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Endocrinology Vol. 143, No. 3 964-969
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Effect of Adenovirus-Mediated Overexpression of Follistatin and Extracellular Domain of Activin Receptor Type II on Gonadotropin Secretion in Vitro and in Vivo

Angela M. O. Leal1, Kazuaki Takabe1, Lili Wang, Cynthia J. Donaldson, Leigh A. MacConell, Louise M. Bilezikjian, Inder M. Verma and Wylie Vale2

Clayton Foundation Laboratories for Peptide Biology (A.M.O.L., K.T., C.J.D., L.A.M., L.M.B., W.V.) and Laboratory of Genetics (L.W., I.M.V.), The Salk Institute for Biological Studies, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie Vale, Ph.D., Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: . vale{at}salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activins are dimeric proteins that stimulate the synthesis and secretion of pituitary FSH by interacting with two classes of receptors, type I and type II, to initiate their intracellular signaling cascade. The extracellular domain of type II activin receptor (ActRII-ECD) contains all structural determinants sufficient for high affinity ligand binding. A soluble recombinant ActRII-ECD has been reported to attenuate FSH secretion from cultured rat anterior pituitary cells in response to exogenous activin A or endogenous activin B. Follistatin is a binding protein that acts as an extracellular factor to bind and inactivate activin. We constructed adenoviral vectors able to mediate expression of follistatin 288 (AdexCAFS288) and ActRII-ECD (AdexCAECD) and tested their biological activities both in vitro and in vivo. The data show that adenovirus-mediated overexpression of either ActRII-ECD or follistatin was able to attenuate FSH secretion by cultured rat anterior pituitary cells. However, AdexCAFS288 overexpression of follistatin was more effective than adenovirus-mediated overexpression of ActRII-ECD. In vivo, a single ip injection of AdexCAFS288 induced the expression of high levels of follistatin and resulted in the suppression of serum FSH levels in castrated male rats for up to 12 d postinjection. Infection with AdexCAFS288 had no effect on LH secretion in vitro or in vivo, demonstrating its selectivity. In conclusion, the results demonstrate the effectiveness of adenovirus-mediated overexpression of follistatin and ActRII-ECD to regulate FSH secretion and the potential of using this strategy as a tool to further define the critical role of activin/inhibin/follistatin circuitry in the modulation of the reproductive system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTIVINS AND INHIBINS, members of the TGFß superfamily of growth and differentiation factors, were identified based upon their ability to stimulate and inhibit, respectively, the release of pituitary FSH (1, 2). Activins are dimeric proteins composed of two closely related ß-subunits, ßA and ßB, linked by a disulfide bridge. Two additional ß-subunits, ßC and ßD, have been reported, but their biological functions are not yet fully characterized (3, 4). Inhibins are related heterodimers comprising one {alpha}- and one ß-subunit (5, 6). Activins, inhibins, and other members of the TGFß family interact with two classes of structurally related receptors, type I and type II (7, 8, 9, 10). Both receptor types have a single transmembrane domain, an extracellular ligand-binding domain, and a cytoplasmic portion that has serine/threonine protein kinase activity. Two type II receptors (ActRII and ActRIIB) and a single type I receptor (ActRIB or ALK-4) (7, 11, 12) have been characterized and shown to be required for activin-specific signal transduction. Upon binding activin, the type II receptor phosphorylates the type I receptor, thus initiating an intracellular signaling cascade. The extracellular domain of type II activin receptor (ActRII-ECD) contains all structural determinants sufficient for high affinity ligand binding. A soluble recombinant ActRII-ECD has been reported to attenuate FSH secretion from cultured rat anterior pituitary cells in response to exogenous activin A or endogenous activin B (13).

Follistatin is a glycosylated, cysteine-rich, monomeric binding protein, structurally unrelated to activins and inhibins, that acts as an extracellular factor to bind and inactivate activin (14, 15). By binding to the ß-subunit, follistatin (FS) blocks the ability of activin to stimulate FSH synthesis and secretion (16, 17, 18).

