins
FST concentrations in media and concentrated affinity-purified eluates were established by two independent immunological assays: 1) a two-site solid-phase immunochemiluminescent assay (29) and 2) a solution-phase assay directed toward the C-terminal Myc tag (28). The concentrations obtained by the two methods were usually in good agreement for all FST preparations. FSTL3 preparations were quantified by the Myc RIA. Concentration and purity of affinity-purified proteins were verified by SDS-PAGE and silver staining (Bio-Rad Laboratories, Hercules, CA) or Western blot as described below.
Western blot analysis
Proteins were separated on 12% Tris-HCl Ready-Gel system (Bio-Rad), transferred to polyvinylidene difluoride membrane (Bio-Rad), blocked in 10% nonfat dry milk, and probed with anti-Myc (1 µg/ml, clone 4A6; Upstate Biotechnology, Lake Placid, NY) and goat antimouse horseradish peroxidase-conjugated (1:7500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Immunoreactivity was visualized using Western Lightning chemiluminescence reagent (PerkinElmer, Boston, MA).
Iodination
Human activin A was iodinated by the lactoperoxidase method and purified by electrophoresis as described previously (30).
Binding to cell-surface heparan-sulfate proteoglycans
COS cells were cultured in DMEM supplemented with 10% FBS (Invitrogen) and plated into 24-well plates and cultured to confluence. FST isoforms or FSTL3 was added at 50 ng/ml for 1 h at 25 C in fresh medium containing 0.1% BSA, washed, and replaced with medium containing 75,000 cpm [125I]activin A alone or together with 10 µg/ml heparan sulfate (Sigma Chemical Co., St. Louis, MO), which displaces FST-activin complexes. After 1 h incubation at 25 C, cells were rinsed, harvested, and counted to detect FST-bound activin.
Activin-binding kinetics
Binding kinetics of FST isoforms to radiolabeled activin was determined by solid binding assay as described (31) using 25 ng/well of each isoform passively adsorbed to microtiter plates. After blocking the wells with 3% BSA/0.01% Tween in 10 mM PBS, 75,000 cpm radiolabeled activin was added. To determine the association rate, the binding reactions were terminated at the indicated times, the wells were washed, and bound radioligand was counted in a 
-counter. For dissociation measurements, the binding reactions were allowed to continue to steady state (3 h), after which unbound radioligand was removed and 100-fold excess of unlabeled ligand was added. At the indicated times, the wells were washed and counted as before. Maximum binding in a typical experiment reached 10–15% of total counts. Observed association rate constants (Kon) and dissociation rate constants (Koff) were calculated by fitting an exponential association equation and exponential decay model, respectively, using Prism4 (GraphPad Software Inc., San Diego, CA). Binding kinetics was assayed in at least three independent experiments for each isoform.
FST and FSTL3 specificity assays
Nonequilibrium competitive binding assays were used to determine isoform specificity as described (31). Microtiter plates were prepared with FST isoforms or FSTL3 as described above. Unlabeled activin A or heterologous competitors at doses indicated in the figures were individually mixed with 75,000 cpm [125I]activin A in 150 µl assay buffer for 1 h. This nonequilibrium assay format was used to compensate for the heavily favored and nearly irreversible activin-binding kinetics (31) that would have displaced competitors if the assay had been allowed to reach steady state (>2 h). At the end of the 1-h incubation, the wells were washed and counted. In a typical experiment, about 10–15% of the added total counts were bound in the absence of unlabeled competitor. Resulting inhibition curves were analyzed using the four-parameter logistic model. Each ligand was assayed in at least three independent experiments.
