Etienne Labbé

Smad2 and Smad3 Positively and Negatively Regulate TGFβ-Dependent Transcription through the Forkhead DNA-Binding Protein FAST2

We identify a mammalian forkhead domain protein, FAST2, that is required for induction of the goosecoid (gsc) promoter by TGF beta or activin signaling. FAST2 binds to a sequence in the gsc promoter, but efficient transcriptional activation and assembly of a DNA-binding complex of FAST2, Smad2, and Smad4 requires an adjacent Smad4 site. Smad3 is closely related to Smad2 but suppresses activation of the gsc promoter. Inhibitory activity is conferred by the MH1 domain, which unlike that of Smad2, binds to the Smad4 site. Through competition for this shared site, Smad3 may prevent transcription by altering the conformation of the DNA-binding complex. Thus, we describe a mechanism whereby Smad2 and Smad3 positively and negatively regulate a TGF beta/activin target gene.

Transcriptional Cooperation between the Transforming Growth Factor-β and Wnt Pathways in Mammary and Intestinal Tumorigenesis

Transforming growth factor-beta (TGF-beta) and Wnt ligands function in numerous developmental processes, and alterations of both signaling pathways are associated with common pathologic conditions, including cancer. To obtain insight into the extent of interdependence of the two signaling cascades in regulating biological responses, we used an oligonucleotide microarray approach to identify Wnt and TGF-beta target genes using normal murine mammary gland epithelial cells as a model. Combination treatment of TGF-beta and Wnt revealed a novel transcriptional program that could not have been predicted from single ligand treatments and included a cohort of genes that were cooperatively induced by both pathways. These included both novel and known components or modulators of TGF-beta and Wnt pathways, suggesting that mutual feedback is a feature of the coordinated activities of the ligands. The majority of the cooperative targets display increased expression in tumors derived from either Min (many intestinal neoplasia) or mouse mammary tumor virus (MMTV)-Wnt1 mice, two models of Wnt-induced tumors, with nine of these genes (Ankrd1, Ccnd1, Ctgf, Gpc1, Hs6st2, IL11, Inhba, Mmp14, and Robo1) showing increases in both. Reduction of TGF-beta signaling by expression of a dominant-negative TGF-beta type II receptor in bigenic MMTV-Wnt1/DNIIR mice increased mammary tumor latency and was correlated with a decrease in expression of Gpc1, Inhba, and Robo1, three of the TGF-beta/Wnt cooperative targets. Our results indicate that the TGF-beta and Wnt/beta-catenin pathways are firmly intertwined and generate a unique gene expression pattern that can contribute to tumor progression.

Transcriptional Regulation by Smads: Crosstalk between the TGF-β and Wnt Pathways

Background: Several studies have shown that cooperation between transforming growth factor beta (TGF-&bgr;) and Wnt/wingless signaling pathways plays a role in controlling certain developmental events. These factors elicit their biological effects through distinct pathways in which TGF-&bgr; and Wnt signaling induce activation of the transcriptional regulators Smads and lymphoid enhancer binding factor/T-cell-specific factor (LEF/TCF), respectively. To understand the mechanism for cooperativity between these pathways, we have investigated the molecular mechanism for this synergistic effect. Methods: Transcriptional assays were conducted by transient transfection of HepG2 cells with use of luciferase reporter constructs. Protein/protein interaction studies were conducted in vitro with the use of glutathione-S-transferase pull-down assays and in intact cells by immunoprecipitation and immunoblotting. Results: We show that Smads physically interact with LEF1/TCF transcription factors and that specific DNA binding sites in the Xenopus twin promoter are required for synergistic activation by TGF-&bgr; and Wnt pathways. In addition, we demonstrate that TGF-&bgr;-dependent activation of LEF1/TCF target genes can occur independently of &bgr;-catenin, an essential component of the Wnt signaling pathway. Conclusions: TGF-&bgr; and Wnt signaling pathways can independently or cooperatively regulate LEF1/TCF target genes. This suggests that the cooperation between these pathways may be important for the specification of cell fates during development.