Adenoviral vectors have been proven to be an efficient means of introducing and expressing transgenes in a wide variety of cells and species, both in vitro and in vivo. By infecting both dividing and nondividing cells, recombinant adenoviral vectors have been useful in achieving high expression levels of transgenes. The establishment of the COS/TPC method, which uses cassette cosmids, has permitted the construction of recombinant adenoviruses with approximately 100-fold higher efficiency than conventional methods (19).

For this study we constructed two replication-defective adenoviral vectors expressing human follistatin-288 (FS288) and ActRII-ECD under a powerful constitutive promoter. Here we demonstrate that the adenovirus-mediated overexpression of ActRII-ECD or FS288 suppresses FSH secretion in vitro, which is induced by endogenous and exogenous activin. Overexpression of FS288 also suppressed plasma FSH levels in vivo, presumably by interfering with the biological effects of activin. Our results indicate that this method can be used to further define the critical roles of the activin/inhibin/FS circuitry in modulation of the reproductive system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Replication-defective recombinant adenovirus constructs
Replication-defective E1 and E3 recombinant adenoviral vectors expressing FS288, ActRII-ECD, or green fluorescent protein (GFP) were prepared as previously described (20, 21). FS288 was chosen as a transgene because it has higher heparin-binding capacity and may be more potent than FS315 (22, 23). Briefly, FS288, ActRII-ECD, or GFP cDNA was placed into a cassette cosmid vector, pAdex1Cawt, under a CAG promoter comprising a cytomegalovirus, i.e. enhancer, and a chicken ß-actin promoter (24) (pAdexCAFS288, pAdexCAActRIIECD, and pAdexCAGFP). A recombinant adenovirus was constructed by in vitro homologous recombination in HEK 293 cells using pAdexCAFS288, pAdexCAActRIIECD, or pAdexCAGFP and the adenovirus DNA-terminal protein complex by the COS/TPC method, previously described (19). The desired recombinant adenovirus (AdexCAFS288, AdexCAECD, or AdexCAGFP) was purified by cesium chloride density gradient centrifugation, followed by extensive dialysis (25). Viral titer was determined by a plaque assay using HEK 293 cells and expressed as plaque formation units (PFU)/ml. Infection of cells with adenoviral vectors was carried out by incubating confluent cells with vectors in DMEM with 5% FBS.

Cell culture and treatments
Anterior pituitary glands of adult male Sprague Dawley rats (200–225 g) were dispersed by treatment with collagenase. The cells [3.3 x 105/well of 48-well Costar (Cambridge, MA) plates] were allowed to recover for 3 d in medium (ßPJ) with 2% FBS. Before the experiment, the cells were washed three times with the same medium and infected with different concentrations [expressed as multiplicity of infection (MOI)] of AdexCAFS288, AdexCAECD, or AdexCAGFP. After 24 h, cells were washed twice and treated either with or without recombinant human activin A (rh activin A) for 48 h. AdexCAGFP was used as a control. Cells were maintained at 37 C in 7.5% CO2 and 92.5% air.

Animal procedures
Adult male Sprague Dawley rats (200–225 g), obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN), were castrated bilaterally and allowed to recover for 4 d. On the fifth day animals were weighed and treated with a single ip injection of 1 ml vehicle (PBS), AdexCAFS288, or AdexCAGFP (3.9 x 1010 PFU/ml each). Rats were maintained in a temperature-controlled, artificially illuminated room with free access to regular laboratory chow and water. Animals were examined daily. Blood was collected by eye-bleed, under isoflurane anesthesia, on d 3, 6, and 9 postinjection. On d 12 postinjection, animals were decapitated, and blood and tissues were collected. All animal procedures were performed in accordance with protocols approved by The Salk Institute animal care and use committee.

Hormone measurements
Serum and cell culture medium FSH, LH, and ACTH concentrations were quantified by RIA, using reagents provided by the National Hormone and Pituitary Program of the NIDDK, as previously described (26, 27). Serum FS was measured using the Quantikine human FS immunoassay kit (R\|[amp ]\|D Systems, Inc., Minneapolis, MN).

