Activin

Activin Teresa K. Woodruff* Department of Neurobiology and Physiology, Northwestern University, 2153 N. Campus Drive, Evanston, IL 60208-2850, USA * corresponding author tel: 847-491-2666, fax: 847-491-2224, e-mail: [email protected] DOI: 10.1006/rwcy.2000.08008.

SUMMARY Activin has been identified as a cytokine, growth and differentiation factor, and endocrine hormone. It is produced by a wide variety of cells in the body including the pituitary, brain, gonads, and bone marrow. Activin belongs to the TGF superfamily of growth and differentiation factors. The TGF superfamily includes bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), mullerian inhibiting substance (MIS), the Drosophila decapentaplegic gene product (dpp), and the Xenopus Vg1 gene product (Vale et al., 1988; Massague et al., 1992; Hogan, 1996). A variety of factors regulate activin production both in vitro and in vivo. Follistatin, a bioneutralizing binding protein of activin, is produced coincidently with activin and control of this protein determines the local bioavailability of the ligand (DePaolo et al., 1991). In addition, the action of activin is opposed by the endocrine hormone inhibin. Inhibin and activin were first discovered on the basis of their ability to regulate pituitary FSH release from the anterior pituitary, but a number of additional functions have been assigned to activin, including regulation of cell differentiation. Male and female activin-deficient mice are not viable and die within the first 24 hours after birth, primarily due to craniofacial defects and the lack of a lower incisor (Matzuk et al., 1995a,b,c). Recombinant or native sources of activin have been used to expand the types of physiological functions directed by activin. Activin regulates developmental programs and proliferation state, directs cellular differentiation, and initiates time- or cycle-dependent secretory events. This chapter collates the major biological, molecular, and structure±function information available regarding activin. Because of the vast numbers of activities regulated by activin, this review

will focus on the more well-known activities of the ligand. Over time, additional biological activities and functions will be added to the online version of this chapter.

BACKGROUND

Discovery In the course of purifying proteins that contribute to a wide range of biological functions, the molecule known as activin was `discovered' at least five times: as a pituitary follicle-stimulating hormone (FSH) releasing hormone, as an erythroid differentiating factor, as a nerve survival factor, as a noncompetitive inhibitor of the IL-6 receptor, and as a mesoderm stimulating factor. Activin was first purified from ovarian follicular fluid based upon its ability to stimulate pituitary FSH release, the opposite activity of the endocrine hormone, inhibin (Vale et al., 1988). Inhibin is a heterodimer of a unique  subunit and the activin subunit. Inhibin inhibits pituitary FSH release from anterior pituitary cells. A gonadal-derived protein hormone that could specifically inhibit the pituitary hormone FSH without affecting LH had been predicted by a number of physiological studies dating back to the early 1930s. Following the isolation of inhibin from ovarian follicular fluid, activin was discovered as a side fraction that caused FSH release. (Note: While inhibin is not the focus of this chapter, there are numerous physiological circumstances that are more easily described by comparison of activin action with inhibin action. For these physiological circumstances, inhibin reference will be made, however, no attempt has been made to provide a complete picture of this ligand in this chapter.)

820 Teresa K. Woodruff Activin was also isolated from bone from a human monocytic leukemic cell line based on its ability to induce differentiation and hemoglobin accumulation in erythroleukemia cell lines in vitro. The activity was named `erythroid differentiating factor' or EDF. The accumulation of hemoglobin in human or mouse erythroleukemic cell lines is the basis of one activin bioassay. The search for nerve growth factors led to the rediscovery of activin and the description of the ligand as a nerve cell survival factor. Subsequent to this discovery, a whole class of TGF superfamily ligands have been described that act specifically in neuronal and glial cells, including glial-derived nerve growth factor (GDNF). Activin was also purified as a noncompetitive inhibitor of the IL-6 receptor. Last, activin participates in mesoderm development and the discovery of activin in early cell fate decisions has provided a great number of functional tools in the understanding of activin signal transduction events.

