Regulable Expression of Inhibin A in Wild-Type and Inhibin Null Mice

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Regulable Expression of Inhibin A in Wild-Type and Inhibin ␣ Null Mice

Tyler Mark Pierson, Yaolin Wang, Francesco J. DeMayo, Martin M. Matzuk, Sophia Y. Tsai, and Bert W. O’Malley Department of Molecular and Cellular Biology (T.M.P., F.J.D., M.M.M., S.Y.T., B.W.O.) Department of Pathology (M.M.M.), and Department of Molecular and Human Genetics (M.M.M.) Baylor College of Medicine Houston, Texas 77030 Schering-Plough Corp. Research Institute (Y.W.) Kenilworth, New Jersey 07033

Exogenous regulation of protein expression creates the potential to examine the consequences of homeostatic Dysregulation in many physiological systems and, when used in transgenic mice, provides the capability of restoring a gene product to its knockout background without antigenicity issues. In this study, we used a mifeprisone-inducible system (the GeneSwitch system) to regulate the expression of inhibin A from the liver of mice. Inhibin is a heterodimeric protein (␣/␤) wherein one of its subunits (␤) is capable of homodimerizing to form its physiological antagonist, activin (␤/␤). Inhibin is also expressed in two forms, A and B, as determined by the subtype of ␤-subunit that dimerizes with the ␣-subunit (␣/␤A or ␣/␤B). To utilize the GeneSwitch system, transgenic transactivator mice with liver-specific expression of a mifepristone-activated chimeric nuclear receptor (GLVP) were crossed with transgenic target mice containing a GVLP-responsive promoter upstream of poliovirus IRES (internal ribosome entry site)-linked sequences coding for the ␣- and ␤-subunits of inhibin A. This intercross produced “bigenic” mice capable of regulable expression of inhibin A from the liver. Overexpression of inhibin A in wild-type mice produced a phenotype wherein males had decreased testis size and females had a block in folliculogenesis at the early antral stage, findings similar to activin type IIA receptor (ActRIIA) null mice. These phenotypes were most likely due to suppressed serum FSH, confirming that the liverderived inhibin A was secreted into the serum to down-regulate pituitary FSH levels. Furthermore, the generation of bigenic mice in the inhibin ␣ null background allowed for the induction of inhibin A 0888-8809/00/$3.00/0 Molecular Endocrinology 14(7): 1075–1085 Copyright © 2000 by The Endocrine Society Printed in U.S.A.

in inhibin ␣ null male mice with subsequent rescue of these mice from their gonadal tumor-induced lethal phenotype. This work demonstrates the in vivo production of a heterodimeric hormone from a single inducible promoter to study its therapeutic and physiological effects. In addition, these studies are the first example of an inducible system being used to prevent a lethal knockout phenotype in an animal model. (Molecular Endocrinology 14: 1075– 1085, 2000)

INTRODUCTION Inhibins function to negatively regulate the production and secretion of FSH from the anterior pituitary, regulate intragonadal events including follicle development and steroidogenesis, and act as tumor suppressors in the gonads and adrenal cortex (1–5). At the level of the pituitary, inhibins function to down-regulate the production and the secretion of FSH in a cycle-dependent manner in females and in a tonic pattern in males (5). Interestingly, the expression patterns of the two inhibins, inhibin A or inhibin B, are sexually, spatially, and temporally dimorphic (5) and have been shown to be differentially regulated by various hormonal stimuli in vitro (6). When the inhibin ␣-subunit is deleted in mice, inhibin-deficient mice form gonadal sex cord-stromal tumors with 100% penetrance as early as 4 weeks of age (3). Soon after the development of these tumors, the mice acquire a cancer cachexia-like syndrome and die (4). This syndrome, the result of large amounts of activins secreted by the tumors (7), consists of a block in gastric epithelial cell differentiation, severe weight loss, hepatocellular degeneration due to apoptosis, and anemia (4, 7, 8). 1075