Statistical analysis and reagents
In vitro experiments were performed in triplicate, each repeated at least three times. Data were analyzed using ANOVA, followed by the Bonferroni-Dunn multiple comparison test (P < 0.01 was considered significant). In vivo data were analyzed using ANOVA, followed by t test or Tukey’s test and Spearman’s rank correlation test when appropriate. P < 0.05 was considered significant. FS288 cDNA was provided by Dr. Sunichi Shimasaki (University of California-San Diego, La Jolla, CA). GFP cDNA was provided by M. Pando, and rh activin A was provided by Dr. Wolfgang Fischer (The Salk Institute, La Jolla, CA); rh FS288 was obtained through the National Hormone and Pituitary Program of NIDDK.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of adenovirus-mediated expression of FS and ActRIIECD in vitro
Infection of cells with AdexCAFS288 or AdexCAECD suppressed FSH secretion from cultured rat anterior pituitary cells in a dose-dependent manner. Infection with AdexCAGFP was used as a control and was determined to have no effect on FSH secretion. Infection with surprisingly low amounts of AdexCAFS288 suppressed basal FSH secretion, which is presumed to be dependent on endogenous activin secretion (28), and attenuated the response to exogenous activin (0.3 nM; Fig. 1AGo). Infection with 0.01 MOI AdexCAFS288 (Fig. 1BGo) inhibited FSH secretion by cultured rat anterior pituitary cells for up to 3 d, whereas lower amounts of AdexCAFS288, PBS, or AdexCAGFP had no effect. As shown in Fig. 1CGo, 2 nM rh FS288 completely suppressed the effect of submaximal concentrations of activin (up to 0.2 nM), but its effect was reversed by 1 nM activin. Likewise, 0.002 or 0.01 MOI AdexCAFS288 was effective in suppressing the effect of up to 1 nM activin, but its effect could be overcome by higher concentrations of activin. To examine the selective effect of adenovirus-mediated expression of FS on gonadotropin secretion, we measured the LH concentration and observed no effect of infection with AdexCAFS288 or AdexCAGFP on LH secretion by cultured rat anterior pituitary cells (data not shown). Finally, to check the effect of adenovirus-mediated expression of FS on other anterior pituitary cell types, we examined the effect of AdexCAFS288 on ACTH secretion by cultured rat anterior pituitary cells. We have previously shown that exogenous activin suppresses ACTH secretion by cultured pituitary cells (26). The suppression of ACTH secretion by 0.03 nM activin was completely blocked by the lowest MOI (0.25) of AdexCAFS288 tested (data not shown).



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  		Figure 1. AdexCAFS288 suppresses the effects of exogenous and endogenous activin on FSH secretion from cultured rat anterior pituitary cells. Cultured rat anterior pituitary cells were treated with different concentrations of AdexCAFS288 ({bullet}), or AdexCAGFP ({circ}) either with ({blacksquare} or {square}) or without 0.3 nM activin A (A). Time course of the effect of 0.01 MOI AdexCAFS288 or AdexCAGFP and 0.5 nM rhFS288 on FSH secretion from cultured rat anterior pituitary cells (B). Cultured rat anterior pituitary cells were treated with different doses of activin (basal, {diamondsuit}) after treatment with either 0.002 or 0.01 MOI rAdexFS288 ({blacksquare} and {bullet}) and AdexCAGFP ({square} and {circ}) or 2 nM recombinant FS ({diamond}, C). Results are shown as the mean ± SEM of a representative experiment performed in triplicate and replicated at least three times. x, P < 0.01; *, P < 0.0001.

 
Infection with AdexCAECD produced similar effects on FSH secretion, but required much higher MOI concentrations (Fig. 2Go). This is consistent with the lower affinity of recombinant ActRII-ECD compared with that of FS288 for activin A (13). Infection with 6.25 MOI of AdexCAECD was also effective in suppressing the effect of up to 1 nM activin on FSH secretion (data not shown).



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  		Figure 2. AdexCAECD suppresses the effect of endogenous and exogenous activin-A. Cells were treated with different concentrations of AdexCAECD ({bullet}), or AdexCAGFP ({circ}) either with ({blacksquare} or {square}) or without 0.3 nM activin A. Results are shown as the mean ± SEM of a representative experiment performed in triplicate and replicated at least three times. x, P < 0.01; *, P < 0.0001.