Alternative names As is clear from the discovery list, there are several names for the molecule that causes FSH secretion, erythroid differentiation, nerve cell survival, and IL-6 receptor antagonist activity. The accepted name for each of these activities is activin. Occasional reference to erythroid-differentiating factor (EDF) persists in the literature. However, a consensus document regarding the naming of activin was published and the majority of publications have adopted the nomenclature described in that document (Burger and Igarashi, 1988). Occasional confusion exists when database searches for `activin' pull out the term `T-activin'. T-activin refers to a thymic hormonal factor (MW 1±6 kDa), not the TGF superfamily member, activin (MW 28 kDa).

Structure The subunits of activin are called subunits. The subunits are similar in overall structure to the TGF superfamily of growth and differentiation factors. Activin is synthesized as a homo- or hetero-dimer of subunits ( A, B, etc.) resulting in a variety of possible molecular species. The term `beta' derives from the antecedent ligand purified from ovarian follicular fluid called inhibin. Inhibin is a dimer of two

dissimilar subunits; the  subunit and the subunit. Inhibin nomenclature reflects the type of subunits in the heterodimer: inhibin A (ÿ A) and inhibin B (ÿ B) are the two forms that have been isolated from native sources and produced by recombinant DNA technology. Five isotypes of the independent chains of activin have been cloned from various cellular sources. Association of the various subunits in homodimers or heterodimers is named as follows: activin A ( Aÿ B); activin B ( B± B); activin AB ( Aÿ B). Other homo- or hetero-dimers would follow suit.

Main activities and pathophysiological roles Activin has a variety of functions that depend on the cell system examined, the dose of the ligand, and the route of delivery. Activin stimulates FSH production by pituitary gonadotropes (in vitro and in vivo), stimulates erythrocyte cell differentiation (in vitro and in vivo), stimulates bone resorption (in vitro), regulates cell cycle genes, and stimulates liver apoptosis (in vitro and in vivo). The list of disease processes in which activin has been implicated as causal is lengthy and includes various types of cancers, preterm labor, neural tube defects, inflammatory disease, infertility, anemias, and cardiovascular disease. Additional functions for activin that have been patented are listed in Table 1.

GENE AND GENE REGULATION

Accession numbers Inhibin  subunit: Human: M13981 Rat: M36453 Activin A subunit: Human: M13436 Rat: M37482 Activin B subunit: Human: M31668 Activin C subunit (liver): Human: 669155 Mouse: 1150506; NCBI: CAA62347 Activin D subunit: African clawed frog: 1085213 Activin E subunit (liver): Mouse: 2072522

Activin

821

Table 1 Patented functions for activin Patent no.

Title

Assignee

05387576

Treatment of hyperproliferative epidermal conditions with activin A

Yissum Research Development Co.

05413989

Method and activin compositions for inducing bone growth

Celtrix Pharmaceuticals Inc.

05753612

Pharmaceutical composition and method for inhibiting hair growth by administration of activin or activin agonists

Yissum Research Development Co.

05563059

Use of human inhibin and human activin to increase the number of mature primate oocytes

Genentech Inc.

05658876

Activin antagonists as novel contraceptives

The General Hospital Corporation

05824637

Activin antagonists as novel contraceptives

The General Hospital Corporation

04997815

Method for augmenting fetal hemoglobin by treatment with activin and /or inhibin

Children's Hospital Medical Center

05208219

Method for inducing bone growth

Celtrix Pharmaceuticals Inc.

05102868

Method for inhibiting follicular maturation

Genentech Inc.

05654404

Protection against liver damage by HGF

Genentech Inc.

05166190

Method for increasing fertility in males

Genentech Inc.

05545616

Method for predicting and/or preventing preterm labor

Genentech Inc.