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Inhibin and activin physiology are interwoven at several levels. Molecularly, activins are homodimers of the ␤-subunit, while inhibins consist of ␣/␤-subunit heterodimers. In order for a cell to produce the inhibin heterodimer without concurrent activin production, the ␣-subunit must be produced in excess of the ␤subunit (creating a situation wherein the ␣-subunit monomer is also secreted in substantial quantities from an inhibin-producing cell) (9). Inhibins and activins also have been shown to have overlapping binding sites in a number of tissues (10, 11). This overlapping pattern and in vitro data indicate that the inhibins, and in particular inhibin A, may have a dominantnegative effect upon activin signaling via the activin type II receptor (12–14). However, inhibin-specific binding sites in the gonads, pituitary, and gonadaltumor tissue of inhibin ␣ null mice also have been observed (10, 15–17). An especially intriguing aspect in the study of inhibin function is that an inhibinspecific receptor has not been elucidated in great detail and so the mechanism by which inhibin transduces its signal and/or antagonizes activin’s signal remains unknown (18). The use of ligand-inducible systems has been very productive in the study of protein function and the generation of animal models of disease or disease therapy (19–23). One ligand-inducible system, which can be used to address in vivo questions, is the GeneSwitch system. This system utilizes a regulator protein, GLVP (a chimeric VP16-GAL4-PR⌬42), whose gene is expressed from one promoter, that is able to induce (in the presence of its activator, mifepristone) the expression of another gene via binding to an upstream GAL4 (promoter) response element. We successfully used this bigenic GeneSwitch system to regulably express inhibin A from the livers of either wild-type mice or mice with a knockout of the inhibin ␣ gene. By using the liver, we could determine the efficacy of our system in the delivery of hormones into the bloodstream from “exogenous” tissues (i.e. a tissue that does not normally express inhibins) and the potential use of these “exogenous” tissues in future gene therapy protocols involving the GeneSwitch system. In this manuscript, we show that we can induce the production of inhibin A into the circulation from a source (i.e., the liver) that normally does not synthesize inhibin. In addition, we demonstrate an endocrine effect of the inhibin A at the level of the pituitary (in wild-type mice) and at the level of the testes (in inhibin ␣ knockout mice). In the latter case, regulable production of inhibin A blocks the development of testicular tumors in the inhibin ␣ knockout, confirming previous studies that inhibin is a secreted tumor suppressor protein. The successful generation of mice capable of producing bioactive inhibin A from the liver in response to mifepristone treatment has broad implications in the study of reproductive physiology and tumorigenesis, the development of novel contraceptive technologies, and the further use of similar inducible systems for gene therapy.

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RESULTS Development of an Inducible System for the Study of Inhibin Function To study the in vivo consequences of overexpression of inhibin A in mice, we used the bigenic GeneSwitch system. The GeneSwitch system consists of a chimeric regulator (GLVP) composed of a mutated progesterone receptor ligand-binding domain (PR⌬42) that binds and is activated by mifepristone (MFP), but not by endogenous steroids (24) (Fig. 1). The DNA binding domain of the yeast GAL4 transcription factor and the transcriptional activation domain of the herpes virus VP16 protein are fused to the mutant progesterone receptor ligand-binding domain. The GLVP is responsive to mifepristone and activates transcription from a target locus consisting of GAL4 response elements (17 mer sequences) positioned upstream from a target gene or genes (19, 24–27) (in our case, there were four copies of the 17 mer sequence; see below). The production of transgenic mice expressing the

Fig. 1. Strategy and Transgenes for the Inducible Expression of Inhibin A This figure represents the method of activation of the inducible inhibin A GeneSwitch system by MFP the liver cells of inh/glvp mice. GLVP expression is driven by a liver-specific transthyretin enhancer/promoter (TTR) that is flanked by chromosomal insulators (27). In the presence of MFP, the expressed GLVP dimerizes and binds to a GAL4 response element located (in four copies) upstream of the inhibin ␣ and ␤A coding sequences, activating transcription of the bicistronic mRNA. The cap-dependent translation of the ␣-subunit is more efficient than the IRES-dependent translation of the ␤A-subunit producing an excess of ␣-subunit in comparison to ␤A-subunit. This relative excess of ␣-subunit favors formation of inhibin A over activin A (9) (see Figs. 2B and 5C). The inh mice contain the X3 transgene, which consists of four copies of the GAL4 DNA response element (17 mer), a minimal thymidine kinase promoter (tk), SV40 splice acceptor and donor sites, the murine inhibin ␣ subunit cDNA, the poliovirus IRES, murine inhibin ␤A cDNA, and the SV40 polyadenylation site.