 
Effect of adenovirus-mediated expression of FS in vivo
We also conducted experiments in adult castrated male rats to examine the effectiveness and longevity of AdexCAFS288 on plasma FSH levels, in vivo. Adenoviral vectors can infect cells in vivo, causing them to express very high levels of the transgene. However, due to epissomal localization of the vector and the virus-induced immune response, this expression usually lasts for only a short time (5–10 d postinfection) (29). We have therefore assessed the duration and levels of FS expression 3, 6, 9, and 12 d after delivery of adenovirus constructs. Infection with AdexCAFS288 resulted in high serum levels of FS. The levels of serum FS were highest 3 d after infection and progressively decreased at later time points, but remained detectable even on d 12 (Fig. 3Go). Serum FS levels were undetectable in control animals that received PBS or control rAdexCAGFP injection at all time points. Infection with AdexCAFS288 significantly decreased serum FSH levels to less than 50% of those in animals that received PBS or control AdexCAGFP injection. Suppression of FSH level was observed as early as d 3 and was maintained through the 12-d period (Fig. 4Go). The overall relationship between FS288 and FSH levels is shown in Fig. 5Go. To examine the selective effect of AdexCAFS288 infection on gonadotropin secretion, we also measured serum LH concentration. As shown in Fig. 6Go, LH levels were not significantly different between rats infected with AdexCAFS288 and control rats injected with PBS, but were slightly elevated in control rats infected with AdexCAGFP. There was no effect of infection with AdexCAFS288 or AdexCAGFP on the body weights of animals during the 12 d postinjection of adenovirus (Table 1Go). Furthermore, based on thorough daily examinations of individual animals, no abnormalities of fur or surgical scars were observed. This observation contrasts with the fur abnormalities observed in transgenic mice that overexpressed FS (30).



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  		Figure 3. Serum FS levels on d 3, 6, 9, and 12 postinjection (ip) of 3.9 x 1010 PFU AdexCAFS288 in male castrated rats. Data are the mean ± SEM (n = 10 animals/group).

 


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  		Figure 4. Serum FSH levels on d 3, 6, 9, and 12 postinjection (ip) of PBS ({blacksquare}) or 3.9 x 1010 PFU AdexCAFS288 ({square}) or AdexCAGFP () in male castrated rats. Data are the mean ± SEM (n = 8–10 animals/group). *, P < 0.05 compared with PBS or AdexCAGFP groups.

 


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  		Figure 5. Relationship between serum FS and FSH levels postinjection (ip) of 3.9 x 1010 PFU AdexCAFS288 in male castrated rats.

 


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  		Figure 6. Serum LH levels on d 12 postinjection (ip) of PBS ({blacksquare}) or 3.9 x 1010 PFU AdexCAGFP ({square}) or AdexCAFS288 () in male castrated rats. Data are the mean ± SEM (n = 8–10 animals/group). *, P < 0.05 compared with PBS or AdexCAGFP groups.

 

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  		Table 1. Body weight (grams) before (initial) and 12 d (final) postinjection (ip) of PBS or 3.9 x 1010 PFU AdexCAFS288 or AdexCAGFP in male castrated rats

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro and in vivo results presented here demonstrate the effectiveness of adenovirus-mediated overexpression of FS and ActRII-ECD to suppress FSH secretion. The in vivo effect was long-lasting, up to 12 d, and was seen in gonadectomized rats. The mechanism of FSH suppression probably involves binding and neutralization of activin by either FS or ActRII-ECD. The suppression of FSH by adenovirus-mediated overexpression of FS in castrated rats indicates that FS acts independently of any gonadal steroids or proteins. This reinforces the importance of activin stimulatory tone in sustaining FSH secretion. In fact, it has been reported that GnRH stimulation of FSHß mRNA is activin dependent and that continuous GnRH treatment suppresses FSHß mRNA levels in vivo by stimulating FS production (31). The selective ability of the overexpressed FS to suppress FSH is demonstrated by the absence of an effect on LH secretion in vitro. The slightly lower levels of LH observed in rats infected with rAdexCAFS compared with rAdexCAGFP could be attributable to the greater variability of circulating LH levels due to the more pronounced pulsatile pattern of LH secretion in castrated rats (32). However, an effect of adenovirus-mediated overexpression of FS in vivo cannot be excluded. Previous observations on the effect of FS on LH secretion in vivo suggest that FS does not play a significant role in the negative feedback regulation of LH (33, 34, 35, 36). It is noteworthy that despite the large amount of adenovirus injected, general health and body weight were not affected up to 12 d postinjection.

