Genetic evidence that SMAD2 is not required for gonadal tumor development in inhibin-deficient mice

Background  

Inhibin is a tumor-suppressor and activin antagonist. Inhibin-deficient mice develop gonadal tumors and a cachexia wasting syndrome due to enhanced activin signaling. Because activins signal through SMAD2 and SMAD3 in vitro and loss of SMAD3 attenuates ovarian tumor development in inhibin-deficient females, we sought to determine the role of SMAD2 in the development of ovarian tumors originating from the granulosa cell lineage.     

Cyclin D2 and p27 are tissue-specific regulators of tumorigenesis in inhibin alpha knockout mice

Inhibins are heterodimeric (alpha:betaA and alpha:betaB) endocrine, paracrine, and autocrine factors of the TGFbeta superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin alpha (Inhalpha) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhalpha knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1-->S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhalpha mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhalpha knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhalpha lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin alpha single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin alpha knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhalpha double knockouts. After gonadectomy, 50% of cyclin D2, Inhalpha double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin alpha double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhalpha single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhalpha knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.     

Activin induces x-zone apoptosis that inhibits luteinizing hormone-dependent adrenocortical tumor formation in inhibin-deficient mice

Inhibin and activin are members of the transforming growth factor beta (TGF-beta) family of ligands produced and secreted primarily by the gonads and adrenals. Inhibin-null (INH(-/-)) mice develop gonadal tumors and-when gonadectomized-adrenocortical carcinoma. The mechanisms leading to adrenal tumorigenesis have been proposed to involve the lack of a gonadal factor and/or a compensatory increase in gonadotropins. In order to achieve elevation of gonadotropins without the concomitant loss of a gonadal hormone, we crossed INH(-/-) mice with a transgenic mouse strain that has chronically elevated luteinizing hormone (LH) levels (LH-CTP). Compound INH(-/-)-LH-CTP mice die within 6 weeks of age from severe cancer cachexia induced by large, activin-secreting ovarian tumors. Unexpectedly, INH(-/-)-LH-CTP mice not only fail to develop adrenal tumors but have smaller adrenals, with a regressed x zone, indicating that elevated LH levels are not sufficient to induce adrenal tumor formation. However, following gonadectomy, INH(-/-)-LH-CTP mice develop large, sex steroid-producing adrenal tumors that arise from the x zone, indicating a growth-promoting effect of high levels of LH on the adrenal cortex in the absence of ovarian tumors. In addition, in vivo and in vitro data indicate that activin induces apoptosis specifically in the adrenal x zone. The restricted expression of activin receptor subunits and Smad2 in cells of the adrenal x zone, together with the elevated activin levels in INH(-/-)-LH-CTP mice, supports the conclusion that activin inhibits adrenal tumor growth by inducing x-zone regression.     

Inhibin and p27 interact to regulate gonadal tumorigenesis

Tumor suppressors function as antiproliferative signaling proteins, and defects in these genes lead to uncontrolled cell proliferation and cancer. For example, absence of the tumor suppressor p27(Kip1), a cyclin-dependent kinase inhibitor (CKI), results in increased body size, hyperplasia of several organs including the testes, and cancer in mice. Similarly, lack of inhibins, alpha/beta heterodimeric members of the transforming growth factor-beta (TGFbeta) superfamily, causes testicular and ovarian tumors of the granulosa/Sertoli cell lineage beginning at 4 weeks of age and adrenal tumors in gonadectomized mice. Neither the cell cycle alterations in the absence of inhibin nor the cause of the increased testis size in the p27 knockout mice is known. To study the molecular (cell cycle) changes that result from absence of inhibins, we analyzed the regulation of cell cycle proteins in gonadal tumors derived from inhibin alpha knockout mice (Inha(-/-)). Northern blot analyses demonstrate that cyclin-dependent kinase 4 (Cdk4) and cyclin D2 mRNA levels are elevated, and immunohistochemistry shows that p27 protein levels are decreased in both ovarian and testicular tumors from Inha(-/-) mice. These findings suggest that increased Cdk4/cyclin D2 (positive) activity and decreased p27 (negative) activity is causal for gonadal tumor formation. To test this hypothesis, we generated double mutant mice lacking both p27 and inhibin alpha to determine whether the tumor suppressors p27 and inhibin have additive suppressor activity in the gonads. Like Inha(-/-) mice, p27(-/-)Inha(-/-) mice demonstrate elevated serum activin levels, ovarian and testicular tumors, and a resultant lethal cachexia-like syndrome. However, whereas 95% of the Inha(-/-) female mice die by 18 weeks of age, 100% of the p27(-/-)Inha(-/-) female mice are dead by 8 weeks. Similarly, 95% of the Inha(-/-) single mutant males die by 13 weeks while 100% of the p27(-/-)Inha(-/-) male mice die by 10 weeks. Moreover, tumor foci in p27(-/-)Inha(-/-) mice can be observed as early as 2 weeks of age in males and as early as 4 weeks in females. These findings demonstrate that absence of both inhibin and p27 in mice causes earlier development of ovarian and testicular tumors and earlier death compared with absence of inhibin alone.     

Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.     

Changes in the reproductive functions of mice due to injection of a plasmid expressing an inhibin alpha-subunit into muscle: a transient transgenic model

Inhibin is a gonadal hormone composed of an a-subunit and one of two beta-subunits (betaA, betaB), and its primary role is to inhibit FSH secretion by the pituitary. To investigate the roles of inhibin alpha in the reproductive system, an expression plasmid, pCMV-rINA, with the rat inhibin alpha cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Inhibin alpha mRNA was detected in the muscle by RT-PCR and Southern blot analysis. Inhibin protein was also detected, and Western blot analysis revealed a relatively high level of serum inhibin, but not of activin betaA. The estrous cycle of the pCMV-rINA-injected mice was extended, but there was no change in levels of pituitary FSH mRNA or serum FSH and no ovarian cysts were observed. When injected female mice were mated with males of proven fertility, litter size increased. Surprisingly, the embryos of pregnant females injected with pCMV-rINA, were retarded in growth and had defects in internal organs. When male mice were injected, testicle weight increased slightly without any noticeable change in the histology of the seminiferous tubules. Taken together, our data indicate that the inhibin alpha subunit influences a number of the reproductive functions of female mice.     

Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta

SMAD3 is one of the intracellular mediators that transduces signals from transforming growth factor-beta (TGF-beta) and activin receptors. We show that SMAD3 mutant mice generated by gene targeting die between 1 and 8 months due to a primary defect in immune function. Symptomatic mice exhibit thymic involution, enlarged lymph nodes, and formation of bacterial abscesses adjacent to mucosal surfaces. Mutant T cells exhibit an activated phenotype in vivo, and are not inhibited by TGF-beta1 in vitro. Mutant neutrophils are also impaired in their chemotactic response toward TGF-beta. Chronic intestinal inflammation is infrequently associated with colonic adenocarcinoma in mice older than 6 months of age. These data suggest that SMAD3 has an important role in TGF-beta-mediated regulation of T cell activation and mucosal immunity, and that the loss of these functions is responsible for chronic infection and the lethality of Smad3-null mice.     

Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

Background  

Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA.     

Genetic removal of Smad3 from inhibin-null mice attenuates tumor progression by uncoupling extracellular mitogenic signals from the cell cycle machinery

Inhibin and activin are members of the TGFbeta family that perform mutually antagonistic signaling roles in the anterior pituitary, gonads, and adrenal gland. Unopposed activin signaling in inhibin-null (Inha-/-) mice causes the formation of granulosa cell tumors in the gonads and adrenal cortex, which depend upon FSH for efficient growth and progression. In this study, we demonstrate that Smad3, a key effector of activin signaling, is expressed at high levels and is constitutively activated in tumors from these mice. Removal of Smad3 from Inha-/- mice by a genetic cross to Smad3-null (Madh3-/-) mice leads to a significant decrease in cyclinD2 expression and a significant attenuation of tumor progression in the gonads and adrenal. The decrease in cyclinD2 levels in compound knockout mice is related to a reduction in mitogenic signaling through the phosphoinositide-3-kinase (PI3-kinase)/Akt pathway, which is required for normal cell cycle progression in tumor cells. Loss of PI3-kinase/Akt signaling cannot be attributed to alterations in IGF expression, suggesting instead that signaling through the FSH receptor is attenuated. Gene expression profiling in the ovaries of Madh3-/- and Inha-/-:Madh3-/- compound knockout mice supports this hypothesis and further suggests that Smad3 is specifically required for FSH to activate PI3-kinase/Akt, but not protein kinase A. Together these observations imply that activin/Smad3 signaling is necessary for efficient signaling by FSH in Inha-/- tumor cells and that interruption of this pathway uncouples FSH from its intracellular mitogenic effectors.     

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA)

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.     