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GLVP specifically in the liver has been previously reported (glvp transactivator line) (26). To produce the GLVP-responsive inhibin A target locus used in the present study, an inhibin A target (transgene) construct (X3-inh) was constructed with four copies of the 17 mer-Gal4 responsive elements upstream of a minimal thymidine kinase promoter. Downstream of this promoter is an intron containing SV40 splice donor and acceptor sites followed by the cDNA for the murine inhibin ␣-subunit, the poliovirus internal ribosome entry site (IRES), the cDNA for the murine inhibin/activin ␤A subunit, and an SV40 polyA addition consensus sequence (Fig. 1). When activated, this locus would produce a bicistronic mRNA, in which the poliovirus IRES links the inhibin ␣ and ␤A subunit cDNAs (28). This strategy would permit a relative excess production of the inhibin ␣ subunit in comparison with the inhibin/activin ␤A subunit, since the inhibin ␣ subunit is translated by a more efficient cap-dependent translational mechanism whereas the inhibin/activin ␤A subunit would be translated by a less-efficient IRES-dependent mechanism. By biasing our translational efficiency in this manner, our goal was to produce a ratio of the two subunits (i.e., ␣ ⬎⬎ ␤A) that would favor the formation of inhibin A (␣/␤A heterodimer) over activin A (␤A:␤A homodimer) (9, 28). Mifepristone-Inducible Inhibin A Production in Tissue Culture and in Vivo Before induction of inhibin A from the liver of mice, transient transfection studies in HepG2 cells were performed to test the inducibility of the X3-inh transgene in a hepatocyte cell line. In these assays we used another transactivator, GLp65, which differs from GLVP in that the transactivation domain is derived from the p65 protein (a member of the NF-␬B family) (27). With all functional assays the GLp65 works similarly to GLVP with the exception that in transient transfections the GLp65 has a much lower basal activity than the GLVP. The X3-inh was transfected into HepG2 cells in the presence or absence of the GLp65 encoding plasmid. Cells were subsequently treated with or without mifepristone. The X3-inh-transfected cells expressed inhibin A only when both the GLp65 transactivator and MFP were present. In contrast, there was no basal level of inhibin A expression in the absence of MFP (Fig. 2A). Thus, our bigenic system was functional in transient transfection assays. Based on the above findings in the hepatocyte cell line, the X3-inh transgene was subsequently microinjected into mouse embryos, and nine founder target lines (X3 mice) were generated. These founder target lines were then crossed with transactivator mice (glvp mice) to produce bigenic mice (X3/glvp mice). Since inhibin A is not expressed at detectable levels in the serum of adult male rodents (5), serum samples from the males were assayed to avoid any confounding endogenous inhibin signal. Male X3/glvp mice generated from the nine X3 founder mouse lines were sub-

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sequently tested for MFP-induced inhibin A secretion. One of the X3 founder lines (line 3065, hereafter referred to as the inh transgenic mouse line) was capable of expressing inhibin A at high levels in the presence of MFP and the GLVP. When male inh/glvp mice from this line were ip injected with MFP (250 and 500 ␮g/kg dosages), inhibin A expression was dependent on the MFP dose and expressed at either physiological (436 ⫾ 155 pg/nl when 250 ␮g/kg MFP was injected) or supraphysiological concentrations (1,210 ⫾ 232 pg/nl when 500 ␮g/kg MFP was injected) relative to inhibin B levels in the male rat (5) (Fig. 2B). To confirm that the immunoreactive inhibin A secreted from the liver was also bioactive, male inh/glvp mice were injected with high-dose MFP for 7 days, and the effect of the induced inhibin A on serum FSH levels was determined. These bigenic males expressing the induced inhibin A had a 2.6-fold reduction in serum FSH levels (Fig. 2C). Monogenic mice (inh or glvp mice) treated with MFP did not have altered serum levels of FSH (data not shown). The need to perform long-term studies facilitated the use of mifepristone-containing time-release pellets. These pellets were designed to release MFP in measured dosages over a 60-day period. Male inh/ glvp mice receiving pellets that released appropriately 6 ␮g MFP a day had inhibin A levels of 1,803 ⫾ 258 pg/ml after 1 week of treatment. Eight weeks after treatment, serum FSH levels in MFP-treated inh/glvp male mice were significantly reduced in comparison to placebo-treated inh/glvp males [126.3 ⫾ 22.1 (n ⫽ 4) vs. 197.8 ⫾ 16.0 ng/ml, (respectively; n ⫽ 5, P⬍0.02)]. These findings demonstrate that inhibin A (␣:␤A) secretion from the liver must predominate over the activin A (␤A:␤A) levels (see below and Fig. 5C). Thus, the GS system can regulate the expression of inhibin A in cell culture and in vivo, and the induced inhibin A is bioactive and capable of reducing serum FSH levels. Effects of Overexpression of Inhibin A in WildType Mice By overexpressing inhibin A in bigenic mice, a greater understanding of inhibin signaling and physiology can be achieved. Long-term studies were initiated by implanting timed-release MFP (6 ␮g/day) or placebo pellets into 18- to 21-day-old bigenic and monogenic mice of both sexes. These mice were weighed weekly and killed 8 weeks after pellet placement. Blood, livers, and gonads were harvested. Male inh/glvp mice that received MFP were found to have significantly reduced testis weights as compared with inh/glvp males treated with placebo or monogenic males receiving MFP or placebo (monogenic controls) (Fig. 3A). These mice also had a decrease in seminiferous tubule volume and diameter as compared with bigenic males receiving placebo pellets and monogenic controls (Fig. 3B). There were no significant differences in body weight over time between the treatment groups (data not shown). Although the mifepristone-treated inh/