05693534

Enhancement of fertilization capability of oocytes

Genentech Inc.

05196192

Actions of hormones

Biotechnology Australia Pty Ltd

05216004

Method for preventing malaria

Children's Hospital Medical Center

05693481

Inhibitor of cytokine activity and applications thereof

Chiron Corporation

Regulatory sites and corresponding transcription factors The A subunit promoter is responsive to forskolin and 12-O-tetradecanoylphorbol-13-acetate (TPA) (Ardekani et al., 1998). A variant cAMP response element has been identified in the proximal A subunit promoter and mutation in the variant CRE reduces the ability of forskolin and TPA to induce promoter activity. Two members of the AP-1 family of transcription factors, jun-B and fos-B, associate with the variant CRE and transfection of either coactivator stimulates promoter activity. Two B subunit mRNAs are observed in ovarian granulosa cells and are differentially regulated by cAMP (Dykema and Mayo, 1994). Two adjacent transcriptional start sites are present in the gene accounting for the 4.3 and 3.3 kb mRNAs. The 4.3 kb mRNA is induced to a greater extent by FSH than is the 3.3 kb mRNA.

PROTEIN

Discussion of crystal structure The crystal structure of activin has not been solved, however, it is likely that it will have similar features to other TGF ligands. Overall homology between TGF family members is as low as 29%, however, analysis of the crystal structures of TGF 2 and OP-1 suggest a three-dimensional fold that may confer topological similarity between superfamily members. A central cysteine knot is generated by disulfide bridges between six of the seven conserved cysteine amino acids in the mature (C-terminal) portion of the protein (Daopin et al., 1992). Four antiparallel sheets emanate from the knot, suggesting finger-like projections. Facing perpendicular to and opposite the cysteine knot is an  helix which is the anthropomorphic `heel' of an overall structure that can be described as a left hand. The individual subunits of OP-1 or TGF 2

822 Teresa K. Woodruff Figure 1 Important homologies. The sequences compared were human except where noted: (c) C. elegans; (x) Xenopus; (m) mouse; (b) bovine; (z) zebrafish; (d) Drosophila; (s) sea urchin.

are joined by the seventh unpaired cysteine and fit together as two hands shaking. It is proposed that all members of the TGF superfamily will have a similarly ordered three-dimensional structure. Activin receptor extracellular domains have been crystallized.

Important homologies The activin subunits are homologous to the TGF superfamily based on a conserved cysteine residue structure. Similarities between the amino acids of the mature domains of TGF family members are shown in Figure 1. The activin subfamily is most closely related to TGF and BMP.

Posttranslational modifications Activin is synthesized as a glycosylated prepro-ligand and cleaved into the mature dimer, which is not glycosylated. Mature inhibin is glycosylated. The protease that cleaves activin or inhibin into mature, active protein has not been identified. The TGF protease is a furin-like protein and it is likely that activin is cleaved by a member of this protease family. Processing mutants have been generated and demonstrate that unprocessed inhibin A is biologically active Figure 2 Members of the inhibin/activin family.

