Ligand-receptor interactions and signaling cross-talk in the TGF-b superfamily

The transforming growth factor-beta (TGF-beta) superfamily of secreted polypeptide growth factors forms part of an evolutionary conserved signaling pathway, present in diverse organisms from flies to humans, used in intercellular communication. The TGF-beta family comprises more than 40 members in vertebrates, Caenorhabditis elegans and Drosophila melanogaster. Based on sequence similarities, this family can be divided into three main subfamilies, the TGF-betas, the activins, and the bone morphogenetic proteins (BMPs). Members of this family regulate a wide number of events during embryonic development and in adult life, including proliferation, apoptosis, differentiation and cell fate.

The members of the TGF-beta family transmit signals through a receptor complex on the cell surface, containing two types (type I and II) of transmembrane receptor serine/threonine kinases (RSTKs). The receptors transmit the signal to a class of intracellular signaling intermediates, the Smad proteins, which then translocate to the nucleus where they interact with DNA and other transcription factors to regulate gene transcription.

The aim of this thesis is to identify components involved in TGF-beta superfamily signal transduction, with special focus on molecules involved in signaling through the receptor activin receptor-like kinase 7 (ALK7). ALK7 is a type I receptor with a specific expression pattern in the nervous system, pancreas and brown fat. At the time the present work commenced, the cognate ligand, type II receptor, and downstream signaling pathways of ALK7 were unknown.

Smad2 and Smad3 were initially established as intracellular mediators of ALK7. We then identified Xenopus Nodal-related 1 (Xnr1) and mouse Nodal proteins as ligands for ALK7, in collaboration with the type II receptor, ActRIlB, and the co-receptor, Cripto. Nodal proteins have instructive roles in mesendoderm formation and left-right patterning during vertebrate development, for which no receptor or downstream signal transduction mechanism were known.

In search for novel interactors of Smad proteins, we discovered direct protein-protein interaction events between Smads and GATA-3, the Notch intracellular domain (NICD), and Phox2. GATA-3 is a zinc-finger transcription factor with a specific expression pattern in different neuronal subtypes and in the immune system, where it is an important regulator of T helper cell development. In particular, we have shown that TGF-beta upregulates interleukin-10 expression, in T helper cells in a GATA-3 dependent manner. The conserved Notch signal transduction pathway controls cell fate decisions in many different cell types, such as for example neural stem cells. Synergistic interactions between NICD and Smad3 were found to regulate the expression of Hes-1, a transcription factor known to be a direct target of the Notch pathway.

By blocking Notch function using a dominant negative form of CSL, a DNAbinding co-factor of Notch, we could inhibit the ability of TGF-beta to upregulate Hes-1, demonstrating a direct link between the two pathways. The Phox2 proteins are homeodomain transcription factors with specific expression pattern in noradrenergic neurons, where they function as key regulators of the dopamine beta hydroxylase (DBH) gene. Both TGF-beta and BMP signaling has been implicated in DBH expression and we could show that this can in part be mediated through a direct interaction between Smads and Phox2 proteins.

In conclusion this thesis work has characterized a signal transduction pathway from receptor-ligand interactions to transcriptional regulation, and the cross-talk with other signaling pathways.

List of scientific papers

I. Jornvall H, Blokzijl A, ten Dijke P, Ibanez CF (2001). The orphan receptor serine/threonine kinase ALK7 signals arrest of proliferation and morphological differentiation in a neuronal cell line. J Biol Chem. 276(7): 5140-6.
https://pubmed.ncbi.nlm.nih.gov/11084022

II. Reissmann E, Jornvall H, Blokzijl A, Andersson O, Chang C, Minchiotti G, Persico MG, Ibanez CF, Brivanlou AH (2001). The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. Genes Dev. 15(15): 2010-22.
https://pubmed.ncbi.nlm.nih.gov/11485994

III. Blokzijl A, ten Dijke P, Ibanez CF (2002). Physical and functional interaction between GATA-3 and Smad3 allows TGF-beta regulation of GATA target genes. Curr Biol. 12(1): 35-45.
https://pubmed.ncbi.nlm.nih.gov/11790301

IV. Blokzijl A, Reissmann E, Ibánez CF (2003). Physical and functional interactions between Smad proteins and Phox2 homeodomain transcription factors. [Manuscript]

V. Blokzijl A, Oberg C, Reissmann E, Falk A, Moliner A, Lendahl U, Ibánez CF (2003). Cross-talk between the Notch and TGF-beta signaling pathways mediated by interaction of the Notch intracellular domain with Smad3. [Submitted]