Robert J. Lechleider

Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.

SMAD3 is one of the intracellular mediators that transduces signals from transforming growth factor‐β (TGF‐β) and activin receptors. We show that SMAD3 mutant mice generated by gene targeting die between 1 and 8 months due to a primary defect in immune function. Symptomatic mice exhibit thymic involution, enlarged lymph nodes, and formation of bacterial abscesses adjacent to mucosal surfaces. Mutant T cells exhibit an activated phenotype in vivo, and are not inhibited by TGF‐β1 in vitro. Mutant neutrophils are also impaired in their chemotactic response toward TGF‐β. Chronic intestinal inflammation is infrequently associated with colonic adenocarcinoma in mice older than 6 months of age. These data suggest that SMAD3 has an important role in TGF‐β‐mediated regulation of T cell activation and mucosal immunity, and that the loss of these functions is responsible for chronic infection and the lethality of Smad3‐null mice.

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.

Smad3/AP-1 interactions control transcriptional responses to TGF-β in a promoter-specific manner

Smad proteins transduce signals from TGF-β receptors and regulate transcription of target genes either directly or in combination with other sequence-specific transcription factors. AP-1 sites and their cognate transcription factors also play important roles in the gene regulatory activities of TGF-β. In this report, we have investigated the functional interactions of the Smad and AP-1 transcription factors. We demonstrate that Smad and AP-1 complexes specifically bind to their cognate cis-elements and do not interact with each other on-DNA, whereas off-DNA interactions occur between Smad3 and both c-Jun and JunB. Using both artificial constructs specific for either the Smad or AP-1 signaling pathways or natural promoters known to be TGF-β-responsive, we have determined that Jun family members downregulate Smad3-mediated gene transactivation whereas AP-1-dependent promoters are synergistically activated by Smad3 and Jun proteins. We propose a model where the presence of Smad- and/or AP-1-specific cis-elements within TGF-β-responsive genes allows dynamic modulation of gene expression, in contrast to the existing model where interactions between Smad and AP-1 proteins are merely an on/off mechanism to regulate TGF-β/Smad targets.

RhoA Modulates Smad Signaling during Transforming Growth Factor-β-induced Smooth Muscle Differentiation

We recently reported that transforming growth factor (TGF)-β induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-β actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-β-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-β. We demonstrate here that RhoA signaling is critical to TGF-β-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle α-actin, SM22α, and calponin in TGF-β-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-β-treated cells. RhoA signaling was activated as early as 5 min following TGF-β addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smad-dependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-β-induced SMC differentiation.

Serine Phosphorylation, Chromosomal Localization, and Transforming Growth Factor-β Signal Transduction by Human bsp-1

The transforming growth factor-β (TGF-β) superfamily regulates a multitude of cellular and developmental events. TGF-β family ligands signal through transmembrane serine/threonine kinase receptors whose downstream effectors are largely unknown. Using genetic data from the fruit fly, we have identified a downstream effector of TGF-β-induced signaling. TGF-β signaling protein-1 (BSP-1) is rapidly phosphorylated in response to TGF-β. Localization of bsp-1 to chromosome 4q28 suggests a role in carcinogenesis. These data suggest that BSP-1 is the prototype of a new class of signaling molecules.

Transcriptional Control of Expression of the TGF‐βs

In recent years, there has been an exponential increase in understanding of the chemistry and biology of the family of peptides called transforming growth factor-S (TGFp). The discovery and characterization of five distinct, highly conserved, yet functionally similar TGF-Ps has added an unexpected level of complexity to the problems of defining the roles of the different TGF-Ps in normal and pathological physiology! Furthermore, understanding of the biology of the TGF-ps, once narrowly defined by their ability to confer a transformed phenotype on nonneoplastic NRK fibroblasts, has now expanded to include effects on almost every cell type and to range from processes such as control of steroidogenesis or control of epithelial cell growth and differentiation, to more complex processes involving multiple cell types such as wound healing, bone remodeling, hemopoiesis, or specific morphogenetic and histogenetic events in embryonic development ! Given the many cell types sensitive to TGF-p action, it is clear that multiple levels of control of its activity must exist. Though control of TGF-P receptor expression, receptor signalling, and activation of the latent forms of the various TGF-Ps are without doubt important, this review will be limited to a discussion of transcriptional regulation of the activity of mF-0 in a variety of in vitro and in vivo systems and of evidence suggesting that expression of the five TGF-ps is under different celland tissue-specific transcriptional control.

SNIP1 Is a Candidate Modifier of the Transcriptional Activity of c-Myc on E Box-Dependent Target Genes

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

Suv39h histone methyltransferases interact with Smads and cooperate in BMP-induced repression

Smad proteins transduce signals from transforming growth factor-β (TGF-β) superfamily ligands to regulate the expression of target genes. In order to identify novel partners of Smad proteins in transcriptional regulation, we performed a two-hybrid screen using Smad5, a protein that is activated predominantly by bone morphogenetic protein (BMP) signaling. We identified an interaction between Smad5 and suppressor of variegation 3-9 homolog 2 (Suv39h2), a chromatin modifier enzyme. Suv39h proteins are histone methyltransferases that methylate histone H3 on lysine 9, resulting in transcriptional repression or silencing of target genes. Biochemical studies in mammalian cells demonstrated that Smad5 binds to both known mammalian isoforms of Suv39h proteins, and that Smad proteins activated by the TGF-β signaling pathway, Smad2 and Smad3, do not bind with significant affinity. Functional studies using the muscle creatine kinase (MCK) promoter, which is suppressed by BMP signaling, demonstrate that Suv39h proteins and Smads cooperate to repress promoter activity. These data suggest a model where association of Smad proteins with Suv39h methyltransferases can repress or silence genes involved in developmental processes, and argues that inefficient gene repression may result in the alteration of the differentiated phenotype. Thus, examination of the Smad–Suv interaction may provide insight into the mechanism of phenotypic determination mediated by BMP signaling.

Histone Methyltransferase and Smad Interactions in BMP Signaling

Although the role of transforming growth factor-β (TGF-β) signaling in the development and progression of cancer is now beginning to be understood, the importance of signaling by bone morphogenetic protein (BMP) ligands is just becoming clear, and much less is known about the mechanisms by which BMP signaling may mediate carcinogenesis. We have shown that the Smad proteins which mediate signals from the BMP cell surface receptors can interact with histone methyltransferases of the Suv39h family, and that this interaction can contribute to transcriptional repression mediated by BMP signaling. This provides the first evidence that BMP signaling can modulate chromatin structure through methylation, and may provide a mechanism for maintenance of suppression originally initiated by Smad signals. Aberrant regulation of this phenomenon is likely to be important both during tumorigenesis and subsequent malignant progression.