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Nodal signaling uses activin and transforming growth factor-beta receptor-regulated Smads
A Kumar1, V Novoselov, A J Celeste
1Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
Nodal, a member of the transforming growth factor beta (TGF-beta) 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-beta. 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-beta 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-beta signaling.
Insights
Nodal signaling in early vertebrate development involves Smad2 and Smad3, distinct from bone morphogenetic proteins. Extracellular cripto protein is essential for this pathway, differentiating it from activin or TGF-beta signaling.
Area of Science:
- Developmental Biology
- Molecular Signaling
- Cell Biology
Background:
- Nodal, a TGF-beta superfamily member, is crucial for early vertebrate development.
- Key roles include mesoderm formation, anterior patterning, and left-right axis specification.
Purpose of the Study:
- To elucidate the intracellular signaling pathway activated by Nodal.
- To determine the specific Smad proteins and co-factors involved in Nodal signaling.
Main Methods:
- Utilized luciferase reporter assays (pAR3-Lux, pTlx2-Lux, p(CAGA)(12)) in P19 embryonal carcinoma cells.
- Employed dominant-negative Smad2 expression and assessed Smad2 phosphorylation.
- Investigated the role of extracellular cripto protein.
Main Results:
- Nodal activated reporters for activin/TGF-beta (pAR3-Lux) and Smad3 (p(CAGA)(12)), but not BMPs (pTlx2-Lux).
- Dominant-negative Smad2 inhibited Nodal-induced reporter activity.
- Nodal signaling led to rapid Smad2 phosphorylation.
- Extracellular cripto protein was required for Nodal signaling.
Conclusions:
- Nodal signaling is mediated by Smad2 and Smad3, aligning with the activin-TGF-beta pathway.
- Nodal signaling is biochemically distinct from activin or TGF-beta due to the requirement of cripto protein.