Cell culture and reporter assays
The capacity of FST isoforms and FSTL3 to inhibit bioactivity of activin and related TGFß family ligands was determined by reporter assays in human HepG2 cells (for activin A and BMPs), and human embryonic kidney (HEK) 293 cells (for activin A and myostatin), because myostatin responses were relatively weak in HepG2 cells. HepG2 cells were maintained in MEM supplemented with Earle’s salts, nonessential amino acids, sodium pyruvate, and 10% fetal bovine serum (FBS) (Life Technologies, Inc., Rockville, MD). Cells were cultured in 24-well trays and transiently transfected with the smad1/5-responsive reporter BRE-Luc (32) for BMP activity or the smad2/3-responsive reporter CAGA-luc (33) for activin A activity, along with pRL-TK (Promega Corp., Madison, WI) and the indicated doses of FST isoform cDNAs (for experiments examining bioactivity of endogenous FST or FSTL3) or empty vector, using Lipofectamine 2000 (Invitrogen). To examine the effect of isoforms on endogenous activin, 1 ng pINHßA cDNA construct (kindly provided by Genentech Inc., San Francisco, CA) was included in the transfection mix. After 20 h, 0.2 nM activin A (for experiments on exogenous activin) was added, or was premixed (60 min at 25 C) with increasing amounts of purified FST isoforms or FSTL3 (for exogenous FST/FSTL3 experiments). After 16 h of treatment, the cells were washed, lysed with Passive lysis buffer (Promega) and assayed for luciferase activity using the dual luciferase reporter assay kit (Promega). Interwell variations in transfection efficiency were corrected by normalizing to Renilla luciferase activity. For each ligand, experiments were repeated at least three times, and the mean and SE of representative experiments are reported.
HEK 293 cells were maintained in RPMI 1640 medium containing 10% FBS (Life Technologies). Transient transfections and reporter assays were performed as described above except for using Effectene as the transfection reagent (QIAGEN) and included the CAGA-luc reporter and pRL-TK control cDNA, as well as FST or FSTL3 expression constructs and pINHßA cDNA for endogenous FST/FSTL3 or activin experiments, respectively.
To examine the effect of cell-surface association on FSTL3 ability to block endogenous activin, FSTL3 coding sequence without the signal peptide was cloned in frame with the transmembrane domain of pDisplay (Promega) and tested in CAGA-luc reporter assays in HEK 293 as described above.
To verify that transfected FST isoforms and FSTL3 were expressed at similar levels, 10 µl cell extract and 20 µl conditioned medium from the highest-dose wells were subjected to reduced SDS-PAGE and Western analysis as described above.
TT cells transfection and proliferation assays
TT cells (provided by Dr. Aaron Hsueh, Stanford University, Stanford, CA) are a clonal, testicular tumor cell line derived from a p53/
-inhibin subunit-deficient mouse line (34). Cells were cultured in DMEM-Ham’s F-12 (1:1) supplemented with 10% heat-inactivated FBS (Invitrogen), 2 mM L-glutamine, and antibiotics.
TT cells were transfected with expression constructs containing full-length FST288 or FS315 cDNAs (kindly provided by Dr. S. Shimasaki, University of California, San Diego, La Jolla, CA) in pCDNA3 vector (Invitrogen). Stable colonies were isolated and screened for FST secretion using our two-site solid-phase immunochemiluminescent assay for free (unbound to activin) FST as previously described (29). Cellular proliferation was assessed using the CellTiter 96 AQueous One Solution Reagent (Promega) colorimetric assay according to the manufacturer’s recommendations.
Generation of recombinant adenovirus and proliferation of infected TT cells
Adenoviruses encoding FST288 and FST315 were constructed using a previously described two-cosmid adenoviral system (35). Briefly, full-length FST288 and FST315 cDNAs were cloned into the pLEP plasmid and then ligated to a cosmid containing the adenoviral genome (pREP7). The ligation product was packaged in phage packaging extracts (MaxPlax; Epicenter Technologies) infected into host bacteria, and hybrid cosmids were selected, amplified, and transfected into HEK 293 cells. High-titer stock solutions of adenovirus were obtained by repeated amplifications in HEK 293 cells.
For cell proliferation studies with FST viruses, early-passage stocks of TT cells were plated into 60-mm culture dishes. After 24 h incubation, 500 µl of HEK 293-conditioned medium containing a high titer of FST288-AdV, FST315-AdV, or empty virus, which contained no cDNA, was added. The next day, the cells were washed and seeded into 96-well plates. Cell proliferation assays were then preformed as described above for transfected cells.