TGFβ and Wnt pathway cross-talk

Transforming growth factor-βs (TGFβ) and Wnts represent two distinct families of secreted molecules each of which utilizes different signaling pathways to elicit their biological effects. These factors regulate numerous developmental events and mutations in components of both pathways have been described in human cancers including colorectal carcinomas. Several studies have demonstrated that TGFβ and Wnt ligands can cooperate to regulate differentiation and cell fate determination by controlling gene expression patterns. In addition, their cooperation in promoting tumorigenesis in mice has been described. Here, we focus on reviewing our current understanding of the molecular mechanisms that may mediate these cooperative effects.

The transcriptional role of Smads and FAST (FoxH1) in TGFβ and activin signalling

The Smad family of proteins are critical components of the TGFbeta superfamily signalling pathway. Ligand addition induces phosphorylation of specific receptor-regulated Smads, which then form heteromeric complexes with the common mediator Smad, Smad4. This complex then translocates from the cytoplasm into the nucleus. Once there, the R-Smad/Smad4 complex interacts with a variety of DNA binding proteins and is thereby targetted to a diverse array of gene promoters. The Smad-containing DNA binding complex can then positively or negatively regulate gene expression through the recruitment of co-activators and co-repressors. Xenopus FAST (now known as FoxH1) was the first Smad DNA binding partner identified and the FoxH1 family now includes related proteins from mouse, human and Zebrafish. In all organisms examined, FoxH1 is expressed primarily during the earliest stages of development and thus FoxH1 is thought to play a critical role in mediating TGFbeta superfamily signals during these early developmental stages. Other Smad partners range from those that are ubiquitously expressed to others that are present only in specific cell types or developmental stages. Thus, it is the interaction of Smads with a wide range of specific transcriptional partners that is important for the generation of diverse biological responses to TGFbeta superfamily members.

Embryonic cortical neural stem cells migrate ventrally and persist as postnatal striatal stem cells

Embryonic cortical neural stem cells apparently have a transient existence, as they do not persist in the adult cortex. We sought to determine the fate of embryonic cortical stem cells by following Emx1IREScre; LacZ/EGFP double-transgenic murine cells from midgestation into adulthood. Lineage tracing in combination with direct cell labeling and time-lapse video microscopy demonstrated that Emx1-lineage embryonic cortical stem cells migrate ventrally into the striatal germinal zone (GZ) perinatally and intermingle with striatal stem cells. Upon integration into the striatal GZ, cortical stem cells down-regulate Emx1 and up-regulate Dlx2, which is a homeobox gene characteristic of the developing striatum and striatal neural stem cells. This demonstrates the existence of a novel dorsal-to-ventral migration of neural stem cells in the perinatal forebrain.

Foxh1 recruits Gsc to negatively regulate Mixl1 expression during early mouse development

Mixl1 is a member of the Mix/Bix family of paired‐like homeodomain proteins and is required for proper axial mesendoderm morphogenesis and endoderm formation during mouse development. Mix/Bix proteins are transcription factors that function in Nodal‐like signaling pathways and are themselves regulated by Nodal. Here, we show that Foxh1 forms a DNA‐binding complex with Smads to regulate transforming growth factor β (TGFβ)/Nodal‐dependent Mixl1 gene expression. Whereas Foxh1 is commonly described as a transcriptional activator, we observed that Foxh1‐null embryos exhibit expanded and enhanced Mixl1 expression during gastrulation, indicating that Foxh1 negatively regulates expression of Mixl1 during early mouse embryogenesis. We demonstrate that Foxh1 associates with the homeodomain‐containing protein Goosecoid (Gsc), which in turn recruits histone deacetylases to repress Mixl1 gene expression. Ectopic expression of Gsc in embryoid bodies represses endogenous Mixl1 expression and this effect is dependent on Foxh1. As Gsc is itself induced in a Foxh1‐dependent manner, we propose that Foxh1 initiates positive and negative transcriptional circuits to refine cell fate decisions during gastrulation.

Integration of Signaling Pathways Via Smad Proteins

TGF-β ligands are involved in most aspects of embryonic development and post-natal homeostasis and numerous examples of crosstalk with other signaling pathways have been described. The Smad proteins are the most extensively characterized signal transducers downstream of the TGF-β superfamily of secreted growth factors. In most cases, Smad proteins have been shown to mediate this crosstalk via the formation of DNA-bound transcriptional complexes with transducers of other pathways, thereby modifying promoter selectivity and transcriptional output. We will examine the ability of Smads to integrate the signaling output of TGF-β ligands with other signaling pathways through direct interactions with other signal transducers