Activin in rat pituitary cell bioassays while unprocessed activin A is not biologically active (Mason et al., 1996). See Figure 2.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Activin subunit mRNA has been detected in many cells. A survey of cells types that produce activin is listed below with primary references. Examples from humans or rodents are provided. Activin production has been measured in tissues from other species such as bovine, ovine, goldfish, stallion, and Xenopus. These species are not cataloged in the summary below. Also, the reference links are not exhaustive. Where several laboratories described similar findings, the most comprehensive paper was selected as a link. A comparison of , A, and B subunit mRNA between a variety of rat tissues has been done (Meunier et al., 1988) with expression of the subunits being very broadly distributed through almost all tissues tested. The next challenge is to determine whether these various sources of ligand contribute to cellular homeostasis or other physiological or developmental pathways. Activin is produced by the brain, including the nucleus of the solitary tract and the dorsal and ventral medullar reticular nuclei ( A); perifornical neurons in the hypothalamus ( B) (Roberts et al., 1996); the rat pituitary (Roberts et al., 1992; Wilson and Handa, 1998); normal human pituitary/pituitary adenomas (Demura et al., 1996): compare with Xenopus distribution (Uchiyama et al., 1996); bone (Hosoi et al., 1996; Nagamine et al., 1998): bone marrow monocytoid cells (human) (Wada et al., 1996), not in myoid cells (Sensebe et al., 1997); salivary gland ( B) (Tuuri et al., 1994); heart: cardiac cushion tissue (becomes the septa and valves) (Moore et al., 1998); pancreas: insulin-positive B cells (human) (Wada et al., 1996); spleen: the c isoform (Loveland et al., 1996); adrenal (human) (Spencer et al., 1992); developing kidney and lung (Ritvos et al., 1995); human ovary (Roberts et al., 1993), rat ovary (Meunier et al., 1988; Woodruff et al., 1988); uterus (Otani et al., 1998); placenta (Petraglia et al., 1987); developing embryo gonads, heart, skin, hair, whiskers, cartilage, bone, tendons, teeth, salivary gland, brain (Feijen et al., 1994); fetal gonads (Rabinovici et al., 1991); testis (Roberts et al., 1989; Vliegen et al., 1993; Anderson et al., 1998); prostate (Thomas et al., 1998); muscle ± developing mesoderm including muscle in Xenopus

823

(Thomsen et al., 1990); pituitary adenoma (Ying et al., 1996); gonadotroph adenoma (Haddad et al., 1994).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Follistatin is a bioneutralizing binding protein of activin and pituitary expression of follistatin is regulated by the hypothalamic releasing peptide, gonadotropin-releasing hormone (GnRH). GnRH is released from the hypothalamus in a pulsatile fashion and an increase in pulse frequency leads to LH secretion, whereas a slowing of the GnRH pulse favors FSH secretion. Pituitary follistatin is responsive to the changing pulse frequency of GnRH: a fast GnRH pulse frequency favors follistatin expression whereas a slow GnRH pulse frequency results in low follistatin expression (Besecke et al., 1996). The changing concentration of follistatin results in an increase in bioavailable activin at times when FSH is produced. Activin is also capable of downregulating its own receptor, thus establishing an ultra short loop feedback system within the pituitary (Trudeau et al., 1994).

RECEPTOR UTILIZATION See the chapter on Activin receptors 1 and R.

IN VITRO ACTIVITIES

Bioassays used There are two main bioassays for activin: FSH pituitary bioassay (activin stimulates FSH) and the K562 bioassay (activin stimulates hemoglobin accumulation in undifferentiated erythroleukemic cell lines) (Schwall and Lai, 1991; Balen et al., 1995).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Activin stimulates FSH secretion in vivo (rats, monkeys, sheep, cows). FSH is suppressed when the