Immunocytochemistry
HepG2 cells were grown on coverslips, fixed in 4% paraformaldehyde in PBS for 20 min, and either left nonpermeabilized or permeabilized with 0.1% Triton X-100. Myc-tagged FST isoforms or FSTL3 (1 µg/ml) was then incubated with cells for 1 h at room temperature in the absence or presence of heparin sulfate (100 µg/ml). After washing, cells were treated with anti-Myc monoclonal 4A6 (Upstate, Charlottesville, VA) and antimouse-tetraethylrhodamine isothiocyanate second antibody (Jackson ImmunoResearch, West Grove, PA).
Statistics
Activin-binding kinetic data were analyzed by one-way ANOVA for differences between isoforms followed by Tukey test. Differences of P < 0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Under reducing conditions, FST288, FST303, and FST315 migrated at apparent molecular weights (Mr) ranging from 38,000–50,000 and appeared as doublets or triplets, whereas purified FSTL3 migrated as a single molecular species at 36,000 Mr. These apparent molecular weights are consistent with previously reported estimates for these proteins, taking into account the size of the Myc-His tag and variable degrees of n-glycosylation. Moreover, relative concentrations of the purified proteins were verified by Western blot with anti-Myc antibody because all four proteins could be simultaneously analyzed with a single antibody. These Myc-tagged proteins had identical activin binding, cell-surface binding, and biological activity to untagged proteins (data not shown).
|
) with nearly undetectable dissociation rates (Fig. 2B), similar to previous observations for untagged FST288 and FSTL3 (21). The different levels of total activin binding among the isoforms may represent differential isoform association with the solid-phase support. Nevertheless, dissociation constants determined as Koff/Kon demonstrated high-affinity activin binding that was not significantly different among the four proteins (Table 1). Thus, there does not appear to be a significant difference in direct binding to soluble activin between the FST isoforms.
|
|
). FSTL3 did not bind to cell-surface proteoglycans as previously reported (23). Only a small fraction of FST315, which contains a full-length C-terminal tail, bound to cell-surface proteoglycans (<50% above basal), whereas FST303, with about half the C-terminal tail removed, bound 5-fold more activin compared with basal but less than half of FST288, indicating an intermediate cell-surface binding ability.
|
, row A). All FST was displaced in the presence of heparin (Fig. 3B, row B), indicating that this cell-surface-associated FST was reversibly bound to proteoglycans. Interestingly, when cells were first permeabilized, substantially more FST288 was detectable bound to membranes. This staining appeared to be intracellular as well as on the plasma membrane (Fig. 3B, row C), and the FST288 was again displaceable by heparin (Fig. 3B, row D), suggesting that FST288 may bind to membrane proteoglycans both outside and inside cells. Permeabilized cell staining was much reduced for FST303 and absent for FST315, consistent with binding to nonpermeabilized cells. However, slight staining was observed for FSTL3 in permeabilized cells that was not displaced by heparin (Fig. 3B, rows C and D), suggesting that Triton permeabilization treatment may have exposed a binding moiety for FSTL3 that is not used by FST isoforms. Taken together, these results demonstrate that the FST isoforms and FSTL3 have distinct capacities to interact with cell-surface proteoglycans and accumulate at the cell surface in correspondence with the presence of an HBS and the length of the C-terminal tail.
Differential inhibition of exogenous vs. endogenous activin by FST isoforms
To determine whether differences in cell-surface binding translated into differential bioactivity, the FST isoforms and FSTL3, added exogenously or expressed endogenously, were examined for their ability to inhibit endogenous vs. exogenous activin in HepG2 hepatoma cells, which have a robust response to activin using the CAGA-luc reporter but do not express FST or detectable activin. When individual FST isoforms or FSTL3 was added with activin as exogenous treatments to the cells, the isoforms were equivalent in their ability to neutralize this activin (Fig. 4A), consistent with their comparable affinities for activin (see Table 1). In contrast, when the FST isoforms and FSTL3 were transfected, i.e. produced endogenously, but the activin treatment was exogenous, FST288 was clearly superior among the isoforms in inhibiting exogenous activin (Fig. 4B), and the order of their activity is consistent with the order of cell-surface association (see Fig. 3A). This pattern was also observed when FST isoforms and FSTL3 were added exogenously but the activin produced endogenously (Fig. 4C) as well as when both FST isoforms and FSTL3 along with activin were produced endogenously (Fig. 4D). To ensure that bioactivity differences were not a result of differential production of FST isoforms or FSTL3, we examined conditioned medium and extracts from transfected HepG2 cells (Fig. 5E). When analyzed by Western blot, all proteins were detected in both conditioned medium and cell extracts. Interestingly, FSTL3 was expressed to a greater degree than any of the other proteins, although it had the lowest bioactivity in Fig. 4, B–D. Conversely, FST288 was expressed at the lowest levels but was the most active. These results indicate that increased activity of FST288 was not because of superior protein biosynthesis. Thus, differential cell-surface binding among the FST isoforms and FSTL3 correlates with differential bioactivity when the FST/FSTL3, activin, or both are derived from endogenous sources, as would be expected when FST and/or activin are acting in an autocrine mode.