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glvp males had smaller testes, spermatozoa were present in the lumens of the seminiferous tubules and, when paired with females, these mice were found to be fertile. Female inh/glvp mice receiving MFP also had defects in the gonads, demonstrating an arrest in folliculogenesis at the early antral follicle stage and a lack of corpora lutea (Fig. 3C). In contrast, female inh/glvp mice receiving placebo and monogenic female controls receiving MFP or placebo had ovaries that underwent normal folliculogenesis and contained numerous corpora lutea; this indicated that the MFP dosage did not antagonize oogenesis, folliculogenesis, and ovulation (Fig. 3C). Thus, overexpression of inhibin A leading to reduced serum FSH levels in male and female mice resulted in gonadal defects reminiscent of activin receptor type II knockout mice, which also have reduced FSH levels (see Discussion) (30). Regulable Rescue of Male Inhibin ␣ Null Mice

Fig. 2. Inhibin A Is Induced in Vitro and in Vivo A, Inducible expression of inhibin A in HepG2 cells. Target vector DNA (0.3 ␮g pX3) was transiently transfected into HepG2 cells with or without transactivator plasmid (0.2 ␮g pSBC-1 or pSBC-GLp65). Either MFP or vehicle was administered to cells. Only cells transfected with the target and transactivator constructs and treated with MFP expressed inhibin A (155.7 ⫾ 7.6 pg/ml). B, Dosage-dependent expression of inhibin A in inh/glvp mice. Serum inhibin A levels were measured before (0 h) and after (12 h) ip injection of either 250 ␮g/kg or 500 ␮g/kg of MFP (n ⫽ 3). C, Effects of overexpression of inhibin A on serum FSH levels. Male inh/glvp mice were given 500 ␮g/kg MFP for 7 days, and serum was collected pre- and posttreatment. Inhibin A and FSH levels were measured from the serum of each mouse. Inhibin A levels

Inhibin ␣ knockout mice develop gonadal sex cordstromal tumors as early as 4 weeks of age (3). More than 95% of the ␣ inhibin homozygote knockout male and female mice will die by 12 and 17 weeks of age, respectively, of a cancer cachexia-like syndrome due to high levels of tumor-secreted activins signaling through activin receptor type IIA in the liver and glandular stomach (4, 7, 8). Because of the earlier death of male inhibin ␣ null mice, we addressed whether overproduction of inhibin A can rescue the male inhibin ␣ null lethal phenotype. Bigenic (inh/glvp␣⫺/⫺ mice) and monogenic (inh␣⫺/⫺ or glvp␣⫺/⫺ mice; called monogenic ␣⫺/⫺ controls below) 18- to 21 day-old inhibin ␣ knockout males were treated with MFP or placebo timed-release pellets, which should be active over an 8-week period. Bigenic inhibin ␣ null male mice treated with MFP did not exhibit cachexia or tumors when examined up to 11 weeks of age. Body weights for inh/glvp␣⫺/⫺ male mice given MFP were comparable to monogenic inhibin ␣ heterozygote mice treated with placebo (Fig. 4A). In contrast, the inh/glvp␣⫺/⫺ male mice given placebo underwent weight loss, and many of these mice died in a manner similar to monogenic inhibin ␣ null male mice receiving MFP or placebo (monogenic␣⫺/⫺ controls) (Fig. 4A). Thus, overexpression of inhibin A can prevent the inhibin knockout mice from dying of the cachexia-like syndrome. To determine how overexpression of inhibin A was rescuing the inhibin ␣ knockout mice, morphological and histological analysis was performed. Special attention was paid to the livers of these mice since activin-induced hepatocellular necrosis is a major finding in the inhibin ␣ knockout mice (4). Livers from inh/glvp␣⫺/⫺ male mice treated with MFP were normal

were induced whereas FSH levels were significantly reduced in male inh/glvp mice before and after MFP treatment (423.7 ⫾ 113.5 vs. 161.4 ⫾ 48.7 ng/ml; respectively, P ⬍ 0.04, paired one-tailed Student’s t test, n ⫽ 3).

