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Endocrinology Vol. 144, No. 5 1671-1674
Copyright © 2003 by The Endocrine Society

Differential Binding and Neutralization of Activins A and B by Follistatin and Follistatin Like-3 (FSTL-3/FSRP/FLRG)

Alan Schneyer, Amy Schoen, Alicia Quigg and Yisrael Sidis

Reproductive Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Alan Schneyer, Reproductive Endocrine Unit, BHX-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: schneyer.alan{at}mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Modulation of activin and other TGFß superfamily signaling is the primary mechanism of action for both follistatin (FS) and FS-like 3 (FSTL-3). However, most studies of these ligands use activin A due to its wide availability. We have now tested the ability of FS288 and FSTL-3 to bind and neutralize activin B relative to activin A. Activin B bound to both FS and FSTL-3 at a potency approximately 10-fold lower than that of activin A. Moreover, whereas both activins had similar biological activity in 293 cell reporter assays, FS and FSTL-3 were approximately 3-fold more effective in neutralizing activin A relative to activin B. These results suggest that neutralization of activins A and B by FS and FSTL-3 are not identical, so that the relative activity of each activin in tissues where both are produced, such as in the ovary, could be quite different. In addition, biological systems that use primarily activin B, but which have been examined in vitro using activin A, may need to be reevaluated to determine the actual physiologic roles of FS or FSTL-3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
ACTIVIN IS A HOMODIMERIC member of the TGFß superfamily that has putative biological actions in a wide variety of adult and embryonic tissues including pituitary, bone, gonad, hematopoeitic cells, liver, and kidney (see Ref. 1). Like other members of this family, activin signals through a complex of two types of transmembrane serine/threonine kinase receptors that then activate Smad second messengers that transmit the signal to the nucleus (2). At present, five different genes can give rise to activins, labeled A–E, and the proteins are named for the gene products contained in the dimer. Thus, activin A is composed of two activin ßA-subunits, whereas activin B is a dimer of two ßB-subunits (3). Activins C and E have been identified in liver, but no physiological role for these activin isoforms has yet been discovered. Activin D has only been identified in Xenopus (reviewed in Ref. 4).

The wide distribution of both activin and its receptors (5) suggests that activin signaling is regulated by a number of extracellular and intracellular mechanisms (4). Among these regulators are members of the follistatin (FS)-related gene family, including FS itself and FS-like 3 [FSTL-3; formerly known as FS-related protein (FSRP) and FS-related gene (FLRG)], both of which bind activin largely irreversibly and inhibit its binding to activin receptors (reviewed in Ref. 1). Thus, both FS and FSTL-3 are likely to be of major physiological importance in tissues where activin is active.

Genetic studies in mice indicate that activin A and B knockout phenotypes do not entirely overlap (6) and further, that activin B cannot completely rescue the defects seen in activin A-deficient mice (7). Together, these studies suggest that the biological actions and relative potencies of these two activins, or their sensitivity to modulators such as FS or FSTL-3, may not be identical in vivo. Because many tissues use primarily activin B in vivo, including the pituitary (8), it is critical to determine whether these two activins have identical biological actions and are modulated by regulators such as FS and FSTL-3 in an identical fashion. Yet the majority of in vitro activin experiments used activin A as the ligand. With the recent availability of pure, recombinant activin B, we sought to determine the relative ability of FS and FSTL-3 to bind and neutralize activin A vs. B. Our results indicate that FS and FSTL-3 differentially regulate the activins, raising the possibility that they contribute to nonoverlapping activities of activins A or B in tissues where FS or FSTL-3 are present. These findings could also have implications for interpretation of primary culture experiments that used activin A in vitro for tissues that normally rely on activin B in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Materials
Activins A and B were purchased from R&D Systems (Minneapolis, MN). Purified recombinant human follistatin 288 was obtained from Dr. Parlow and the National Hormone and Peptide Program (National Institutes of Health, Torrance, CA). Purified recombinant FSTL-3-Fc was kindly provided by Millennium Pharmaceuticals (Cambridge, MA) and prepared as previously described (9).