|
|
25%; P < 0.01), whereas proliferation in FST288-transfected lines was suppressed by more than 50% (P < 0.001) (Fig. 5A).
To verify this differential activity in unselected cells, we generated adenoviruses expressing FST288 or FST315 under the control of the cytomegalovirus promoter and used them to infect different pools of wild-type TT cells derived from an identical stock. Free FST could easily be detected in conditioned medium of FST288 and FST315 virus-infected cells 24 h after infection (54 and 76 ng/ml, respectively). As observed in the stable cell lines, expression of FST315 by viral infection significantly enhanced proliferation (25%; P < 0.05), whereas FST288 significantly suppressed proliferation (25–40%; P < 0.01) (Fig. 5B). These observations demonstrate that FST288 and FST315 differentially regulate TT cell proliferation, likely reflecting their differential ability to suppress endogenous activin activity as described in Fig. 4.
Cell-surface binding of FSTL3 increases biological activity
To further test the hypothesis that cell-surface binding is responsible for the superior inhibition of endogenous activin by FST288, we expressed FSTL3 as a fusion protein with the transmembrane domain of the pDisplay vector, which anchors FSTL3 to the exterior surface of the plasma membrane (anchored FSTL3). When tested with exogenous activin, 1–10 ng of both wild-type and anchored FSTL3 suppressed more than 75% of activin’s activity (Fig. 6A). However, 10 ng of wild-type FSTL3 suppressed less than 50% of endogenous activin activity, whereas the same amount of anchored FSTL3 suppressed 90%, reaching similar levels of activity as FST288. To verify that anchored FSTL3 was not secreted and thus must have been acting at the cell surface, we compared FSTL3 in cell extracts and conditioned medium by SDS-PAGE and Western blot. In cell extracts, both wild-type and anchored FSTL3 were detected whereas in conditioned medium, only wild-type FSTL3 was seen (Fig. 6B), and the protein concentration of wild-type FSTL3 in both compartments was similar to membrane-bound anchored FSTL3. Taken together, these results demonstrate that localization at the cell surface, as occurs for FST288 because of its HBS, and in anchored FSTL3, greatly enhances the ability of FST and FSTL3 to suppress endogenous activin activity.
|
, A–C, and Table 2). In contrast, whereas myostatin bound to FSTL3 at a relative potency comparable to the FST isoforms, BMP6 and BMP7 as well as BMP2 and BMP4 were inactive (Fig. 7D and Table 2). Thus, although there was little difference in TGFß-family ligand specificity among the FST isoforms, BMP binding to FSTL3 was substantially reduced compared with FST isoforms.
