Nodal Signaling Uses Activin and Transforming Growth Factor-β Receptor-regulated Smads

Abstract

Nodal, a member of the transforming growth factor ␤ (TGF-␤) superfamily, is implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Here we define the intracellular signaling pathway induced by recombinant nodal protein treatment of P19 embryonal carcinoma cells. Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-␤. However, nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. We also demonstrate that nodal induces p(CAGA) 12 , a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduces the level of luciferase reporter activity induced by nodal treatment. Finally, we show that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide the first direct biochemical evidence that nodal signaling is mediated by both activin-TGF-␤ pathway Smads, Smad2 and Smad3. We also show here that the extracellular cripto protein is required for nodal signaling, making it distinct from activin or TGF-␤ signaling. Members of the transforming growth factor ␤ (TGF-␤)1 superfamily of intercellular signaling factors regulate cell fate and behavior during development and in the adult (1). The three major subgroups based on sequence similarity are the TGF-␤s, activins and inhibins, and bone morphogenetic proteins (BMPs; Ref. 1). Nodal and related factors form a separate subgroup and are implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification (2).Signaling by TGF-␤ and related ligands uses two types of receptors, type I and type II transmembrane serine-threonine kinases. Ligand binding results in the formation of heteromeric receptor complexes, in which type II receptors phosphorylate type I receptors (1, 3). Downstream signal transduction events are mediated by the intracellular Smad proteins. One class, the receptor-regulated Smads (R-Smads), are directly phosphorylated by activated type I receptors on a C-terminal SSXS motif. Upon phosphorylation, R-Smads form complexes with the coSmad, Smad4 and then translocate to the nucleus and regulate transcription of target genes. Biochemical and biological studies have established that the R-Smads used by TGF-␤ and activin signaling, Smad2 and Smad3, are distinct from those used by BMP signaling, Smad1, Smad5, and Smad8 (1, 3, 4).The nodal signaling pathway awaits characterization at the biochemical level. However, mutational studies in the mouse and zebrafish and ectopic expression studies in Xenopus and zebrafish suggest that the nodal and activin signaling pathways may share receptors and Smads. Targeted mutations in the mouse Smad2 gene (5-8) and the activin type IB receptor gene (9) and combined mutations of the activin type IIA and IIB receptor genes (10) show gastrulation phenotypes resem...

Nodal enhances the activity of FoxO3a and its synergistic interaction with Smads to regulate cyclin G2 transcription in ovarian cancer cells

“…The canonical Nodal signaling resembles the TGF-b/ Smad pathway (Kumar et al, 2001;Derynck and Zhang, 2003;Massague et al, 2005). Nodal binds to a membrane serine/threonine kinase receptor complex consisting of type I (ALK4 or ALK7) and type II (ActRIIA or ActRIIB) serine/theronine kinase receptors to activate Smad2 and Smad3 (Kumar et al, 2001;Reissmann et al, 2001).…”

“…Nodal binds to a membrane serine/threonine kinase receptor complex consisting of type I (ALK4 or ALK7) and type II (ActRIIA or ActRIIB) serine/theronine kinase receptors to activate Smad2 and Smad3 (Kumar et al, 2001;Reissmann et al, 2001). The phosphorylated Smad2/3 proteins bind to Smad4 and subsequently undergo nuclear translocation, allowing for the assembly of active transcription complexes on the promoter of Nodal target genes (Schier, 2003;Massague et al, 2005).…”

Nodal and ALK7 Inhibit Proliferation and Induce Apoptosis in Human Trophoblast Cells

“…First, Nodal can bind to the activin receptor complex containing activin receptors IIB and IB (ALK4) (19,23,24). Signaling of Nodal through this receptor complex requires Cripto since, in the absence of Cripto, Nodal loses its signaling activity through the activin receptor complex (19,(23)(24)(25). The other receptor complex that mediates Nodal action is activin receptor IIB-ALK7 (19).…”

“…Since ALK7 has been shown to mediate the effect of Nodal in mesoderm induction (19) and to activate Smad2 and Smad3 (23,25), we also examined mRNA expression of Nodal, Smad2, and Smad3. Similar to our previous studies that showed that Nodal mRNA is expressed in JEG-3 cells and in normal placenta (12), RT-PCR detected Nodal mRNA in JEG-3, JAR, JAR-Smad3/c, and HTR8/SVneo cells (Fig.…”

NODAL signaling components regulate essential events in the establishment of pregnancy

“…The EGF-CFC co-receptor is essential for NODAL signaling by recruiting the convertases to the receptor complex site and facilitating NODAL binding to the ACVR1B/ACVR1C type I receptor (Blanchet et al 2008). Upon receptor complex formation, ACVR2A/ACVR2 phosphorylates ACVR1B/ACVR1C, which in turn phosphorylates the intracellular signal transducers SMAD2 or SMAD3 (Kumar et al 2001). SMAD2/3 then interacts with SMAD4 and additional transcription factors in the nucleus (e.g.…”