Smad3-deficient Mice Research Articles

Smad signalling in the ovary

It has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGF-beta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.

Helicobacter Infection Is Required for Inflammation and Colon Cancer in Smad3-Deficient Mice

Accumulating evidence suggests that intestinal microbial organisms may play an important role in triggering and sustaining inflammation in individuals afflicted with inflammatory bowel disease (IBD). Moreover, individuals with IBD are at increased risk for developing colorectal cancer, suggesting that chronic inflammation may initiate genetic or epigenetic changes associated with cancer development. We tested the hypothesis that bacteria may contribute to the development of colon cancer by synergizing with defective transforming growth factor-beta (TGF-beta) signaling, a pathway commonly mutated in human colon cancer. Although others have reported that mice deficient in the TGF-beta signaling molecule SMAD3 develop colon cancer, we found that SMAD3-deficient mice maintained free of the Gram-negative enterohepatic bacteria Helicobacter spp. for up to 9 months do not develop colon cancer. Furthermore, infection of SMAD3(-/-) mice with Helicobacter triggers colon cancer in 50% to 66% of the animals. Using real-time PCR, we found that Helicobacter organisms concentrate in the cecum, the preferred site of tumor development. Mucinous adenocarcinomas develop 5 to 30 weeks after infection and are preceded by an early inflammatory phase, consisting of increased proliferation of epithelial cells; increased numbers of cyclooxygenase-2-positive cells, CD4(+) T cells, macrophages; and increased MHC class II expression. Colonic tissue revealed increased transcripts for the oncogene c-myc and the proinflammatory cytokines interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IFN-gamma, and tumor necrosis factor-alpha, some of which have been implicated in colon cancer. These results suggest that bacteria may be important in triggering colorectal cancer, notably in the context of gene mutations in the TGF-beta signaling pathway, one of the most commonly affected cellular pathways in colorectal cancer in humans.

Adenoviral Gene Transfer Allows Smad-Responsive Gene Promoter Analyses and Delineation of Type I Receptor Usage of Transforming Growth Factor-β Family Ligands in Cultured Human Granulosa Luteal Cells

In the human ovary, cell growth and differentiation are regulated by members of the TGF-beta superfamily, including growth differentiation factor-9 (GDF9), TGF-beta, and activin. TGF-beta and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-beta, and activin responses. In rodent granulosa cells, TGF-beta and GDF9 signal through the TGF-beta type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase reporter activity. A similar response to Alk5 overexpression was observed for TGF-beta, but not for activin. Adenoviral overexpression of the activin type IB receptor Alk4 in hGL cells specifically potentiated activin signaling, but not GDF9 or TGF-beta signaling. Alk5 overexpression in hGL cells also potentiated the GDF9 response when inhibin B production was used as the read-out. These results indicate that the CAGA-luciferase adenovirus can be used to study Smad3 signaling in primary cultures of human cells, and that adenoviral overexpression of wild-type receptors of the TGF-beta superfamily can be used to amplify the cellular response to ligands such as GDF9, TGF-beta, and activin. Furthermore, these studies indicate the involvement of Alk5 in GDF9 signaling in human cells and therefore, along with other recent studies, highlight how a limited number of type I and II receptors cooperate to generate specificity of action within the TGF-beta superfamily.

Ovarian follicle development requires Smad3.

Smad3 is an important mediator of the TGF beta signaling pathway. Interestingly, Smad3-deficient (Smad3-/-) mice have reduced fertility compared with wild-type (WT) mice. To better understand the molecular mechanisms underlying the reduced fertility in Smad3-/- animals, this work tested the hypothesis that Smad3 deficiency interferes with three critical aspects of folliculogenesis: growth, atresia, and differentiation. Growth was assessed by comparing the size of follicles, expression of proliferating cell nuclear antigen, and expression of cell cycle genes in Smad3-/- and WT mice. Atresia was assessed by comparing the incidence of atresia and expression of bcl-2 genes involved in cell death and cell survival in Smad3-/- and WT mice. Differentiation was assessed by comparing the expression of FSH receptor (FSHR), estrogen receptor (ER) alpha, ER beta, and inhibin alpha-, beta(A)-, and beta(B)-subunits in Smad3-/- and WT mice. Because growth, atresia, and differentiation are regulated by hormones, estradiol, FSH, and LH levels were compared in Smad3-/- and WT mice. Moreover, because alterations in folliculogenesis can affect the ability of mice to ovulate, the number of corpora lutea and ovulated eggs in response to gonadotropin treatments were compared in Smad3-/- and WT animals. The results indicate that Smad3 deficiency slows follicle growth, which is characterized by small follicle diameters, low levels of proliferating cell nuclear antigen, and low expression of cell cycle genes (cyclin-dependent kinase 4 and cyclin D2). Smad3 deficiency also causes atretic follicles, degenerated oocytes, and low expression of bcl-2. Furthermore, Smad3 deficiency affects follicular differentiation as evidenced by decreased expression of ER beta, increased expression of ER alpha, and decreased expression of inhibin alpha-subunits. Smad3 deficiency causes low estradiol and high FSH levels. Finally, Smad3-/- ovaries have no corpora lutea, and they do not ovulate after ovulatory induction with exogenous gonadotropins. Collectively, these data provide the first evidence that reduced fertility in Smad3-/- mice is due to impaired folliculogenesis, associated with altered expression of genes that control cell cycle progression, cell survival, and cell differentiation. The findings that Smad3-/- follicles have impaired growth, increased atresia, and altered differentiation in the presence of high FSH levels, normal expression of FSHR, and lower expression of cyclin D2, suggest a possible interaction between Smad3 and FSH signaling downstream of FSHR in the mouse ovary.

