Lawrence A. Wolfraim

Smad4 signalling in T cells is required for suppression of gastrointestinal cancer

SMAD4 (MAD homologue 4 (Drosophila)), also known as DPC4 (deleted in pancreatic cancer), is a tumour suppressor gene that encodes a central mediator of transforming growth factor-β signalling. Germline mutations in SMAD4 are found in over 50% of patients with familial juvenile polyposis, an autosomal dominant disorder characterized by predisposition to hamartomatous polyps and gastrointestinal cancer. Dense inflammatory cell infiltrates underlay grossly normal appearing, non-polypoid colonic and gastric mucosa of patients with familial juvenile polyposis. This prominent stromal component suggests that loss of SMAD4-dependent signalling in cells within the epithelial microenvironment has an important role in the evolution of intestinal tumorigenesis in this syndrome. Here we show that selective loss of Smad4-dependent signalling in T cells leads to spontaneous epithelial cancers throughout the gastrointestinal tract in mice, whereas epithelial-specific deletion of the Smad4 gene does not. Tumours arising within the colon, rectum, duodenum, stomach and oral cavity are stroma-rich with dense plasma cell infiltrates. Smad4-/- T cells produce abundant TH2-type cytokines including interleukin (IL)-5, IL-6 and IL-13, known mediators of plasma cell and stromal expansion. The results support the concept that cancer, as an outcome, reflects the loss of the normal communication between the cellular constituents of a given organ, and indicate that Smad4-deficient T cells ultimately send the wrong message to their stromal and epithelial neighbours.

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation†

Oval cells are hepatocytic precursors that proliferate in late‐stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor‐β (TGF‐β) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF‐β levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF‐β signaling in vivo in oval cells, we analyzed livers of rats fed a choline‐deficient, ethionine‐supplemented (CDE) diet for phospho‐Smad2. Phospho‐Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF‐β signaling and proliferation in oval cells and hepatocytes, we examined TGF‐β signaling in vitro. TGF‐β caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF‐β–induced growth inhibition may result from the absence of Smad6 in these cells. Conclusion: Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF‐β–induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation. (HEPATOLOGY 2007;45:31–41.)

p21Cip1 and p27Kip1 Act in Synergy to Alter the Sensitivity of Naive T Cells to TGF-β-Mediated G1 Arrest through Modulation of IL-2 Responsiveness

Induction of G1 arrest by TGF-β correlates with the regulation of p21Cip1 and p27Kip1, members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-β1-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-β is diminished in T cells from mice deficient for both p21Cip1 and p27Kip1 (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8+ T cells from DKO mice, TGF-β is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15Ink4b in T cells stimulated in the presence of TGF-β is not essential, as TGF-β also efficiently suppressed proliferation of T cells from p15Ink4b−/− mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-β, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3−/− T cells to the induction of growth arrest by TGF-β. In summary, the growth suppressive effects of TGF-β in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-β by lowering thresholds for a maximal mitogenic response.

Cutting Edge: TCR-Induced NAB2 Enhances T Cell Function by Coactivating IL-2 Transcription

TCR engagement leads to the up-regulation of genetic programs that can both activate and inhibit T cell function. The early growth receptor (Egr) proteins Egr-2 and Egr-3 have recently been identified as TCR-induced negative regulators of T cell function. NAB2 (NGFI-A-binding protein 2) is both a coactivator and a corepressor of Egr-mediated transcription and has been implicated in regulating Schwann cell myelination. In this report we demonstrate that NAB2 is induced by TCR engagement and that its expression is enhanced by the presence of costimulation. The overexpression of NAB2 enhanced IL-2 production while small interfering RNA to NAB2 markedly inhibited IL-2 expression. Mechanistically, we demonstrate that NAB2 enhances IL-2 transcription by acting as a coactivator for Egr-1. Indeed, chromatin immunoprecipitation analysis reveals that NAB2 is recruited to the Egr-1 binding site of the IL-2 promoter. Taken together, our findings identify NAB2 as a novel coactivator of T cell function.

Cutting Edge: p27Kip1 Deficiency Reduces the Requirement for CD28-Mediated Costimulation in Naive CD8+ but Not CD4+ T Lymphocytes

Cell cycle re-entry of quiescent T cells is dependent upon cyclin-dependent kinase 2. Inhibition of cyclin-dependent kinase 2 by p27Kip1 is believed to be the principal constraint on S-phase entry in T cells. We report that deficiency for p27Kip1 has a more pronounced effect on the expansion of murine naive CD8+ T cells and that this disparity is due to a reduced requirement for CD28-mediated costimulation in CD8+ but not CD4+ T cells lacking p27Kip1. These data highlight a previously unappreciated difference in the way CD28 signaling is coupled to the core cell cycle machinery in these two T cell subsets.

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.

Perturbations of TGF-β Signaling in Leukocytes as Drivers of Leukemogenesis and Epithelial Tumorigenesis

The inactivation of specific components of the transforming growth factor-beta (TGF-β) signaling pathway has been implicated in many types of hematological malignancies. These range from alterations at the level of TGF-β receptors to mutations, deletions or functional inactivation of downstream signaling components such as members of the Smad family of proteins. It is becoming increasingly apparent that, in addition to playing a role in the progression of certain leukemias, disruption of TGF-β signaling in the lymphoid compartment also has profound effects on tumor progression of epithelial cells. In this respect, the use of conditional knockout murine models has been particularly instructive. We review here well-documented examples where TGF-β signaling is thought to control leukemogenesis. More recent data from our laboratory and others are lighlighted in support of a role for T-cell TGF-β signaling in regulating epithelial tumor progression. Finally, we review the link between TGF-β, regulatory T cells (Treg) and tumor immunotherapies, an understanding of which has significant therapeutic relevance.

Targeting the TGF-β Pathway In Vivo

Transforming growth factor-s1 (TGF-(s1) is a 25-kDa homodimeric peptide and the prototype in a family of structurally related but functionally distinct regulatory proteins. These TGF-s isoforms (TGF-s1, -s2, and -s3 in mammals) bear some structural relationship to a much larger family of peptide signaling molecules, with over 45 known members in this superfamily. The high degree of similarity that exists at the structural level among the isoforms of these growth factors is also accompanied by a significant overlap in function, as defined by many in vitro model systems. Moreover, the signaling pathway is not strictly linear in that there is extensive crosstalk with components of other signaling cascades (Fig. 1), with the TGF-ss typically influencing the manner in which cells interpret other signals in their environment. The evolution of more sophisticated functional genomics approaches has been instrumental in generating unique perspectives into the mechanisms governing the activity of the members of the TGF-s family. The studies outlined in this review serve to demonstrate how these models are more clearly defining the function of this pathway in immune homeostasis, wound healing, and carcinogenesis.