Smad7-induced beta-catenin degradation alters epidermal appendage development

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.     

Inflammatory monocytes and neutrophils are licensed to kill during memory responses in vivo

Immunological memory is a hallmark of B and T lymphocytes that have undergone a previous encounter with a given antigen. It is assumed that memory cells mediate better protection of the host upon re-infection because of improved effector functions such as antibody production, cytotoxic activity and cytokine secretion. In contrast to cells of the adaptive immune system, innate immune cells are believed to exhibit a comparable functional effector response each time the same pathogen is encountered. Here, using mice infected by the intracellular bacterium Listeria monocytogenes, we show that during a recall bacterial infection, the chemokine CCL3 secreted by memory CD8+ T cells drives drastic modifications of the functional properties of several populations of phagocytes. We found that inflammatory ly6C+ monocytes and neutrophils largely mediated memory CD8+ T cell bacteriocidal activity by producing increased levels of reactive oxygen species (ROS), augmenting the pH of their phagosomes and inducing antimicrobial autophagy. These events allowed an extremely rapid control of bacterial growth in vivo and accounted for protective immunity. Therefore, our results provide evidence that cytotoxic memory CD8+ T cells can license distinct antimicrobial effector mechanisms of innate cells to efficiently clear pathogens.     

Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the plasma membrane

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-beta type I receptor (TbetaR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1.Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(deltaC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(deltaC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TbetaR-II.TbetaR-I complex. Moreover, although Smurf1(deltaC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TbetaR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-beta signaling pathway.     

Bax: Addressed to kill

The pro-apoptototic protein Bax (Bcl-2 Associated protein X) plays a central role in the mitochondria-dependent apoptotic pathway. In healthy mammalian cells, Bax is essentially cytosolic and inactive. Following a death signal, the protein is translocated to the outer mitochondrial membrane, where it promotes a permeabilization that favors the release of different apoptogenic factors, such as cytochrome c. The regulation of Bax translocation is associated to conformational changes that are under the control of different factors. The evidences showing the involvement of different Bax domains in its mitochondrial localization are presented. The interactions between Bax and its different partners are described in relation to their ability to promote (or prevent) Bax conformational changes leading to mitochondrial addressing and to the acquisition of the capacity to permeabilize the outer mitochondrial membrane.     

Liver fibrogenesis due to cholestasis is associated with increased Smad7 expression and Smad3 signaling

BACKGROUND/AIMS: Profibrogenic TGF-beta signaling in hepatic stellate cells is modulated during transdifferentiation. Strategies to abrogate TGF-beta effects provide promising antifibrotic results, however, in vivo data regarding Smad activation during fibrogenesis are scarce. METHODS: Here, liver fibrosis was assessed subsequent to bile duct ligation by determining liver enzymes in serum and collagen deposition in liver tissue. Activated hepatic stellate cells were identified by immunohistochemistry and immunoblots for alpha smooth muscle actin. Cellular localization of Smad3 and Smad7 proteins was demonstrated by immunohistochemistry. RTPCR for Smad4 and Smad7 was conducted with total RNA and Northern blot analysis for Smad7 with mRNA. Whole liver lysates were prepared to detect Smad2/3/4 and phospho- Smad2/3 by Western blotting. RESULTS: Cholestasis induces TGF-beta signaling via Smad3 in vivo, whereas Smad2 phosphorylation was only marginally increased. Smad4 expression levels were unchanged. Smad7 expression was continuously increasing with duration of cholestasis. Hepatocytes of fibrotic lesions exhibited nuclear staining Smad3. In contrast to this, Smad7 expression was localized to activated hepatic stellate cells. CONCLUSIONS: Hepatocytes of damaged liver tissue display increased TGF-beta signaling via Smad3. Further, negative feedback regulation of TGF-beta signaling by increased Smad7 expression in activated hepatic stellate cells occurs, however does not interfere with fibrogenesis.     

Antagonistic effects of Smad2 versus Smad7 are sensitive to their expression level during tooth development

Members of the transforming growth factor-beta (TGF-beta) superfamily regulate cell proliferation, differentiation, and apoptosis, controlling the development and maintenance of most tissues. TGF-beta signal is transmitted through the phosphorylation of Smad proteins by TGF-beta receptor serine/threonine kinase. During early tooth development, TGF-beta inhibits proliferation of enamel organ epithelial cells but the underlying molecular mechanisms are largely unknown. Here we tested the hypothesis that antagonistic effects between Smad2 and Smad7 regulate TGF-beta signaling during tooth development. Attenuation of Smad2 gene expression resulted in significant advancement of embryonic tooth development with increased proliferation of enamel organ epithelial cells, while attenuation of Smad7 resulted in significant inhibition of embryonic tooth development with increased apoptotic activity within enamel organ epithelium. These findings suggest that different Smads may have differential activities in regulating TGF-beta-mediated cell proliferation and death. Furthermore, functional haploinsufficiency of Smad2, but not Smad3, altered TGF-beta-mediated tooth development. The results indicate that Smads are critical factors in orchestrating TGF-beta-mediated gene regulation during embryonic tooth development. The effectiveness of TGF-beta signaling is highly sensitive to the level of Smad gene expression.     

Smad7: a new key player in TGF-beta-associated disease

Smad7 is a major inhibitory regulator of transforming growth factor (TGF)-beta signaling. Smad7 expression is induced by TGF-beta itself and other signaling pathways, indicating a key role for Smad7 in feedback or cross-talk control of TGF-beta signaling. Recent reports have implicated Smad7 as a crucial regulator of TGF-beta activity in human disease; aberrant expression of Smad7 is involved in inflammatory bowel disease and scleroderma. Thus, modulation of Smad7 expression could provide a novel therapeutic basis for TGF-beta-associated disorders.     

Repression of endogenous Smad7 by Ski

The Ski protein has been proposed to serve as a corepressor for Smad4 to maintain a transforming growth factor-beta (TGF-beta)-responsive promoter at a repressed, basal level. However, there have been no reports so far that it indeed acts on a natural promoter. We have previously cloned the human Smad7 promoter and shown that it contains the 8-base pair palindromic Smad-binding element (SBE) necessary for TGF-beta induction. In this report, we have characterized the negative regulation of Smad7 promoter basal activity by Ski. We show that Ski inhibits the Smad7 promoter basal activity in a SBE-dependent manner. Mutation of the SBE abrogates the inhibitory effect of Ski on the Smad7 promoter. Moreover, mutation of the SBE increases the Smad7 promoter basal activity. Using the chromatin immunoprecipitation assay, we further show that Ski together with Smad4 binds to the endogenous Smad7 promoter. Finally, we show that RNAi knockdown of Ski increases Smad7 reporter gene activity in transient transfection assays as well as elevating the endogenous level of Smad7 mRNA. Taken together, our results provide the first evidence that Ski is indeed a corepressor for Smad4, which can inhibit a natural TGF-beta responsive gene at the basal state.