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in appearance and weight in contrast to the small, pale livers of inh/glvp␣⫺/⫺ male mice treated with placebo or monogenic␣⫺/⫺ controls (Fig. 4, B and C). Bigenic male inh/glvp␣⫺/⫺ mice treated with MFP exhibited normal liver architecture, while inh/glvp␣⫺/⫺ males treated with placebo had pale livers with extensive lymphocytic infiltration and hepatocellular degeneration around the central vein similar to monogenic null controls (Fig. 4C). Thus, overexpression of inhibin A prevents the liver pathology seen in the inhibin ␣ knockout mice. We next determined the long-term effect of overexpression of inhibin A on testicular tumorigenesis in the inhibin ␣ knockout mice. Testes from inh/glvp␣⫺/⫺ male mice treated with MFP appeared grossly tumor free in contrast to inh/glvp␣⫺/⫺ males treated with placebo or monogenic␣⫺/⫺ controls, both of which exhibited grossly hemorrhagic tumors (Fig. 5, A and B). Male inh/glvp␣⫺/⫺ mice treated with MFP exhibited normal testicular cytoarchitecture and had spermatozoa in the lumen of their seminiferous tubules (Fig. 5B) consistent with the normal fertility of these mice (see below). In contrast, inh/glvp␣⫺/⫺ males treated with placebo and monogenic ␣⫺/⫺ controls had testes that had disorganized testicular cytoarchitecture, hemorrhage, and tumor cells of sex cord-stromal origin (Fig. 5B). Furthermore, male inh/glvp␣⫺/⫺ mice treated with MFP were fertile, while inh/glvp␣⫺/⫺ males given placebo and monogenic null controls were infertile. Thus, high level production of inhibin A is sufficient to prevent testicular tumor development in our inhibin ␣ knockout mice. In inhibin ␣ null mice, high activin secretion from the testicular tumors directly causes the cachexia-like syndrome (4, 7, 8). Consistent with this data, serum activin A levels were undetectable in inh/glvp␣⫺/⫺

Fig. 3. Inhibin A Overexpression Mimics the Activin Type IIA Receptor Null Phenotype A, Testis size in bigenic and monogenic mice (⬃11 weeks of age). Testis size was significantly reduced in male inh/glvp mice treated with 6 ␮g/day MFP as compared with male inh/glvp mice treated with placebo [68.8 ⫾ 5.4 mg vs. 102.5 ⫾ 6.2 mg, respectively, P ⬍ 0.006, one-tailed Student’s t test] or monogenic male controls. B, Gross and microscopic analysis of testes from placebo (left) or MFP-treated (right) male inh/glvp mice. Male inh/glvp mice receiving MFP had smaller testes that contained seminiferous tubules with a narrower cross-sectional diameter as compared with male inh/glvp mice receiving placebo or monogenic controls. Mature spermatozoa were evident in the tubules of all mice. C, Microscopic analysis of ovaries from female inh/glvp mice. The ovaries of the majority of female inh/glvp mice treated with MFP (right panels) had a block in folliculogenesis at the early antral follicle stage, had a number of atretic follicles, and did not contain corpora lutea (4 of 7), whereas ovaries from female inh/glvp mice treated with placebo (left panel) underwent normal folliculogenesis and contained numerous corpora lutea (*) (8 of 8). Monogenic female controls treated with MFP also underwent normal folliculogenesis and contained corpora lutea (8 of 8 each) (data not shown).

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Fig. 4. Male Bigenic Mice Expressing Inhibin A in the Inhibin ␣ Null Background (inh/glvp␣⫺/⫺ ) Do Not Exhibit the Cancer Cachexia-Like Syndrome A, Growth rates for inhibin ␣ null males. Male inh/glvp␣⫺/⫺ mice (open circles) treated with MFP had normal growth rates similar to monogenic␣⫹/⫺ males (male mice expressing inhibins along with one of the two transgenes) treated with placebo (closed diamond). However, male inh/glvp␣⫺/⫺ mice treated with placebo (closed circles) had reduced growth rates similar to monogenic inhibin ␣ null males given MFP (open triangles) or placebo (closed triangles). B, Liver weights for inhibin ␣ null males (⬃ 11 weeks of age). Male inh/glvp␣⫺/⫺ mice treated with MFP had livers that were similar to monogenic inhibin ␣ wild-type mice (data not shown). However, male inh/glvp␣⫺/⫺ mice treated with MFP had livers that were significantly larger than livers from male inh/glvp␣⫺/⫺ mice treated with placebo (1.05 ⫾ 0.06 g vs. 0.63 ⫾ 0.04 g, respectively; P ⬍ 0.001, one-tailed Student’s t test,

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males treated with MFP and lacking testicular tumors, while placebo-treated inh/glvp␣⫺/⫺ males and monogenic␣⫺/⫺ controls with testicular tumors had similar elevated serum activin levels (Fig. 5C). These results confirm that gonadal tumorigenesis is required for the elevation of serum activins in inhibin ␣ null mice, while also verifying that our system does not produce significant amounts of activin A as a by product of the inducible inhibin A expression. Rescue of Inhibin ␣ Null Male Mice Is Reversible To determine whether the preventative effects of overexpression of inhibin A on the tumorigenesis process is reversible, we analyzed the inh/glvp␣⫺/⫺ after cessation of MFP treatment. When the inh/glvp␣⫺/⫺ males are treated for 60 days with MFP, with subsequent cessation of MFP treatment, the testicular tumors and the cancer cachexia-like syndrome did not occur within the first 12 weeks after MFP removal. However, by the 13th week after the MFP removal, many of these inh/glvp␣⫺/⫺ males began to exhibit the phenotypic characteristics of the cancer cachexia-like syndrome and demonstrated obvious testicular tumors (Fig. 5D). Thus, high level inhibin A from 3–11 weeks of age does not preclude the eventual development of testicular cancer in these mice. The reason why the tumorigenesis and the cancer cachexia-like syndrome developed so slowly after MFP withdrawal is not clear (see below).

