SIGNAL TRANSDUCTION PATHWAYS OF TRANSFORMING GROWTH FACTOR-BETA

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The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. Recently, a short-circuit signal transduction pathway from these receptors 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. We have identified a 45 amino acid region of the middle linker region of the common mediator, Smad4, called the Smad activation domain (SAD). We have shown the SAD to be a proline-rich transcriptional activation domain which interacts with the co-activator p300/CBP and is both necessary and sufficient for activity of the Smad transcriptional complex. Other studies are focused on characterization of two novel clones we have isolated from a yeast two-hybrid system using Smad1 as bait. One of these proteins, a nuclear transcriptional repressor which we have called SNIP1, interacts principally with Smad4 and with p300/CBP in such a manner as to block the activating interaction of Smad4 with these coactivators. Another protein has recently been found to have homology to other sorting nexins and appears to bind both to receptors of the TGF-b family and to other receptors including the EGF and insulin receptors. A third protein, Trap1, identified by interaction cloning using an activated TGF-b receptor, appears to act as a shuttle between Smad2 and Smad4, possibly facilitating the association of these two Smads in the cytoplasm. The characterization of these proteins and their functional activity suggests additional complexity to the Smad signal transduction pathways. To delineate the role of Smad-mediated signal transduction in vivo, we have generated both transgenic and knockout mice. To study the TGF-b-activated Smad3 pathway, we have collaborated with Chuxia Deng (NIDDK) on characterization of a Smad3 knockout mouse. Study of wound healing in these mice has shown that deletion of this signaling intermediate has selective effects on TGF-b-dependent gene targets, resulting in narrower wounds that epithelialization more rapidly and have reduced inflammatory cell infiltratrates and reduced granulation tissue. This has possible implications for healing of human wounds and for fibrosis. To study the BMP-activated Smad1 pathway, we also generated mice null for Smad1, but these mice die at embryonic day 9.5 from an inability to form extraembryonic circulation. To circumvent this early embryonic lethality and to study the effect of deletion of Smad1 in the adult, we are in the process of generating a Smad1 conditional knockout mouse, which will be crossed with various mice expressing Cre recombinase in a tissue-specific manner. We have also generated mice expressing a dominant negative from of Smad1. These mice are being backcrossed both with mice heterozygous for Smad1, to reduce the quantity of the active Smad, and with mice overexpressing Smad6, an inhibitor of this signaling pathway. The phenotypes of these mice are still under investigation. - growth factors, receptors, signaling, TGF beta, Tumor Suppressor,