SMAD3 regulates gonadal tumorigenesis

Inhibin is a secreted tumor suppressor and an activin antagonist. Inhibin alpha null mice develop gonadal sex cord-stromal tumors with 100% penetrance and die of a cachexia-like syndrome due to increased activin signaling. Because Sma and Mad-related protein (SMAD)2 and SMAD3 transduce activin signals in vitro, we attempted to define the role of SMAD3 in gonadal tumorigenesis and the wasting syndrome by generating inhibin alpha and Smad3 double mutant mice. Inhibin alpha and Smad3 double homozygous males were protected from early tumorigenesis and the usual weight loss and death. Approximately 90% of these males survived to 26 wk in contrast to 95% of inhibin-deficient males, which develop bilateral testicular tumors and die of the wasting syndrome by 12 wk. Testicular tumors were either absent or unilaterally slow growing and less hemorrhagic in the majority of double-knockout males. In contrast, development of the ovarian tumors and wasting syndrome was delayed, but still occurred, in the majority of the double-knockout females by 26 wk. In double mutant females, tumor development was accompanied by typical activin-induced pathological changes. In summary, we identify an important function of SMAD3 in gonadal tumorigenesis in both sexes. However, this effect is significantly more pronounced in the male, indicating that SMAD3 is the primary transducer of male gonadal tumorigenesis, whereas SMAD3 potentially overlaps with SMAD2 function in the ovary. Moreover, the activin-induced cachexia syndrome is potentially mediated through both SMAD2 and SMAD3 or only through SMAD2 in the liver and stomach. These studies identify sexually dimorphic functions of SMAD3 in gonadal tumorigenesis.     

Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression

Ovarian germ cell survival is dependent upon the formation of primordial follicles, which occurs during fetal life in the human. Activin contributes to germ cell proliferation and survival at this time. SMADs2 and 3 are central elements in the activin signalling pathway and thus indicate sites of activin action. We have investigated the expression and localisation of SMADs2 and 3 in the fetal ovary between 14 and 20 weeks gestation, i.e. preceding and during primordial follicle formation. SMAD3 mRNA expression increased 1.9 fold (P = 0.02). SMAD2 and 3 proteins were localised by immunofluorescence to the nuclei of three distinct populations of somatic cells: (a) stromal cells between clusters of germ cells; (b) some somatic cells intermingled with activin βA-expressing germ cells; (c) pre-granulosa cells surrounding primordial follicles. Germ cells did not express SMAD2 or 3. Activin A increased and follistatin decreased phosphorylation of SMAD2/3 in vitro, and activin increased SMAD2 and decreased KITLG mRNA expression. It therefore appears that somatic cells are the targets for activin signalling in the developing ovary. The effects of activin on germ cells are indirect and include mediation by the kit ligand/c-Kit pathway, rather than being an autocrine germ cell effect.     

Disruption of postnatal folliculogenesis and development of ovarian tumor in a mouse model with aberrant transforming growth factor beta signaling

Background

Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes.

Methods

A mouse model containing constitutively active TGFBR1 (TGFBR1^CA) using Gdf9-iCre (termed TGFBR1-CA^G9Cre) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CA^G9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3’-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumor tissues.

Results

TGFBR1-CA^G9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CA^G9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues.

Conclusions

These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.

     

Activin A induces neuronal differentiation and survival via ALK4 in a SMAD-independent manner in a subpopulation of human neuroblastomas

Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-β superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine β-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-β target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-β target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.     

Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid

Background  

The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model.     

Role of inhibin and activin in the modulation of gonadotropin- and steroid-induced oocyte maturation in the teleost Fundulus heteroclitus

Background  

Activin and inhibin are glycoproteins structurally related to the transforming growth factor-beta superfamily. These peptides were first described as factors that regulate the follicle-stimulating hormone (FSH) at the pituitary level. The possible role of inhibin and activin, at the ovarian level, in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) and 17alpha,20beta-dihydroprogesterone (DHP) on oocyte maturation was investigated in this study.     

SMAD7, an antagonist of TGF-beta signaling,is a candidate of prenatal skeletal muscle development and weaning weight in pigs

SMAD7 promotes and enhances skeletal muscle differentiation by inhibiting transforming growth factor beta (TGF-β)/activin signaling and bone morphogenetic protein (BMP) pathways. However, its function, the mechanism regulating its translation, and its association with production meat traits remain unclear in pigs. In this study, we explored SMAD7 gene spatio-temporal and tissue distribution, conducted a single nucleotide polymorphism association analysis, and examined regulation of its expression during skeletal muscle development. We found that SMAD7 was positively related to TGF-β pathway genes and mainly expressed in prenatal developing muscle, and dual luciferase and western blot assays demonstrated that SMAD7 expression was regulated by miRNA-21 at the protein level via inhibition of mRNA translation. Finally, the association analysis showed that a single nucleotide mutation (Exon 4_28816;C/A) was significantly associated with the weaning weight of piglets among Yorkshire pigs. These data indicate that SMAD7 plays a potentially important role in mammalian prenatal skeletal muscle development and is a candidate gene for promoting greater weaning weight in pig breeding.     

The Specificity of the FOXL2 c.402C>G Somatic Mutation: A Survey of Solid Tumors

Background

A somatic mutation in the FOXL2 gene is reported to be present in almost all (97%; 86/89) morphologically defined, adult-type, granulosa-cell tumors (A-GCTs). This FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W). It was also found in a minority of other ovarian malignant stromal tumors, but not in benign ovarian stromal tumors or unrelated ovarian tumors or breast cancers.

Methodology/Principal Findings

Herein we studied other cancers and cell lines for the presence of this mutation. We screened DNA from 752 tumors of epithelial and mesenchymal origin and 28 ovarian cancer cell lines and 52 other cancer cell lines of varied origin. We found the FOXL2 c.402C>G mutation in an unreported A-GCT case and the A-GCT-derived cell line KGN. All other tumors and cell lines analyzed were mutation negative.

Conclusions/Significance

In addition to proving that the KGN cell line is a useful model to study A-GCTs, these data show that the c.402C>G mutation in FOXL2 is not commonly found in a wide variety of other cancers and therefore it is likely pathognomonic for A-GCTs and closely related tumors.