824 Teresa K. Woodruff bioneutralizing binding protein follistatin is infused into rats, supporting the notion that endogenous activin regulates FSH. Activin also has important effects directly on the follicle, oocyte, and corpus luteum of rodents and primates. Activin inhibits and inhibin augments LHand insulin-like growth factor (IGF)-stimulated androgen production by human thecal cells via modulation of 17 -hydroxylase activity. Activin is an autocrine modulator of FSH-induced aromatase activity in nonhuman primate granulosa cells from immature follicles; however, in mature follicles that have acquired LH-responsiveness, activin inhibits steroidogenesis. Dimeric activin A also suppresses progesterone synthesis by luteinizing granulosa cells in vitro. Activin A can direct reorganization of cells from primary rodent follicles to develop into large antral, oocyte-containing follicles and the ability of activin to stimulate immature granulosa proliferation supports a role for activin in early follicular development in vitro. While in vivo evidence points to this ligand as a potent regulator of follicular atresia in rats and monkeys. In the primate, activin suppresses progesterone production by macaque luteal cells in vitro; however, intraluteal infusion of activin A at midluteal phase does not alter luteal function in vivo. Conversely, systemic infusion of rhesus monkeys with activin A during the midluteal phase caused premature luteolysis that was not observed with inhibin A. The ability of activin to act in a dualistic manner in vivo and in vitro likely reflects the differences in comodulating factors available in vivo that are not present in culture. Moreover, the marked differences in activin-regulated events when the route of administration is altered (e.g. intraluteal versus systemic) points out the difficulties in dissecting the physiological versus pharmacological effects of this ligand. Activin is also a potent regulator of erythropoiesis. Activin synergizes with erythropoietin in vitro and in vivo to stimulate the erythroid differentiation pathway of both erythroid and multipotential progenitor cells. In Xenopus, activin is a potent regulator of mesoderm development. Mice deficient in the activin subunits survive embryonic development, thus, the role of activin in mammalian mesoderm development is not essential or there are additional ligands that can fulfill this function.

Species differences The activin subunits are highly conserved between species with no differences at the amino acid level

between rat, cow, sheep, and human A subunit or B subunit.

Knockout mouse phenotypes Activin A-deficient and activin AB-deficient mice develop normally through fetal life, then die at birth with defects in craniofacial development (cleft palate and lack of whiskers). Activin B-deficient mice grow to adulthood and have no eyelids and reduced reproductive function. Curiously, activin RII subunitdeficient mice develop normally through adulthood with reduced fertility due to lower FSH production. These results suggest several possibilities. First, activin may not be essential for prenatal development of all organ systems in which RII/RI is expressed. Second, RII/RI may utilize a ligand other than activin. A candidate ligand that binds the activin RII subunit is OP-1. OP-1 regulates bone formation, is expressed in the embryo, and may account for the normal development of the null mice in the absence of an activin signal. Further adding to the complexity of this system is the phenotype of animals deficient in the activin-binding protein follistatin. Loss of follistatin function results in widespread defects in muscle, skin, bone, and tooth development. Modulation of previously uncharacterized ligands may produce this phenotype. The best example of overlap between receptor, ligand, and predicted physiology is the MIS gene family. MIS RII knockout animals and MIS knockout animals develop into pseudohermaphrodites.

Interactions with cytokine network Activin has been called a cytokine as well as a growth factor, endocrine hormone, and apoptosis-inducing factor. All of which leads to the description of this factor as a `multicrine'. Activin A is stimulated by traditional cytokines (such as GM-CSF and IL-1, TNF, LPS, TPA) and inhibited by glucocorticoids and retinoic acids (particularly in human monocytes). It is also able to regulate traditional cytokines. For example, activin A inhibits the production of IL-1 and stimulates the production of IL-1 receptor antagonists (IL-1Ra). These findings indicate that activin may act as an anti-inflammatory cytokine (Ohguchi et al., 1998). Primary placenta cells are able to make activin and activin is stimulated by IL-1 and TNF. Interestingly, activin A is detected at high levels in women with preterm labor, a condition frequently associated with infection and elevated levels of cytokines (Keelan et al., 1998).

Activin

Endogenous inhibitors and enhancers Two activin-binding proteins have been identified that are present in human serum and follicular fluid. 2-Macroglobulin, a broad-spectrum protease inhibitor and abundant serum protein binds inhibin and activin, but does not alter the biological activity of either ligand (Krummen et al., 1993). Thus, this protein may serve as a carrying protein for the mature dimer. In addition, a 32 kDa protein known as follistatin binds activin and is abundant in follicular fluid and serum. Follistatin inhibits the biological activity of activin in vivo and in vitro and masks many of the available activin immunoassays. Follistatin was originally described as a side-fraction of follicular fluid that inhibits pituitary FSH. Later, it was recognized that this activity is predicated on the ability of follistatin to bind to the locally produced activin. In the pituitary, activin is tonically produced while the binding protein follistatin is exquisitely sensitive to GnRH and serves as the principal biological regulator of activin in this setting.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