Activin iodination
Activins were iodinated using lactoperoxidase and purified by PAGE as previously described (10). Specific activity was approximately 30–35 µCi/µg.

Solid-phase ligand binding assay
Binding of activin to FS and FSTL-3 was assessed as previously described (10) except that FS288 was plated at 10 ng/well, and FSTL-3-Fc was plated at 25 ng/well.

Activin bioassay
Human embryonic kidney 293 cells were maintained in RPMI 1640 medium containing 10% FBS (Life Technologies, Inc., Rockville, MD). Transient transfections were performed in 24-well trays using Effectene (QIAGEN, Valencia, CA) and a total of 200 ng DNA [80 ng of the Smad-responsive reporter CAGA-Luc (a gift from Drs. S Dennler; Ref. 11)], 20 ng pRL-TK (Promega Corp., Madison, WI), and 100 ng of nonspecific plasmid DNA. For activin dose-response assays, media were replaced 16 h post transfection with RPMI + 0.1% BSA and increasing doses of activin A or B (0.001–1 nM). For FS and FSTL-3 bioactivity assays, 0.11 nM activin A or B were preincubated with increasing amounts of FS288 or FSTL-3-Fc for 1 h at 25 C. After treating for 24 h, the cells were lysed and assayed for luciferase activity using the dual luciferase reporter assay kit (Promega Corp.), with results normalized to Renilla luciferase activity.

Data analysis
Ligand binding assay results were analyzed using the NIHRIA program for slope (±SE) and ED50 (±SE). Curves were considered parallel if their slopes differed by less than two SE. Table 1Go lists results from five to eight separate experiments, whereas Fig. 1Go shows one representative assay. Bioassay results in Fig. 2Go are representative of three experiments.


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  		TABLE 1. Mean (±SE) of five to eight separate experiments comparing activin A or B binding to plates coated with FS288 or FSTL-3-Fc

 


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  		FIG. 1. Binding of activins A and B to FSTL-3 and FS. Representative binding experiment testing unlabeled activin A or B against either labeled activin A or B when FSTL-3 (left panel) or FS (right panel) is the solid phase binding protein.

 


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  		FIG. 2. Inhibition of activins A or B by FSTL-3 or FS. Both FS and FSTL-3 inhibit activin A and B biological activity in a 293 cell reporter assay, but inhibition of activin A is more potent than that for activin B (representative of three experiments). Inset, Dose-response assay for activin A and B showing similar biological activity in this reporter assay.

 

   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The ability of FSTL-3 and FS to bind the two forms of activin was examined using a solid phase competition assay. When activin A was the radioligand, unlabeled activin A was nearly 10-fold more potent then activin B (Fig. 1Go, closed vs. open circles) for both FSTL-3 and FS plates. To assess whether this difference was related to the iodination process itself, activin B was also radiolabeled. Again, unlabeled activin A was more potent than activin B for binding to FSTL-3, suggesting that this potency difference reflects actual structural or biochemical differences between the activins (Fig. 1Go, closed vs. open triangles). In contrast, when FS was used as binding agent, activin A was less potent than activin B, and the curve was often not parallel to the other curves (see Table 1Go), indicating that, in this case, the iodination process itself alters binding sites on activin B that have more effect on recognition by FS than by FSTL-3. This finding suggests that one of the three tyrosines found on activin B that are not on activin A could be involved in the FS-activin B contact site, and that the contact sites used by FS and FSTL-3 do not entirely overlap. Further mutational analysis will be required to substantiate this potential difference in activin B contact sites between FS and FSTL-3. Nevertheless, results from five to eight separate experiments, summarized in Table 1Go, indicate that activin B is about 10-fold less potent that activin A for binding to either FSTL-3 or FS.

