Endocrinology Vol. 146, No. 12 5048-5051
Copyright © 2005 by The Endocrine Society
Too Many Follistatins: Racing Inside and Getting Out of the Cell
T. Rajendra Kumar
Departments of Molecular & Integrative Physiology, and Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: T. Rajendra Kumar, Ph.D., Department of Molecular & Integrative Physiology, 3901 Rainbow Boulevard, 3011 WHE, University of Kansas Medical Center, Kansas City, Kansas 66160. E-mail: tkumar{at}kumc.edu.
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Introduction
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During the late 1980s, reproductive endocrinologists witnessed the emergence of a new era with the discovery of potent stimulators and inhibitors of pituitary FSH (1, 2, 3, 4, 5). These novel peptides belong to the transforming growth factor-ß superfamily, the largest family of secreted growth factors. Most important among these are activins, inhibins, and follistatin (FST). Although activins, inhibins, and FST were originally identified in gonadal extracts and the follicular fluid, respectively, subsequent studies have identified widespread expression of these peptides throughout the body (6, 7, 8). Two distinct genes encode activin subunits ßA and ßB that give rise to biologically active and physiologically well-characterized homodimers activin A and activin B, although the physiological actions of the heterodimeric peptide activin AB are less understood (6, 9).
FST is a monomeric glycoprotein coexpressed with activins in many tissues and irreversibly binds activins to block their binding to the cognate receptors. Thus, FST neutralizes the actions of activins via its auto/paracrine effects (6, 7, 10). Genetic approaches using gain- and loss-of-function, and more recently cell-specific gene inactivation mouse models have revealed important physiological and developmental roles of FST in various tissues (9, 11, 12, 13, 14). However, the fundamental aspects of FST cell biology including its biosynthesis, intracellular trafficking, and secretion have never been explored in great detail. In this issue of Endocrinology, Saito et al. (15) describe a very interesting and comprehensive study of biosynthesis and secretion of various forms of FST and the related FST like-3 (FSTL-3) peptide using a variety of biochemical and cell biologic methods.
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FSTs: Structural and Functional Diversity
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The Fst gene consists of six exons and encodes mainly two protein forms (Fig. 1A
); FST315, a full-length 315 amino acid-containing peptide, and another peptide, FST288, produced as a result of the mRNA generated by alternative splicing of intron 5. In addition, FST303, a third peptide produced by proteolytic cleavage of FST315, was originally identified in porcine follicular fluid (1, 2, 4, 5, 16, 17, 18). The characteristic feature of FST is its ability to bind activin with varying degrees through its three 10-cysteine FST domains. Of these, the first FST domain also contains a heparin binding sequence that facilitates the binding to cell surface heparin-sulfated proteoglycans (19, 20, 21). The full-length peptide FST315 contains a C-terminal acidic tail that inhibits FST binding to cell surface proteoglycans via the heparin binding sequence in the first FST domain (22). In contrast, the spliced variant FST288 does not contain this C-terminal tail region and FST303, the truncated form generated from FST315, has intermediate binding properties to cell surface proteoglycans (19, 20, 21). Thus, these biochemical studies suggest differences in cell surface binding and presumably other biological activities among the three FST forms.
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FIG. 1. Summary of the work by Saito et al. A, The Fst gene encodes two forms FST315 and an alternately spliced FST288. FST315 is proteolytically cleaved to generate FST303. In cell transfection assays, FST315 is secreted the fastest, followed by FST303, and FST288 is the slowest form secreted into medium. Although the majority of the FST is secreted, a portion of it is also retained within the cytoplasm. B, The Fstl3 gene produces FSTL3, which is glycosylated and secreted less rapidly than the FST forms. Some of FSTL3 is also observed in the nucleus, and this nuclear form is less glycosylated than the secreted form (sugar residues are denoted by hexagons). Whether the nuclear form of FSTL3 acts via protein-protein interactions or directly at the DNA level is unknown. C, Site-directed mutagenesis of the first methionine residue in Fst and Fstl3 reveal their importance in initiation of translation. The first methionine mutant form of FST288 is unstable; it does not localize to cytoplasm nor gets secreted out of the cell. In contrast, FSTL3 with first methionine mutated is presumably transported from the ER and localized only to the nucleus. Interestingly, this mutant form does not get glycosylated unlike the naturally occurring form observed in HeLa cells.
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Subsequent studies have identified FSTL3, another FST member encoded by a distinct Fstl3 gene that has one less FST domain, but at the protein level shares 45% identity with FST (10, 23, 24, 25, 26, 27, 28). FSTL3 irreversibly binds activin with almost the same affinity as does FST. Thus, there could be some overlap in biological actions of FST and FSTL3. However, one important distinction between the two is the lack of the heparin binding sequence in FSTL3 that prevents its binding to cell surface proteoglycans.
Although several in vitro and classical physiological studies indicated the functional diversity of FST, more convincing in vivo evidence came from Fst gene knockout (ubiquitous and gonad specific) and FST overexpressing mice. These mouse genetic manipulation studies identified the in vivo roles of FST in skin (11), hair follicle (29), tooth (30), testis (12), and early ovarian development and later in folliculogenesis (12, 31). More recently, the Schneyer group (14) also achieved FSTL3 overexpression in the gonads of transgenic mice. These mice have varying degrees of gonad pathology including many degenerating tubules devoid of germ cells, Leydig cell hyperplasia in the testis, and ovarian follicular atresia. Whereas FST is normally expressed in high levels in the ovary and kidney and FSTL3 in the placenta and testis, the mouse models do suggest that both the peptides mostly regulate activin functions in vivo, at least in the tissues examined. Whether this is achieved by the same or different mechanisms is unclear. One clue emerged that the differences in biological functions of FST and FSTL3 could be due to differences in their intracellular behavior based on the earlier studies by Schneyer and colleagues.