825

in the human and nonhuman primate ovary, the cumulus and granulosa cells of growing follicles express the mRNA for the , A, and B subunits. Unlike the rat, human theca cells express the  subunit. In the dominant follicle,  and A subunit are expressed, however, the B subunit is not detected. Polycystic ovarian syndrome (PCOS) is a disease of follicle maturation in the human and the expression pattern of inhibin and activin subunit mRNA has been examined in follicles collected from ovaries with this disease. The follicles have similar subunit mRNA abundance and localization relative to those from normal menstrual cycles, however,  subunit protein is not produced. This suggests that PCOS ovaries produce activin and little inhibin. Whether this imbalance in the local production of the ligands participates in disease remains to be determined. Intriguingly, activin A is detected in a number of pathophysiological conditions including hyperthyroidism, renal failure, inflammatory bowel disease, infertility, atherosclerosis, rheumatoid arthritis, and in some solid cancers. Tests are under way to determine if activin can be diagnostic for fetal anomalies such as Downs syndrome or neural tube defects, may induce some of the processes of preterm labor, or the lack of activin may result in post-date delivery. The role of the ligand in the pathology of the disease and the utility of the assay as a diagnostic tool for disease states are areas of active investigation.

Normal levels and effects

IN THERAPY

Activin A does not appear to change in concentration in men or women except in the later stages of pregnancy when the ligand rises 10- to 22-fold (Petraglia et al., 1993). Activin A has been detected in the rat during the estrous cycle at two times corresponding to the primary and secondary FSH surges; however, attempts to measure free activin A during the normal human menstrual cycle have been unsuccessful (Besecke et al., 1997).

Pharmacokinetics The pharmacokinetic profile of rh-activin A has been described for rats and monkeys (Stouffer et al., 1993; Woodruff et al., 1993). The half-life for activin A was determined in humans following the delivery of the placenta at the end of pregnancy and is very similar to the half-life of the recombinant ligand. Table 2 outlines the pharmacokinetic profile of rh-activin A in female rats.

Role in experiments of nature and disease states

Toxicity

In the rat, inhibin and activin subunit mRNA is expressed exclusively in the granulosa cells of follicles that have been recruited into the preovulatory pool. The abundance of the subunit mRNAs increases during the normal estrous cycle and declines following the preovulatory gonadotropin surge. Similarly,

The major toxicity associated with activin occurs when the ligand is delivered in a continuous dose rather than a single bolus dose. Continuous infusion of activin is associated with massive liver toxicity. The cells around the central vein of the liver undergo rapid (within 24 hours) programmed cell death. Animals

826 Teresa K. Woodruff Table 2 Pharmacokinetic parameters for rh-activin A after bolus intravenous injection Parameter

Value

Cmax (ng/mL)

1900

Tmax (min)

1

V1/W (mL/kg)

63.1

Vss/W (mL/kg)

143±386

AUC (ng/mL/min)

23491

CL/W (mL/min/kg)

5.1

t1/2 (min)

6.1

t1/2 (min)

46

T1 (min)

12

MRT (min)

28±76

maintained on continuous activin A loose weight rapidly and the weight change can be accounted for almost entirely by loss of liver mass (up to two-thirds of the liver within 24±48 hours). The effects on the liver can be completely reversed when the activin is removed from the animal. This effect is recapitulated in the  inhibin knockout mouse model (activin A overexpression), where the observed cancer-related cachexia is directly related to the high levels of activin A affecting liver function. It should be noted that under one physiological condition, namely, pregnancy, activin A levels rise dramatically during the days preceding parturition. Thus, peripheral activin can be tolerated, however, much more information is required to completely understand the toxicological implications of activin.

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