Previous approaches to define the physiological role of FS include the use of transgenic technology to delete or overexpress the FS gene, the injection of recombinant FS, and the determination of FS concentrations in biological fluids (30, 31, 33, 35, 36, 37). In the first approach transgenic mice lacking FS presented multisystemic embryonic defects, including insufficient muscle development and skeletal abnormalities, and died soon after birth (37). By contrast, transgenic mice that overexpressed FS exhibited some interesting phenotypes, such as small testes, suppressed FSH levels, and irregular fur (30). Other striking findings were testicular atrophy with Leydig cell hyperplasia, arrest of spermatogenesis, and seminiferous tubular degeneration leading to infertility. However, serum FSH levels were only reduced in one of the five lines and were not significantly different in gonadectomized wild-type and gain of function mutant male mice that overexpressed FS.

Several in vivo studies have shown that exogenous FS was able to suppress FSH in several species by administration of limited amounts of FS for a short period of time (31, 33, 35, 36). However, the limited supplies of recombinant FS288 have made these studies technically difficult, and the results may have underestimated the effects of FS administration. In this study we have explored the physiological application of adenoviral vectors as useful tools for the induction of FS expression. The results demonstrate that infection with our adenoviral construct successfully induced the expression of FS and resulted in high levels of serum FS. Most importantly, we demonstrate the effectiveness of adenovirus-mediated overexpression of FS to regulate FSH secretion. The longevity of adenovirus-mediated overexpression of FS was assessed by monitoring circulating FS and FSH levels 3, 6, 9, and 12 d after delivery of adenoviruses. Although there was a gradual decline in serum FS levels, at 12 d postinfection FS levels were still high and effective in suppressing FSH. The cause of the decline of adenovirus-mediated overexpression of transgenes over time is unclear; however, virus-induced immune responses may play a part (38).

It has been previously reported that the treatment of cultured rat anterior pituitary cells with a soluble form of ActRII-ECD was able to attenuate FSH secretion in response to exogenous activin A or endogenous activin B (13). The data demonstrated that the soluble ActRII-ECD had structural determinants that were sufficient for high affinity ligand binding. The data presented here show that adenovirus-mediated overexpression of ActRII-ECD was able to attenuate FSH secretion. Compared with adenovirus-mediated overexpression of FS, however, ActRII-ECD was much less potent in inhibiting FSH secretion by cultured rat anterior pituitary cells.

In conclusion, the method we describe here, overexpression of FS and ActRII-ECD by a replication-defective adenoviral vector, is a method that can be used as a tool to further define the critical roles of the activin/inhibin/FS circuitry in the modulation of the reproductive system. This new strategy of a single and noninvasive injection of recombinant adenovirus can provide a sustained level of biologically active FS.


   Acknowledgments
 
Matthew Pando of the Laboratory of Genetics, The Salk Institute, is acknowledged for expert technical support. We also thank Dr. Sunichi Shimasaki of University of California-San Diego for providing FS288 cDNA, Genentech, Inc., for providing recombinant FS, and Yaira Haas for technical assistance.


   Footnotes
 
This work was supported by Americas Fellowship, NIH (to A.M.O.L.); the Yoshida Scholarship Foundation, the Gail I. Zuckerman Foundation, the Kleberg Foundation, and the Adler Foundation (to K.T.); NIH Program Project HD-13527; and in part by the Foundation for Medical Research, Inc.

1 A.M.O.L. and K.T. contributed equally to this work. Back

2 Foundation for Medical Research, Inc., Senior Investigator. Back

Abbreviations: ActRII, Activin receptor type II; ActRII-ECD, extracellular domain of type II activin receptor; FS, follistatin; FS288, follistatin-288; GFP, green fluorescent protein; MOI, multiplicity of infection; PFU, plaque formation units; rh activin, recombinant human activin.

Received June 20, 2001.

Accepted for publication October 3, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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