|
|
). In addition, neither the FST isoforms nor FSTL3 inhibited BMP2 or -4 activity (Fig. 8, B and C), and FSTL3 did not inhibit BMP6 and -7 bioactivity (Fig. 8, D and E). On the other hand, FST288 inhibited BMP6 and -7 bioactivity (Fig. 8, D and E) to a greater degree than the other FST isoforms despite little difference between the isoforms in the binding assay. Similarly, although the three FST isoforms were equally active in inhibiting myostatin bioactivity (Fig. 8F), FSTL3 had about 5-fold less inhibitory activity despite having similar binding activity. Taken together with the binding studies, these bioassay results demonstrate that differential binding specificity of the FST isoforms and FSTL3 for activin, BMPs, and myostatin cannot, by itself, account for differences in inhibition of TGFß ligand biological activity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Inhibition of activin and related TGFß-family ligands by cell-surface-bound FST is analogous to inhibition of autocrine/paracrine actions of these ligands in vivo. Such situations might be encountered in tissue differentiation during embryonic development or in the neonate and adult in regulation of these ligands within tissues. In fact, the observations in TT cells are a model for autocrine/paracrine regulation of activin action because TT cells produce substantial amounts of activin. Thus, we found that endogenous FST288 was effective in inhibiting activin-dependent proliferation in TT cells, whereas FST315 was not. Although we did not test FSTL3 in this context, based on our results in the in vitro bioassays as well as previous studies (27), FSTL3 would be expected to be even weaker than FST315 in inhibiting autocrine activin. Nevertheless, when FSTL3 was anchored to the cell surface by a heterologous transmembrane sequence, its ability to inhibit endogenous activin was greatly enhanced. Our results are therefore consistent with the HBS and cell-surface binding being critical for maximal inhibition of endogenous activin and related ligands so that in vivo, FST288, and to a lesser degree, FST303, would be expected to serve these roles preferentially to FST315 and FSTL3. In vivo examination of this concept is underway.
The seemingly anomalous increase in TT cell proliferation when transfected or infected with FST315 vectors is more challenging to explain. However, it has been shown that increasing doses of activin resulted in receptor down-regulation in TT cells, suggesting that because these cells constitutively produce copious amounts of activin, their signaling system is chronically down-regulated (37). Although our results demonstrate that FST315 is a less effective inhibitor of endogenous activin compared with FST288, transfection with FST315 may still have inhibited activin activity sufficiently to reduce the level of receptor down-regulation, thereby resulting in a more sensitive activin signaling system. These more sensitive TT cells might then respond to the remaining endogenous activin with enhanced proliferation.
This situation in TT cells is somewhat analogous to the pituitary where activin is produced endogenously and constitutively and is regulated by FST (4, 39, 40, 41). It is not presently known which FST isoform is produced within the pituitary, although there is some suggestion that it might be FST315 (42, 43). Thus, one possible mode of FST315 regulation of activin-stimulated FSH biosynthesis in the pituitary may be through reducing down-regulation of gonadotroph activin receptors, thereby producing greater activin sensitivity and FSH release.
In contrast to inhibition of endogenous activin, exogenous FST isoforms and FSTL3 were equally effective at inhibiting exogenous activin. This is equivalent to the in vivo situation of inhibiting activin and related ligands in the circulation. Moreover, to maintain effective serum concentrations of FST and FSTL3, isoforms that do not bind cell-surface proteoglycans would be preferable. Consistent with differential distribution of FST isoforms within the body, we recently identified FST315 as the circulating FST isoform in humans (18), whereas FSTL3, but not FST, was identified as a circulating binding protein for myostatin (24). Thus, our results indicate that the distribution of FST isoforms correlates with their heparin-binding activity so that FST315 and FSTL3 act primarily as regulators of endocrine activin, myostatin, and perhaps BMPs. Moreover, our results are consistent with a compartmentalization of FST isoforms based on cell-surface binding that results in their being responsible for different biological actions in vivo.
In addition to the classical signaling pathway of activin binding to cell-surface receptors and transducing an intracellular signaling cascade, recent evidence has been presented for another pathway where activin or TGFß, bound to type II receptor, is endocytosed to early endosomes. In this model, activin or TGFß signals are transduced via contact with Smad anchor for receptor activation (SARA)-bound Smad proteins that reside in endosomes (44, 45, 46). Within this system, FST could act at the cell surface to antagonize activin binding or enter the cell via endocytosis along with the activin-receptor complex where it could then reduce signaling by sequestering activin as it dissociates from receptors. FST288 has also been reported to bind radiolabeled activin at the cell surface, where it is then internalized and presumably degraded (47). Thus, FST288, by virtue of its greater ability to bind cell-surface proteoglycans, may have greater activin-inhibitory activity by antagonizing activin both at the cell surface and within endosomes, whereas FST315 and FSTL3 can act only on activin that is extracellular. Intracellular localization and possibly activity of FST288 is supported by the immunocytochemical results in this study showing FST288 binding to intracellular membranes that is reversible by heparin treatment, as well as by our previous results demonstrating that some newly synthesized FST288 remains in the cytoplasm for up to 4 h (48). Regardless of where FST288 actually inhibits activin binding to its receptor, it clearly has increased ability to inhibit endogenous activin activity relative to the other isoforms and FSTL3. These results collectively indicate that the different FST isoforms may have different in vivo biological roles and that FST315 will have activities more closely related to FSTL3 because both have low cell-surface binding ability.
