The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. Recently, a direct signal transduction pathway from these receptors to nuclear target genes has been elucidated which involves a novel family of proteins termed Smads. In this pathway, receptor-activated Smads are phosphorylated directly by the type I receptor kinase and, in association with a common mediator Smad4/ DPC4, translocate to the nucleus where they participate in transcriptional complexes. We have taken a multi-faceted approach to delineate the biochemistry of this pathway in vitro and to understand its significance in vivo. One approach has been to identify unique factors which modulate receptor or Smad activity. We have utilized a yeast two-hybrid system using Smad1 as bait to identify two novel clones. One protein, SNX6, a member of the sorting nexin family of receptor interacting proteins, is likely to play a role in trafficking of serine-threonine kinase receptors. The other, SNIP1, is a novel nuclear repressor of Smad signaling, and may integrate cross-talk between different signaling pathways such as NF-kappaB converging on transcriptional complexes dependent on the C/H1 domain of the transcriptional coactivators p300 and CBP. We have also elucidated a novel role for the previously identified TGF-beta-receptor-interacting protein, TRAP1, which acts as a chaperone for the obligate partner, Smad4, presenting Smad4 to receptor-activated Smads. These studies will be expanded to define the roles of these molecules further and to include additional yeast two-hybrid screens to determine whether these proteins, in turn, interact with yet other components of the signaling complex. We are also using microarray analysis to identify cDNAs uniquely induced by Smad3- or Smad2-dependent pathways in mouse embryo fibroblasts. These studies have resulted in identification of targets of these signaling pathways, the characterization of which is certain to provide a new perspective on immediate-early and second-order gene targets of TGF-beta. To complement the above basic science approaches, we have also developed a strong program of research based on the hypothesis that deletion of specific signaling components in vivo should, conceptually, have a less severe and more selective effect than broader-based approaches involving targeted deletion or overexpression of ligand or receptors. We have been fortunate to collaborate with Chuxia Deng, NIDDK, on the characterization of a Smad3 knockout mouse, which is now providing new insights into the roles of TGF-beta in hematopoiesis, in wound healing, and in fibrosis. In collaboration with Drs. Angelo Russo and James Mitchell, we are focusing on protective effects of loss of Smad3 in response to ionizing radiation, and attempting to correlate our findings in mice with studies of effects of irradiation on primary keratinocytes and fibroblasts in vitro. Elucidation of pathogenetic mechanisms of TGF-beta dependent on this specific pathway now suggest that development of a Smad3 inhibitor will have wide-ranging clinical applications. To investigate BMP-dependent signaling pathways, we have created a conditional knockout of the Smad1 gene to overcome the early embryonic lethality of the Smad1 knockout mouse, also generated in our group.
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