Interrelationship of growth differentiation factor 9 and inhibin in early folliculogenesis and ovarian tumorigenesis in mice

To investigate the interrelationship of inhibin alpha and growth differentiation factor 9 (GDF9) during early folliculogenesis, we generated mice lacking both inhibin alpha and GDF9. Our findings on these Inha Gdf9 double-mutant mice are as follows: 1). females develop ovarian tumors and a cachexia-like wasting syndrome, resembling mice lacking inhibin alpha alone. This indicates that the granulosa cells are competent to proliferate despite the lack of GDF9; 2). follicular development progresses to multiple-layer follicle stages before tumorigenesis. This demonstrates that the up-regulation of inhibin alpha in the Gdf9 knockout ovary directly prevents the proliferation of the granulosa cells at the primary follicle stage, an effect that is released in the absence of inhibin alpha; 3). a morphological theca forms around the preantral follicles with no detectable selective theca markers [i.e. 17alpha-hydroxylase (Cyp17), LH receptor (Lhr), and Kit]. These results indicate that the theca recruitment can occur independently of GDF9, but the differentiation of thecal cells is blocked; and 4). inhibin/activin subunits betaA, betaB, and Kit ligand (Kitl) mRNA are highly up-regulated, suggesting that the increased activins and KITL play functional roles in early folliculogenesis. Thus, GDF9 appears to function indirectly to regulate early granulosa cell proliferation and theca recruitment in vivo.     

The rasGAP-binding protein, Dok-1, mediates activin signaling via serine/threonine kinase receptors

Activins, members of the transforming growth factor-beta family, are pleiotropic growth and differentiation factors. Activin A induces B-cell apoptosis. To identify the genes responsible for activin-induced apoptosis, we performed retrovirus-mediated gene trap screening in a mouse B-cell line. We identified the rasGAP-binding protein Dok-1 (p62) as an essential molecule that links activin receptors with Smad proteins. In B cells overexpressing Dok-1, activin A-induced apoptotic responses were augmented. The expression of bcl-X(L) was down-regulated by inhibition of the ras/Erk pathway. Activin stimulation triggered association of Dok-1 with Smad3, as well as association of Smad3 with Smad4. Dok-1 also associated with both the type I and type II activin receptors. Dok-1 has been characterized previously as a tyrosine-phosphorylated protein acting downstream of the protein tyrosine kinase pathway: intriguingly, activin signaling did not induce tyrosine phosphorylation of Dok-1. These findings indicate that Dok-1 acts as an adaptor protein that links the activin receptors with the Smads, suggesting a novel function for Dok-1 in activin signaling leading to B-cell apoptosis.     

Effects of activin and TGFβ on p21 in colon cancer

Activin and TGFβ share SMAD signaling and colon cancers can inactivate either pathway alone or simultaneously. The differential effects of activin and TGFβ signaling in colon cancer have not been previously dissected. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21 (p21(cip1/waf1)). Here, we evaluate activin-specific effects on p21 regulation and resulting functions. We find that TGFβ is a more potent inducer of growth suppression, while activin is a more potent inducer of apoptosis. Further, growth suppression and apoptosis by both ligands are dependent on SMAD4. However, activin downregulates p21 protein in a SMAD4-independent fashion in conjunction with increased ubiquitination and proteasomal degradation to enhance migration, while TGFβ upregulates p21 in a SMAD4-dependent fashion to affect growth arrest. Activin-induced growth suppression and cell death are dependent on p21, while activin-induced migration is counteracted by p21. Further, primary colon cancers show differential p21 expression consistent with their ACVR2/TGFBR2 receptor status. In summary, we report p21 as a differentially affected activin/TGFβ target and mediator of ligand-specific functions in colon cancer, which may be exploited for future risk stratification and therapeutic intervention.     

Regulable expression of inhibin A in wild-type and inhibin alpha null mice

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 (alpha/beta) wherein one of its subunits (beta) is capable of homodimerizing to form its physiological antagonist, activin (beta/beta). Inhibin is also expressed in two forms, A and B, as determined by the subtype of beta-subunit that dimerizes with the alpha-subunit (alpha/betaA or alpha/betaB). 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 polio-virus IRES (internal ribosome entry site)-linked sequences coding for the alpha- and beta-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 liver-derived inhibin A was secreted into the serum to down-regulate pituitary FSH levels. Furthermore, the generation of bigenic mice in the inhibin alpha null background allowed for the induction of inhibin A in inhibin alpha 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.     

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.