Cross-talk between the Notch and TGF-β signaling pathways mediated by interaction of the Notch intracellular domain with Smad3

The Notch and transforming growth factor-β (TGF-β) signaling pathways play critical roles in the control of cell fate during metazoan development. However, mechanisms of cross-talk and signal integration between the two systems are unknown. Here, we demonstrate a functional synergism between Notch and TGF-β signaling in the regulation of Hes-1, a direct target of the Notch pathway. Activation of TGF-β signaling up-regulated Hes-1 expression in vitro and in vivo. This effect was abrogated in myogenic cells by a dominant-negative form of CSL, an essential DNA-binding component of the Notch pathway. TGF-β regulated transcription from the Hes-1 promoter in a Notch-dependent manner, and the intracellular domain of Notch1 (NICD) cooperated synergistically with Smad3, an intracellular transducer of TGF-β signals, to induce the activation of synthetic promoters containing multimerized CSL- or Smad3-binding sites. NICD and Smad3 were shown to interact directly, both in vitro and in cells, in a ligand-dependent manner, and Smad3 could be recruited to CSL-binding sites on DNA in the presence of CSL and NICD. These findings indicate that Notch and TGF-β signals are integrated by direct protein–protein interactions between the signal-transducing intracellular elements from both pathways

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

Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay

Background The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder. Objectives To investigate SOX10 as a potential biomarker for melanoma and vitiligo. Methods In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes. Results The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically. Conclusions We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically

The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development

Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling

PLA-SOX10 vs. Cobas-s100 in 72 MM patients.

<p>PLA-SOX10 vs. Cobas-s100 in 72 MM patients.</p

SOX10 reactivity over time in nine patients during treatment follow up.

<p>DERMA and METRIC study treatment regimens are described under Methods.</p

Stages when 21 MM patients were found to be SOX10 positive.

<p>Stages when 21 MM patients were found to be SOX10 positive.</p

Measurement of SOX10 via solid-phase PLA in sera from vitiligo patients (n = 85) and from controls (healthy blood donors) (n = 40).

<p>Mean Ct (y-axis) values of duplicate samples are shown. For statistical analyses 1-tail two sample heteroscedastic t-test was applied. *** P<0.001. Line indicates cut-off for samples calculated as positive.</p

Measurement of SOX10 via solid-phase PLA in sera from MM patients.

<p>Ct values in sera of 110 patients (in total 148 MM samples) (left) and 45 control sera samples (blood donors) (right). For statistical analyses 1-tail two sample heteroscedastic t-test was applied. * P< 0.05. Line indicates cut-off for samples calculated as positive.</p