To determine whether the differences in relative potency between activin A and B seen in the solid phase binding assays were biologically relevant, we next examined the ability of FS and FSTL-3 to neutralize the biological actions of activin A or B in vitro. Activins A and B exhibited similar biological activity over a two-log order dose range (Fig. 2Go, inset). Whereas both FS and FSTL-3 inhibited activin A and B activities, inhibition of activin A was more than 3-fold greater than activin B (Fig 2Go). Because these experiments did not involve iodinated compounds, this potency difference is likely manifested in the structural differences between activin A and B.

Our results indicate that activins A and B have nearly identical biological activity in a 293 cell-based transcriptional reporter biological assay, an observation with commercially available activins that agrees with earlier studies (12). On the other hand, mouse genetic studies suggest that activins A and B do not have identical biological actions in vivo (7). Several potential explanations emerge for this paradox. First, it is possible that activins A and B differentially use activin signaling pathways that are not homogeneously distributed within the body, thereby effecting differential actions in vivo. Whereas components of the activin signaling system are widely distributed (5), there is some evidence for nonoverlapping distribution of receptors during development (13). Our results suggest an alternative (and not mutually exclusive) explanation. Specifically, binding of activin B to both FS and FSTL-3 was approximately 10-fold lower than activin A, and further, it took at least 3-fold more FS or FSTL-3 to neutralize activin B compared with activin A. Thus, differential sensitivity to activin modulators such as FS or FSTL-3, which are not homogeneously distributed (9), could favor actions of activin B over activin A if both are present at similar concentrations in the same tissue. Our results, therefore, indicate that differential recognition by FS and FSTL-3 is at least one mechanism to explain the nonoverlapping actions of activins A and B in vivo.

Activins A and B can be synthesized in different tissues, or in some cases in the same tissues. For example, the pituitary has been reported to use primarily activin B to regulate FSH biosynthesis (14). Although FS has been shown to regulate this action of activin B, the doses of FS required both in vivo and in vitro have been substantial (15, 16). Despite FS production in both folliculostellate cells and gonadotropes (17, 18), our finding that more FS or FSTL-3 is required to neutralize activin B than for activin A suggests that endogenous FS may play less of a role in regulating activin B action at the pituitary than previously appreciated. Consequently, inhibin could actually be more important because its ability to neutralize activin B could be more potent than that of FS. Finally, it has recently been shown that bone morphogenetic proteins (BMPs), including BMP 6 and 7 (19) and BMP 15 (20) can also regulate FSH biosynthesis in cultured pituitary cells. Moreover, FS and FSTL-3 also bind BMPs but at much lower potency relative to activin A (9, 10). It was previously thought that this large difference in affinity would prevent FS action on BMPs in vivo. Our findings that FS binds less activin B than activin A suggests that the real difference between activin B and BMPs for binding to FS is smaller than currently believed, so that FS could act on both members of the TGFß superfamily in vivo. Alternatively, neutralization of activin B by FS in the pituitary could make the actions of the BMPs more important in regulation of FSH biosynthesis in vivo.

In tissues such as the ovary and testis, both activins are probably synthesized because mRNAs for ßA and ßB-subunits have been identified (reviewed in Ref. 1). However, because no assay for activin B exists, the relative concentration of activins A and B in these organs is not presently known. Furthermore, genetic replacement of activin A with activin B did not completely restore gonadal function (7), suggesting that activins A and B may have differential actions in the gonad. Because both FS and FSTL-3 are synthesized in the gonads (9), our results suggest that these molecules, through their differential binding of activins A and B, could alter the dominant activity of activin in favor of activin B when concentrations are similar. Thus, FS and FSTL-3 could be one mechanism for manifestation of these different biological actions in normal animals. Moreover, whereas the relevance of these in vitro findings remains to be established in vivo, it might be prudent to take these considerations into account in considering activin’s roles in various physiological systems.


   Acknowledgments
 
We great appreciate the efforts of Drs. Jeff Weiss, William Crowley, Herb Lin, Henry Keutmann, and Abir Mukherjee in reviewing the manuscript.


