The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. 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, translocate to the nucleus where they participate in transcriptional complexes. We have taken a multi-faceted approach to gain insight into 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. Having previously identified a role for the TGF-beta-receptor-interacting protein, TRAP1, as a chaperone for the obligate signaling partner, Smad4, we are now characterizing a related protein which we call TRAP1-like protein, or TLP. TLP appears to have the unique feature of being required for signaling from Smad3, but not Smad2. We are investigating whether altering the expression of TLP in cells might modulating the signal transduction pathways and gene targets of TGF-beta in specific cell types or in disease. Among the targets are two Smad3-dependent immediate-early genes which have been implicated in apoptosis and in epithelial-to-mesenchymal transition, respectively, Gadd45beta and Snail. We are also attempting to identify specific targets of Smad2 and Smad3 in cancer cells, by characterizing the outcome of overexpression or suppression of Smad2 or Smad3 in tumorigenesis and metastasis by examining both in vitro and in vivo effects of altering the balance of these two pathways in human breast cancer cells derived from the parental MCF10A line. Initial results surprisingly demonstrate that the Smad pathway mediates both tumor suppressor and pro-metastatic activities of TGF-beta on tumor cells. Other approaches are based on the novel finding that SNIP1, a molecule which we first identified as a suppressor of TGF-beta signaling, interacts with c-Myc and enhances its transcriptional activating activity. It is hoped that ongoing studies examining the expression patterns of SNIP1 in a variety of human cancers will shed light onto the complex role of this molecule in carcinogenesis. To complement the above basic biochemical approaches, we have also developed a strong program of research based on the hypothesis that deletion of specific downstream signaling pathways 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. The Smad3 knockout mouse, developed by Chuxia Deng, NIDDK, is now providing new insights into the roles of TGF-beta in hematopoiesis, in repair of injury, and in fibrosis. In collaboration with Drs. Angelo Russo and James Mitchell, CCR, we are focusing on mechanisms of protective effects of loss of Smad3 in the skin 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. In collaborations with Dr. Shizuya Saika and Akira Ooshima, Wakayama U., we have shown that injury-induced transdifferentiation of epithelial cells to a mesenchymal phenotype post injury, as in injury to the eye or obstructive disease in the kidney, is completely blocked in mice lacking Smad3. Elucidation of pathogenetic mechanisms of TGF-beta dependent on the Smad3 pathway now suggest that development of a Smad3 inhibitor will have wide-ranging clinical applications in acceleration of epithelization of cutaneous wounds, in reducing opacification of lens implants, and in reducing fibrosis resulting from lung injury, retinal detachment, or ureteral obstruction.
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