Essential role for Smad3 in regulating MCP-1 expression and vascular inflammation.

Transforming growth factor (TGF)-beta(1) is a pleiotropic growth factor with known inhibitory effects on immune cell activation. However, the specific mechanism(s) and in vivo significance of the effectors of TGF-beta(1) modulation in the context of vascular inflammation are not well characterized. The chemokine monocyte chemoattractant protein (MCP)-1 is critical for the recruitment of macrophages in inflammatory disease states. In this study, we provide definitive evidence that the ability of TGF-beta(1) to inhibit MCP-1 expression is mediated via its effector Smad3. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous MCP-1. Conversely, TGF-beta(1) inhibition of cytokine-mediated induction of MCP-1 expression was completely blocked in Smad3-deficient macrophages. Consistent with this impaired response, cardiac allografts in Smad3-deficient mice developed accelerated intimal hyperplasia with increased infiltration of adventitial macrophages expressing MCP-1. Previous studies show that MCP-1 inducibility is regulated by an AP-1 complex composed of c-Jun/c-Fos heterodimers. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on AP-1 DNA-protein binding and activity. Thus, Smad3 plays an essential role in modulating vascular inflammation characteristic of transplant-associated arteriopathy, is important in regulating MCP-1 expression, and plays a critical role in the ability of TGF-beta(1) to repress stimuli from a major inflammatory signaling pathway.

Functional analysis of the TGFbeta receptor/Smad pathway through gene ablation in mice.

During recent years, our understanding of TGFbeta signalling through serine/threonine kinase receptors and Smads has increased enormously. Activation of R-Smads by receptor induced phosphorylation is followed by complex formation with co-Smads and translocation to the nucleus, where the transcription of specific genes is affected and ultimately results in changes in cell behaviour. Experimental analysis primarily of epithelial cells in culture has revealed that a number of members of the TGFbeta family are interchangeable in the effect they have on growth and differentiation. On the other hand, different ligands of the TGFbeta superfamily can result in different responses because of cell type specific expression of other components of the signalling pathway. The relative expression levels of receptors and Smads within the cell is an important determinant of TGFbeta induced responses. Functional analysis of genes in the TGFbeta superfamily signal transduction cascade in vivo in mice either lacking entire genes, or expressing dominant negative forms of particular proteins, are providing profound new insights into the signalling cascades, their interaction and their specificity (Table 3). For example, by phenotypical comparison and intercrossing different heterozygous mutants, it has become clear that nodal, until recently an orphan protein without receptor/signal complex, probably signals through the activin type II receptor, ALK-4 and Smad2 (Nomura and Li, 1998; Song et al., 1999). Many of the genes of this cascade that have been targeted in the mouse result in early embryonic lethal phenotypes, demonstrating an important function for the BMP and TGFbeta/activin-activated pathways in mesoderm formation and differentiation, but masking a possible role in later events. For example mutations in BMP2 and 4 are lethal at or soon after gastrulation so that their putative role in skeletogenesis cannot be studied in mice lacking these genes. The difference in severity of the phenotypes between ligand, receptor and Smad deficient mice suggest that other receptors and ligands may partially compensate for the loss of one protein. Chimeric analysis provides one tool for analysing later developmental functions. By rescuing the early defects it was demonstrated that TGFbeta family members have an important function in anterior development and left/right asymmetry. Temporal and spatial specific gene targeting will be a powerful tool for analysing the function of TGFbeta family members in for example, bone formation, angiogenesis and carcinogenesis. Isolation of cells from the different gene targeted mice provides a unique source of material to gain more insight in the biochemical mechanisms of specific pathways. For example, use of cells deficient in Smad2 for biochemical and cell biological assays could give a better view of the function of Smad3. Smad3 deficient mice already demonstrate that there is a clear difference between Smad2 and Smad3 during development. Full descriptions of the remaining gene ablation studies of this signal transduction cascade, namely those for ALK-5, BMPR-II and Smad1 and -7 are eagerly awaited to complete the puzzle. As more of these superfamily of ligands and their signalling pathways have been functionally dissected, it has become evident that this superfamily of growth factors plays a pivotal role in epiblast formation and gastrulation, signalling from both the epiblast as well as the extraembryonic tissues. Furthermore, it becomes clear that TGFbeta is indeed important for proper vessel formation and that it might use endoglin, as well as ALK-1, ALK-5 and Smad5 to mediate this function. Further analyses of these mice should provide a clearer understanding of the mechanism of TGFbeta action in vascular development and remodelling.