DISCUSSION In this study, we used the GeneSwitch system to study the overexpression of a target gene and to rescue a knockout mouse from its lethal phenotype. This regulable gene expression system was capable of the tonic expression of inhibin A, while also allowing for a precise control of temporal activation and deactivation of inhibin A expression that would not be possible using constitutive promoters or daily injections. The use of a regulable transgenic inhibin A expression system in inhibin-deficient mice would also allow these mice to be exposed to inhibin A without activation of the immune system. Thus, the regulable expression of inhibin A in mice allows for several aspects of inhibin physiology to be investigated. For example, in wildtype mice, we tested the effects of increased levels of circulating inhibin on the pituitary physiology without any of its normal homeostatic feedback responses to

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FSH and steroids. This system also allowed for inhibin A production to be initiated just before the onset of tumorigenesis in inhibin-deficient mice with the potential of inhibiting the formation of the sex cord-stromal tumors in a regulable manner. The system also allowed us to determine whether the gonadal cells could initiate the oncogenic process when the inhibin A secretion was “turned off.” Thus, the use of a regulable gene expression system was predicted to provide enough flexibility to alter the exposure strategy to more thoroughly investigate inhibin’s role in the onset of tumorigenesis and cancer cachexia-like syndrome in these mice. In our studies, the poliovirus IRES was shown to be functional for the dual translation of a bicistronic mRNA in transgenic mice. This translation resulted in the production of the two subunits favoring the formation of heterodimeric inhibin instead of homodimeric activin. The inh/glvp male and female mice treated with MFP and overexpressing inhibin A phenocopy the activin receptor type IIA (ActRIIA) null mice (30). Both the ActRIIA null mice and the inhibin A-overexpressing mice have reduced serum FSH levels, which is the most likely cause of both phenotypes [i.e. both mouse models are reminiscent of a theoretical FSH hypomorph since the FSH null mice have a similar, but slightly more dramatic phenotype (31)]. Alternatively, the gonadal phenotypic similarity of the inhibin Aoverexpressing mice to ActRIIA null mice could indicate that inhibin A is functioning to antagonize signaling through this receptor and/or other receptors that interact with the ActRIIA. Unfortunately, the lack of adult phenotypes, due to embryonic or neonatal lethality, for mice deficient in other “activin” receptors [ActRIIB(32), ActRIA/ALK2 (33), and ActRIB/ALK4(34)] or both activin ligands (35) does not allow for a comparison of these mice to the inhibin A-overexpressing mice. The phenotypic similarity of the ActRIIA null mice and the inhibin A-overexpressing mice could also be the result of a combination of effects at the level of the pituitary (decreased FSH secretion) and gonad (inhibin A antagonism of signaling via the activin and/or putative inhibin receptors). Overexpression of inhibin A rescued the inhibin ␣ null male mice from their lethal phenotype. These results are relevant to the fields of physiology, oncology, and gene therapy for several reasons. The induced inhibin A was not expressed in the inhibin ␣ null mice until 18–21 days after birth and yet prevented testicular tumorigenesis. In addition, when the inducing mifepristone is removed, these mice formed gonadal

n ⫽ 6) or monogenic␣⫺/⫺ controls treated with or without MFP. C, Gross and histological analysis of livers from inhibin ␣ null males. Male inh/glvp␣⫺/⫺ mice treated with MFP had livers that were grossly normal (right side of top panel) and were normal histologically (bottom right panel). Male inh/glvp␣⫺/⫺ mice treated with placebo had livers that were grossly paler and smaller (left side, top panel) and had a significant amount of hepatocellular necrosis around the central vein along with lymphocytic infiltration (arrow) (bottom left panel). Male inh/glvp␣⫺/⫺ mice treated with placebo had similar pathology to monogenic null controls (data not shown).