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Intracellular and Secreted Forms of FST and FSTL3
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Two key observations were recently reported that suggest differential regulation of the biosynthesis and intracellular trafficking of FSTL3 and FST. First, although FSTL3 is mostly secreted from several cells or when overexpressed in transfected cell lines, it has also been localized within the nucleus of cell lines, tissue sections, and primary granulosa cells in the ovary (27, 28). Second, whereas FST is also mostly secreted, in contrast to FSTL3, it is localized within the cytoplasm in some cells but not in the nucleus (27, 28).
How are secretory proteins with signal sequences [that normally direct them to the endoplasmic reticulum (ER) pathway] destined to be "cargoed" to nucleus or other intracellular compartments within the cell? At least two mechanisms have been described: 1) alternative splicing can modify signal peptide or a nuclear localization signals in some mRNAs, thus rerouting the proteins to different cellular compartments (32, 33); and 2) translation initiation can occur at different methionines or at non-AUG codons resulting in proteins with variable length or no signal peptides (34, 35, 36). This can lead to truncated versions of some proteins that can, to some extent, remain intracellular. Testing these possibilities experimentally has been feasible for Saito et al., having made an interesting observation that both FST and FSTL3 have a second methionine in their signal sequences. The hypothesis they sought out to test is whether the use of alternate methionines to initiate FST or FSTL3 translation could allow a fraction of translated proteins to be retained inside the cell.
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Highlights of the Work by Saito and Colleagues: Molecular Analyses of the Secretory Fates of FST and FSTL3
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Using metabolic 35S-labeling, pulse-chase experiments, subcellular fractionation, Saito et al. first identified that FST315 is the fastest secreted form followed by FST303, whereas FST288 is secreted more slowly (Fig. 1A
) with some remaining intracellular. They further analyzed the secretion kinetics of endogenous FSTL3 from HeLa cells or in transfected Chinese hamster ovary cells and found that it is secreted the slowest; the newly secreted FSTL3 protein is either secreted or ended up in the nucleus. Glycanase treatment confirmed that both the secreted as well as nuclear forms are glycosylated, although of the two, the nuclear form is less glycosylated (Fig. 1B) and migrated faster when separated electrophoretically. To test the hypothesis that differential usage of the methionines may result in production of different forms of FST288 and FSTL3, the authors have mutated either of the two methionines into alanine residues by site-directed mutagenesis (Fig. 1C). Both FST288 and FSTL3 tolerated the mutation at the second methionine, and shorter forms are observed in cell extracts and in the medium when transfected into cell lines. Whereas mutation of the first methionine resulted in FSTL3 that was present in only cell extracts and no secreted form was observed, FST288 was completely absent both in extracts and the medium. These data suggest that FSTL3 is normally translated from the first or second methionine.
Subsequently, Saito et al. analyzed the bioactivity and immunocytochemical and glycosylation status of the methionine-mutated forms. These led to three important observations. First, in contrast to FST, biologically active (inhibition of activin-induced reporter activity in human embryonic kidney 293 cells) FSTL3 can be translated from the first methionine, and in both cases the second methionine mutants retained the bioactivity. Second, the majority of the immunostaining was localized to the cytoplasm when the second methionine mutants of both FST288 and FSTL3 were separately transfected into Chinese hamster ovary cells. Whereas no immunostaining was detected in case of the first Met mutant of FST288, the similar FSTL3 mutant showed intense staining only in the nucleus. Finally, the nuclear transported first Met mutant form of FSTL3 did not undergo glycosylation, although the second Met mutants of FST288 and FSTL3 are both glycosylated. It is intriguing to note that the endogenous FSTL3 normally detected in the nucleus of HeLa cells is glycosylated, however.
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Existence of Multiple FST and FSTL3 Forms: Final Remarks and Future Directions
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The studies by Saito et al. elegantly demonstrated the diversity of various FST and FSTL3 forms with regard to their secretion kinetics, subcellular localization, the glycosylation status, and the use of alternate methionines to initiate translation to produce some of these forms. These exciting studies opened new avenues of research into exploring the cell biology of FST and FSTL3 secretion and their physiological significance. Several questions must now be addressed: What are the carbohydrate structures on nuclear, cytoplasmic, and secreted forms? Is the nuclear FSTL3 glycosylated on Ser/Thr residues (O-glycosylated)? How does FSTL3 get targeted from the ER to the nucleus? What is the biological role of this nuclear FSTL3? What proteins does it interact with in the nucleus or does it bind DNA directly? Are these different bioactive FST and FSTL3 forms developmentally and physiologically regulated in vivo? Because FST and FSTL3 have a wide tissue distribution of expression, are these various forms tissue/cell specific?
That a secreted glycoprotein enters the nucleus via a short loop from the ER is a thought-provoking and major advancement in cell biology. Unraveling the mechanism by which this phenomenon occurs particularly in case of nuclear FSTL3 will require more sophisticated cell biological and biophysical tools including live cell imaging and fluorescence energy transfer techniques. Finally, understanding the cell biology of FST and FSTL3 secretion should also facilitate delineation of the complex regulation of activin secretion and its action during various developmental, physiological, and pathological contexts (8, 10, 37).
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Footnotes
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Abbreviations: ER, Endoplasmic reticulum; FST, follistatin; FSTL-3, FST like-3.
Received September 20, 2005.
Accepted for publication September 22, 2005.
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Introduction
FSTs: Structural and Functional...