   Footnotes
 
This research was supported by NIH Grants R01-DK-55838 and R01-HD-39777.

Abbreviations: BMP, Bone morphogenetic protein; FLRG, FS-related gene; FS, follistatin; FSRP, FS-related protein; FSTL-3, FS-like 3.

Received December 30, 2002.

Accepted for publication February 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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  2. Heldin CH, Miyazono K, Ten Dijke P 1997 TGF-ß signaling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471[CrossRef][Medline]
  3. Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsak T, Vaughan J, Corrigan A, Bardin W, Sawchenko P, Spiess J, Rivier J 1988 Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res 44:1–34
  4. Phillips DJ 2000 Regulation of activin’s access to the cell: why is mother nature such a control freak? Bioessays 22:689–696[CrossRef][Medline]
  5. DePaolo LV 1997 Inhibins, activins, and follistatins: the saga continues. Proc Soc Exp Biol Med 214:328–339[Medline]
  6. Chang H, Brown CW, Matzuk MM 2002 Genetic analysis of the mammalian transforming growth factor-ß superfamily. Endocr Rev 23:787–823[Abstract/Free Full Text]
  7. Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM 2000 Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions. Nat Genet 25:453–457[CrossRef][Medline]
  8. Bilezikjian LM, Corrigan AZ, Blount AL, Vale WW 1996 Pituitary follistatin and inhibin subunit messenger ribonucleic acid levels are differentially regulated by local and hormonal factors. Endocrinology 137:4277–4284[Abstract]
  9. Tortoriello DV, Sidis Y, Holtzman DA, Holmes WE, Schneyer AL 2001 Human follistatin-related protein: a structural homologue of follistatin with nuclear localization. Endocrinology 142:3426–3434[Abstract/Free Full Text]
  10. Sidis Y, Tortoriello DV, Holmes WE, Pan Y, Keutmann HT, Schneyer AL 2002 Follistatin-related protein and follistatin differentially neutralize endogenous vs. exogenous activin. Endocrinology 143:1613–1624[Abstract/Free Full Text]
  11. Dennler S, Itoh S, Vivien D, Ten Dijke P, Huet S, Gauthier JM 1998 Direct binding of Smad3 and Smad4 to critical TGF ß-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 17:3091–3100[CrossRef][Medline]
  12. Mason AJ, Berkemeier LM, Schmelzer CH, Schwall RH 1989 Activin B: precursor sequences, genomic structure, and in vitro activities. Mol Endocrinol 3:1352–1358[CrossRef][Medline]
  13. Feijen A, Goumans MJ, van den Eijnden-van Raaij AJ 1994 Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. Development 120:3621–3637[Abstract]
  14. Corrigan AZ, Bilezikjian LM, Carroll RS, Bald LN, Schmelzer CH, Fendly BM, Mason AJ, Chin WW, Schwall RH, Vale W 1991 Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 128:1682–1684[Abstract]
  15. Besecke LM, Guendner MJ, Sluss PA, Polak AG, Woodruff TK, Jameson JL, Bauer-Dantoin AC, Weiss J 1997 Pituitary follistatin regulates activin-mediated production of follicle-stimulating hormone during the rat estrous cycle. Endocrinology 138:2841–2848[Abstract/Free Full Text]
  16. Leal AM, Takabe K, Wang L, Donaldson CJ, MacConell LA, Bilezikjian LM, Verma IM, Vale W 2002 Effect of adenovirus-mediated overexpression of follistatin and extracellular domain of activin receptor type II on gonadotropin secretion in vitro and in vivo. Endocrinology 143:964–969[Abstract/Free Full Text]
  17. Kawakami S, Fujii Y, Okada Y, Winters SJ 2002 Paracrine regulation of FSH by follistatin in folliculostellate cell-enriched primate pituitary cell cultures. Endocrinology 143:2250–2258[Abstract/Free Full Text]
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