Although high-affinity binding of FST to activin and myostatin has been previously reported (21, 25, 31, 36, 49), the relative activity of the three FST isoforms, as directly compared here, has not been described. In addition, there is some disagreement over relative binding affinities for the different ligands owing to the wide range of different techniques used (36, 49, 50). For example, different dissociation constants were reported for FST288 and FST315 binding to activin immobilized on a Biacore chip (36), which contrasts with our determination of similar affinities among the FST isoforms. However, the recently reported crystal structure for the FST288-activin complex (51), which found two FST molecules bound to one activin dimer, appeared to require flexibility of the activin dimer because its configuration in complex with FST was distinct from its conformation in complex with its receptor (52). Thus, immobilization of activin during surface plasmon resonance analysis with the Biacore system may inhibit this flexibility, leaving activin in an unnatural conformation that favored one FST isoform over another. This may also be applicable to previously reported affinities for FST binding to BMP4 determined using the same technology (50). In our study, activin was presented in solution and may thus be more physiological because in vivo, TGFß ligands are more likely to contact FST or FSTL3 in solution. Nevertheless, our results indicate that when compared directly in competition assays as in this study, activin appeared to be the preferred ligand for FST isoforms and FSTL3.
FSTL3 has been identified as a circulating binding protein for myostatin in humans and mice (24). However, these investigators were unable to identify any circulating myostatin bound to FST using SDS-PAGE followed by mass spectroscopy. This is unexpected because the FST isoforms appear to neutralize myostatin better than FSTL3. We have previously reported (21, 31), and confirmed here, that activin binding to FST is essentially irreversible, as appears to be the case for myostatin as well (49). Moreover, FST has been shown to inhibit myostatin activity when expressed transgenically in muscle (25). Taken together, these studies indicate that both FST and FSTL3 can bind and neutralize activin and myostatin. The preferred ligand may depend on which ligand gains access first to either FST or FSTL3, which is in turn a function of differential tissue or organ distribution of FST and FSTL3 (21).
In summary, our results clarify the activin-binding affinity among the FST isoforms and FSTL3 and demonstrate that their differential activin-regulating activity is dependent on their relative cell-surface binding activity rather than on differential activin-binding affinity. Our results also define the relative specificity of binding and inhibitory activity for a number of related TGFß-family ligands by the FST isoforms and FSTL3, with FSTL3 being almost completely inactive in regulating BMP ligands, thereby suggesting that in vivo, FSTL3 is unlikely to regulate BMP activity. Finally, our results suggest that the in vivo biological roles of the FST isoforms and FSTL3 are likely to be distinct, dependent on their relative cell-surface binding activity and consequent compartmentalization within the body as well as on colocalization of biosynthesis in different tissues.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Current address for A.D.: Fertility Clinic, Erasme Hospital, Laboratory of Research on Human Reproduction, Universite Libre de Bruxelles, Belgium.
Disclosure: All authors have nothing to declare.
First Published Online April 20, 2006
Abbreviations: BMP, Bone morphogenetic protein; FBS, fetal bovine serum; FST, follistatin; FSTL3, follistatin-like-3; HBS, heparin-binding sequence.
Received January 23, 2006.
