DEDD, A Novel Negative Modulator Of TGF-β Signaling That Interacts With Smad3

Transforming growth factor-β(TGF-β) family members, which include TGF-βs, activins, nodal and bone morphogenetic proteins (BMPs), are secreted cytokines that regulate a broad array of cellular responses including proliferation, differentiation, migration and apoptosis. TGF-P signaling pathway is a key player in metazoan biology, and its misregulation can result in tumor developmen. It is well known that how TGF-βsignaling exerts its function. The active TGF-P dimer signals by bringing together two pairs of receptor serine/threonine kinases known as the typeⅠand typeⅡreceptors, respectively. On binding TGF-P, the typeⅡreceptors phosphorylate and activate the typeⅠreceptors that then propagate the signal by phosphorylating Smad transcription factors. Based on their function, Smad proteins are generally divided into three groups:the receptor-regulated Smads (R-Smads), the common Smads (Co-Smads), and the inhibitory Smads (I-Smads). The typeⅠreceptors phosphorylate R-Smads, which shuttle to the nucleus and form a complex with Smad4, a binding partner common to all R-Smads. The activated heteromeric Smad complexes translocate into the nucleus and cooperate with other nuclear cofactors to regulate the transcription of target genes. Although TGF-β/Smad-mediated signaling is conceptually simple, a number of Smad-interacting proteins provide a high degree of signaling specificity and versatility.Apoptosis signaling is regulated and executed by specialized proteins that often carry protein-protein interaction domains. One of these domains is the Death effector domain (DED) that is predominantly found in components of the death receptor-induced signaling complex, which forms at the members of the death receptor family following their ligation. DED-containing proteins comprise Fas-associated death domain protein (FADD), Caspase-8 and death effector domain containing DNA binding (DEDD), and the like. DEDD plays an important role in CD95-mediated apoptosis through activating caspase3 or caspase6 as a scaffold protein. Recently, the study found that DEDD can suppress the activity of Cdkl/cyclin B1 complexes and keep S6K1 activity, suggesting that DEDD participate in cell cycle and inhibit cell mitosis. However, it is unclear whether DEDD involves in other signaling pathway via interacting with key proteins of major signal transduction pathway, and the pathophysiological functions of DEDD have not been fully clarified. Previously, using a yeast two-hybrid screen designed to identify Smad3-interacting protiens, we identified a novel interaction between Smad3 and DEDD. Here, we report that DEDD interacts with Smad3. The interaction between DEDD and Smad3 was mediated by the DED domain of DEDD. To the best of our knowledge, this is the first report revealing that Smad3 interacts with DED-containing proteins.Furthermore, TGF-P stimulation interfered with the association and the phosphorylation of Smad3 decreased the DEDD-Smad3 interaction. Inhibition of Smad3 by DEDD resulted in a decrease in TGF-β/Smad3-mediated transcription. DEDD reduced Smad3 functions by preventing its phosphorylation, binding to Smad4 and nuclear translocation which led to target gene expression reduction. TGF-βinhibited DEDD expression and Smad3 synergistically enhanced the repression of DEDD inhibited NF-κB-Luc reporter activity. DEDD inhibited TGF-β-mediated cell motility and invasion, but had no effect on TGF-β-induced EMT. DEDD itself also obviously inhibited cell migration, invasion and promoted apoptosis. Finally, DEDD showed different expression in human colon carcinoma and normal colon tissues. Therefore, our findings suggest that DEDD is a novel inhibitor of TGF-βsignaling pathway through interacting with Smad3 and DEDD suppresses invasion and proliferation of tumor cell. As DEDD belongs to the CD95 signaling pathway, our results demonstrated that a possible cross-talk between TGF-βand CD95-mediated signaling pathways.