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Fig. 5. Male inh/glvp Mice Expressing Inhibin A in the Inhibin ␣ Null Background Do Not Undergo Gonadal Tumorigenesis A, Testicular weights for inhibin ␣ null males. Male inh/glvp␣⫺/⫺ mice treated with MFP exhibited testis weights that were similar to monogenic inhibin ␣ wild-type males treated with placebo (data not shown). Male inh/glvp␣⫺/⫺ mice treated with MFP had significantly smaller testes in comparison to male inh/glvp␣⫺/⫺ mice treated with placebo (135 ⫾ 20 vs. 381 ⫾ 78 mg, respectively; P ⬍ 0.001, one-tailed Student’s t test, n ⫽ 6) or monogenic␣⫺/⫺ controls. B, Gross and histological analysis of testes from inhibin ␣ null males. Male inh/glvp␣⫺/⫺ mice treated with MFP had testes that grossly and histologically (bottom right panel) appeared tumor free. Male inh/glvp␣⫺/⫺ mice treated with placebo exhibited hemorrhagic and tumorous testes (top panel, left; bottom left panel) similar to monogenic null controls. Spermatozoa were evident in the seminiferous tubules of male inh/glvp␣⫺/⫺ mice treated with MFP (bottom right panel). C, Serum activin levels in inhibin ␣ null males. Male inh/glvp␣⫺/⫺ mice treated with MFP did not express any detectable serum activin A (n ⫽ 3), while male inh/glvp␣⫺/⫺ mice treated with placebo (127.7 ⫾ 20.5 pg/ml, n ⫽ 3) had serum activin A levels that were high similar to the monogenic␣⫺/⫺ controls. D, Body weights of male inh/glvp␣⫺/⫺ mice during and after MFP treatment (vertical line at 11 weeks represents the end of the MFP treatment); 18- to 21-day-old male inh/glvp␣⫺/⫺ mice were given 60-day time-release pellets delivering 12 ␮g of MFP a day (open circle) or placebo (closed circles). These mice

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tumors (albeit at a slower rate than untreated inhibin ␣ null mice). These results indicate that a lack of inhibin A in either pubertal or adult male mice sets the testis on the pathway to tumorigenesis. In contrast, exposure of inhibin ␣ null mice to inhibin A at 18–21 days of age inhibits the formation of sex cord-stromal tumors in these mice for a period coinciding with inhibin A expression. The results also indicate that the system is reversible and that early inhibin A exposure is not “curative” for gonadal tumorigenesis in the inhibin ␣ null background (i.e., loss of inhibin A expression permits subsequent initiation of gonadal tumorigenesis). This ability to delay the formation of the tumors could be of specific and general value in the study of tumorigenesis. In our case, factors affecting the progression of the gonads into tumorigenesis can now be manipulated and evaluated in this background to determine which factors regulate the rate of tumorigenesis after inhibin A expression is terminated. This study indicates the utility of inducible systems for the expression of therapeutic proteins. Even with a complicated scenario requiring production of a heterodimeric protein (each subunit of which must also be processed from precursors), the GeneSwitch system was capable of producing adequate amounts of the therapeutic protein to rescue a genetic disease. The regulable system was also capable of producing inhibin A in supraphysiological amounts so that concentrations in the gonad could approach normal local tissue concentrations to simulate the effects of the higher concentrations observed in paracrine and autocrine interactions. Because gene therapy vectors may not be capable of transducing certain cell types, the ability to express a protein under an inducible promoter in a different cell type, such as hepatocytes, may prove to be a common method of delivery and therapy. In summary, these results indicate the value of inducible gene systems in future in vivo physiology, tumorigenesis, and gene therapy research.

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inhibin/activin ␤A cDNA was cloned into the pSBC-2 vector (pSBC2-␤A). p17mer ⫻ 4 tk ␣ and pSBC2-␤A were then combined to produce the X3-inh transgene-containing plasmid. The GLp65 has been described previously (27) and was placed into the pSBC-1 vector (pSBC-GLp65). Transient Transfection of HepG2 Cells and Assay of Inhibin A HepG2 cells (600,000/well) were cotransfected with 0.3 ␮g of the pX3-inh transgene-containing plasmid and 0.2 ␮g of pSBC-1 or pSBC-GLp65 using SuperFect reagents (QIAGEN Inc., Valencia, CA). Sixteen hours later the media were removed, and cells were exposed to DMEM (10% FBS) containing either 70% ethanol vehicle or 10⫺8 M mifepristone (Rousell-Uclaf, Paris, France) for 24 h. Conditioned media were then collected and stored at ⫺70 C. One milliliter of conditioned media was then concentrated 10-fold with Ultrafree-4 Centrifugal Filter Devices (Millipore Corp., Bedford, MA) and reconstituted to a 2-fold dilution with FBS. Inhibin A assays were performed using a human inhibin A enzymelinked immunosorbent assay (ELISA) kit (Serotec Inc., Raleigh, NC). Experimental Animals All mouse studies were conducted in accord with the principles and procedures outlined by Molecular Endocrinology’s instructions to authors under “Guidelines for the Care and Use of Experimental Animals.” Generation of Target Transgenic Mice and Bigenic Mice The glvp transactivator mice were produced and screened as described previously (26). The X3 transgene was restriction endonuclease released from pX3 and a 4.3-kb fragment isolated and microinjected into the pronuclei of mouse embryos. Injected embryos were transferred into pseudopregnant female mice and allowed to develop to term. Mice were screened at 2 weeks of age by PCR using genomic tail DNA. PCR analysis of tail DNA to detect the X3 transgene was carried out with primers TT1 (5-CAAAGTGCAGTGTCTTCCTGGCTGTGC-3⬘) and TT2 (5⬘-CGGAACCGACTACTTTGGGTGTC-3⬘). Nine founder X3 lines were generated, with only one having inducible inhibin A expression (line 3065/inh) when crossed with glvp mice and treated with MFP. Generation and Analysis of inh/glvp Mice and inh/glvp␣ⴚ/ⴚ Mice

