W. Tony Parks

Haploid loss of the tumor suppressor Smad4/Dpc4 initiates gastric polyposis and cancer in mice.

The tumor suppressor SMAD4, also known as DPC4, deleted in pancreatic cancer, is a central mediator of TGF-β signaling. It was previously shown that mice homozygous for a null mutation of Smad4 (Smad4−/−) died prior to gastrulation displaying impaired extraembryonic membrane formation and endoderm differentiation. Here we show that Smad4+/− mice began to develop polyposis in the fundus and antrum when they were over 6–12 months old, and in the duodenum and cecum in older animals at a lower frequency. With increasing age, polyps in the antrum show sequential changes from hyperplasia, to dysplasia, in-situ carcinoma, and finally invasion. These alterations are initiated by a dramatic expansion of the gastric epithelium where Smad4 is expressed. However, loss of the remaining Smad4 wild-type allele was detected only in later stages of tumor progression, suggesting that haploinsufficiency of Smad4 is sufficient for tumor initiation. Our data also showed that overexpression of TGF-β1 and Cyclin D1 was associated with increased proliferation of gastric polyps and tumors. These studies demonstrate that Smad4 functions as a tumor suppressor in the gastrointestinal tract and also provide a valuable model for screening factors that promote or prevent gastric tumorigenesis.

A novel mitochondrial septin-like protein, ARTS, mediates apoptosis dependent on its P-loop motif.

Here we describe a protein product of the human septin H5/PNUTL2/CDCrel2b gene, which we call ARTS (for apoptosis-related protein in the TGF-β signalling pathway). ARTS is expressed in many cells and acts to enhance cell death induced by TGF-β or, to a lesser extent, by other apoptotic agents. Unlike related septin gene products, ARTS is localized to mitochondria and translocates to the nucleus when apoptosis occurs. Mutation of the P-loop of ARTS abrogates its competence to activate caspase 3 and to induce apoptosis. Taken together, these observations expand the functional attributes of septins previously described as having roles in cytokinesis and cellular morphogenesis.

The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain.

Transforming growth factor-β (TGF-β) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressorDPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

Loss of TGF-β signaling contributes to autoimmune pancreatitis

Recent observations suggest that immune response is involved in the development of pancreatitis. However, the exact pathogenesis underlying this immune-mediated response is still under debate. TGF-β has been known to be an important regulating factor in maintaining immune homeostasis. To determine the role of TGF-β in the initiation or progression of pancreatitis, TGF-β signaling was inactivated in mouse pancreata by overexpressing a dominant-negative mutant form of TGF-β type II receptor in the pancreas, under control of the pS2 mouse trefoil peptide promoter. Transgenic mice showed marked increases in MHC class II molecules and matrix metalloproteinase expression in pancreatic acinar cells. These mice also showed increased susceptibility to cerulein-induced pancreatitis. This pancreatitis was characterized by severe pancreatic edema, inflammatory cell infiltration, T- and B-cell hyperactivation, IgG-type autoantibodies against pancreatic acinar cells, and IgM-type autoantibodies against pancreatic ductal epithelial cells. Therefore, TGF-β signaling seems to be essential either in maintaining the normal immune homeostasis and suppressing autoimmunity or in preserving the integrity of pancreatic acinar cells.

Incisional wound healing in transforming growth factor‐β1 null mice

Expression of endogenous transforming growth factor‐β1 is reduced in many animal models of impaired wound healing, and addition of exogenous transforming growth factor‐β has been shown to improve healing. To test the hypothesis that endogenous transforming growth factor‐β1 is essential for normal wound repair, we have studied wound healing in mice in which the transforming growth factor‐β1 gene has been deleted by homologous recombination. No perceptible differences were observed in wounds made in 3–10‐day‐old neonatal transforming growth factor‐β1 null mice compared to wild‐type littermates. To preclude interference from maternally transferred transforming growth factor‐β1, cutaneous wounds were also made on the backs of 30‐day‐old transforming growth factor‐β1 null and littermate control mice treated with rapamycin, which extends their lifetime and suppresses the inflammatory response characteristic of the transforming growth factor‐β1 null mice. Again, no impairment in healing was seen in transforming growth factor‐β1 null mice. Instead these wounds showed an overall reduction in the amount of granulation tissue and an increased rate of epithelialization compared to littermate controls. Our data suggest that release of transforming growth factor‐β1 from degranulating platelets or secretion by infiltrating macrophages and fibroblasts is not critical to initiation or progression of tissue repair and that endogenous transforming growth factor‐β1 may actually function to increase inflammation and retard wound closure.

Development and application of fully functional epitope-tagged forms of transforming growth factor-β

Abstract Administration of transforming growth factor-β (TGF-β) has been found to be of therapeutic benefit in various mouse disease models and has potential clinical usefulness. However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-β and related TGF-β isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-β1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-β signaling intermediate, smad 2. Intracellular FLAG- and HA-TGF-β1 can be detected in transfected cells by confocal immunofluorescence microscopy. We also describe sandwich ELISAs designed to specifically detect epitope-tagged TGF-β and demonstrate the utility of these tagged ligands as probes for TGF-β receptor expression by flow cytometry. The design of these fully functional epitope-tagged TGF-β proteins should facilitate studies such as the evaluation of in vivo peptide pharmacodynamics and trafficking of TGF-β ligand–receptor complexes.