Accepted for publication April 10, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and p53-deficient mice: the role of activin as an autocrine growth factor. Mol Endocrinol 8:983–995This article has been cited by other articles:
 |
|
 |
|
  Y. Xia and A. L Schneyer The biology of activin: recent advances in structure, regulation and function J. Endocrinol., July 1, 2009; 202(1): 1 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Gilson, O. Schakman, S. Kalista, P. Lause, K. Tsuchida, and J.-P. Thissen Follistatin induces muscle hypertrophy through satellite cell proliferation and inhibition of both myostatin and activin Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E157 - E164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Beites, P. L. W. Hollenbeck, J. Kim, R. Lovell-Badge, A. D. Lander, and A. L. Calof Follistatin modulates a BMP autoregulatory loop to control the size and patterning of sensory domains in the developing tongue Development, July 1, 2009; 136(13): 2187 - 2197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Abdipranoto-Cowley, J. S. Park, D. Croucher, J. Daniel, S. Henshall, S. Galbraith, K. Mervin, and B. Vissel Activin A Is Essential for Neurogenesis Following Neurodegeneration Stem Cells, June 1, 2009; 27(6): 1330 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Singh, S. Bhasin, M. Braga, J. N. Artaza, S. Pervin, W. E. Taylor, V. Krishnan, S. K. Sinha, T. B. Rajavashisth, and R. Jasuja Regulation of Myogenic Differentiation by Androgens: Cross Talk between Androgen Receptor/ {beta}-Catenin and Follistatin/Transforming Growth Factor-{beta} Signaling Pathways Endocrinology, March 1, 2009; 150(3): 1259 - 1268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Schneyer, Y. Sidis, A. Gulati, J. L. Sun, H. Keutmann, and P. A. Krasney Differential Antagonism of Activin, Myostatin and Growth and Differentiation Factor 11 by Wild-Type and Mutant Follistatin Endocrinology, September 1, 2008; 149(9): 4589 - 4595. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Butler, R. A. Dean, E. M. Tam, and C. M. Overall Pharmacoproteomics of a Metalloproteinase Hydroxamate Inhibitor in Breast Cancer Cells: Dynamics of Membrane Type 1 Matrix Metalloproteinase-Mediated Membrane Protein Shedding Mol. Cell. Biol., August 1, 2008; 28(15): 4896 - 4914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xia, J. L. Babitt, Y. Sidis, R. T. Chung, and H. Y. Lin Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin Blood, May 15, 2008; 111(10): 5195 - 5204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Blount, J. M. Vaughan, W. W. Vale, and L. M. Bilezikjian A Smad-binding Element in Intron 1 Participates in Activin-dependent Regulation of the Follistatin Gene J. Biol. Chem., March 14, 2008; 283(11): 7016 - 7026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Rae, K Hollebone, V Chetty, D Clausen, and J McFarlane Follistatin serum concentrations during full-term labour in women significant differences between spontaneous and induced labour Reproduction, November 1, 2007; 134(5): 705 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Antony, J. J. Bass, C. D. McMahon, and M. D. Mitchell Myostatin regulates glucose uptake in BeWo cells Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1296 - E1302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kashiwagi, C. M. DiGirolamo, Y. Kanda, Y. Niikura, C. T. Esmon, T. R. Hansen, T. Shioda, and J. K. Pru The Postimplantation Embryo Differentially Regulates Endometrial Gene Expression and Decidualization Endocrinology, September 1, 2007; 148(9): 4173 - 4184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. F. Lerch, S. Shimasaki, T. K. Woodruff, and T. S. Jardetzky Structural and Biophysical Coupling of Heparin and Activin Binding to Follistatin Isoform Functions J. Biol. Chem., May 25, 2007; 282(21): 15930 - 15939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. B Lee, V. Khivansara, M. M Santos, P. Lamba, T. Yuen, S. C Sealfon, and D. J Bernard Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone {beta} subunit transcription J. Mol. Endocrinol., February 1, 2007; 38(2): 315 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mukherjee, Y. Sidis, A. Mahan, M. J. Raher, Y. Xia, E. D. Rosen, K. D. Bloch, M. K. Thomas, and A. L. Schneyer FSTL3 deletion reveals roles for TGF-beta family ligands in glucose and fat homeostasis in adults PNAS, January 23, 2007; 104(4): 1348 - 1353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Goswami and N. C. Andrews Hereditary Hemochromatosis Protein, HFE, Interaction with Transferrin Receptor 2 Suggests a Molecular Mechanism for Mammalian Iron Sensing J. Biol. Chem., September 29, 2006; 281(39): 28494 - 28498. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
myostatin 