MATERIALS AND METHODS Plasmid Construction Vectors were constructed using standard cloning procedures. Construction of the pTTR-GLVP transgene-containing plasmid has been described previously (26). The pX3 target transgene-containing plasmid was constructed using a poliovirus IRES cloning system (28). The SV40 promoter/ enhancer in the pSBC-1 vector was removed, and a fragment containing the 17mer ⫻ 4 tk promoter sequences was inserted into the promotorless pSBC-1 vector (p17mer ⫻ 4 tk BC-1). A fragment containing the murine inhibin ␣ cDNA was isolated and subcloned into the p17mer ⫻ 4 tk BC-1 construct (p17mer ⫻ 4 tk ␣). A fragment containing the murine

Transactivator and target mice were mated, and bigenic mice containing both transgenes were genotyped by PCR analysis. Heterozygote inhibin ␣ null mice were bred with both transactivator and target mice to produce monogenic glvp or inh inhibin ␣ heterozygous mice. Crossing of these monogenic inhibin ␣ heterozygotes would produce monogenic or bigenic inhibin ␣ null mice. PCR analysis was also used for determining the genotype at the inhibin ␣ locus. PCR analysis of tail DNA using the primers E2–2 (5⬘-GGTCTCCTGCGGCTTTGCGC-3⬘), E2–3 (5⬘-GGCCTCCCGAGGAACCCGCTG-3⬘), HPRT (5⬘-GGATATGCCCTTGACTATAATG-3⬘), and Intron (5⬘-CCTGGGTGGAGCAGGATATGG-3⬘) determined whether the mouse was wild type, heterozygous, or homozygous null at the inhibin ␣ locus. Male inh/glvp mice had MFP or vehicle injected ip at the specified dosages or released from sc 60-day timed release

continued to maintain body weights similar to monogenic inhibin ␣ wild-type controls (closed diamonds) for up to 12 weeks after removal of MFP (n ⫽ 6). Male inh/glvp␣⫺/⫺ mice treated with placebo (closed circles) lost weight in a manner similar to studies presented in Fig. 3A.

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pellets (Innovative Research of America, Sarasota, FL). Retroorbital or cardiac blood was collected at the specified time intervals before or after treatment and incubated at room temperature for 30 min before isolation of serum in Microtainer tubes (Becton Dickinson and Co., Franklin Lakes, NJ) and stored at ⫺70 C. Inhibin A levels were determined by using a human inhibin A ELISA assay kit. FSH levels were determined with a rat FSH EIA assay kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ). Inhibin Long-Term Overexpression Studies and Rescue of Inhibin ␣ Null Male Mice Male and female inh/glvp mice(18–21 days old) were implanted with 60-day timed-release MFP or placebo pellets. In the rescue experiments, bigenic and monogenic 18- to 21day-old male inhibin ␣ null mice were implanted with 60-day timed-release MFP or placebo pellets. MFP pellets released 6 or 12 ␮g MFP/day for 60 days as specified. Mice were weighed weekly and killed after 8 weeks, and blood, livers, and gonads were harvested. Blood was treated as stated previously, while livers were weighed, sectioned, and stained with hematoxylin and eosin. Testes were weighed, placed into 10% buffered formalin, processed for histology, and stained as described (30). Ovaries were treated similarly to the livers but not weighed. Tumorous testes were sectioned and stained with hematoxylin and eosin. Inhibin A levels were determined by using a human inhibin A ELISA assay kit. Activin A levels were determined by using a human activin A ELISA assay kit (Serotec Inc., Raleigh, NC).

Acknowledgments We thank Dr. J. C. Smith for the generous gift of the inhibin ␣ cDNA; Drs. Mark M. Burcin, Sherry C. Cipriano, and Simona Varani for helpful comments and excellent review of the manuscript; Drs. T. Rajendra Kumar, Jason Yovandich, Dorit Elberg, and Steven Chua for helpful comments and technical assistance; Blake Abbot, Mei-jin Chu, Lei Gong, and Elizabeth Hopkins for technical assistance.

Received February 2, 2000. Revision received March 14, 2000. Accepted March 15, 2000. Address requests for reprints to: Bert W. O’Malley, M.D., Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: [email protected]. These studies were supported by NIH Grant HL-59314 to S.Y.T. M.M.M. acknowledges the support of his NIH Grant (CA-60651). T.M.P. is funded in part by the Edward and Josephine Hudson Scholarship through the M.D./Ph.D. Program at Baylor College of Medicine.

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