Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor beta and bone morphogenetic protein

Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.     

Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor beta1 with higher affinity receptors and an activated Alk1 pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.     

Squamous cell carcinoma and mammary abscess formation through squamous metaplasia in Smad4/Dpc4 conditional knockout mice

Smad4 is a central mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in mammary gland development and neoplasia, we disrupted this gene in mammary epithelium using a Cre-loxP approach. Smad4 is expressed in the mammary gland throughout development; however, its inactivation did not cause abnormal development of the gland during the first three pregnancies. Instead, lack of Smad4 gradually induced cell proliferation, alveolar hyperplasia and transdifferentiation of mammary epithelial cells into squamous epithelial cells. Consequently, all mutant mice developed squamous cell carcinoma and/or mammary abscesses between 5 and 16 months of age. We demonstrated that absence of Smad4 resulted in beta-catenin accumulation at onset and throughout the process of transdifferentiation, implicating beta-catenin, a key component of the Wnt signaling pathway, in the development of squamous metaplasia in Smad4-null mammary glands. We further demonstrated that TGFbeta1 treatment degraded beta-catenin and induced epithelial-mesenchymal transformation in cultured mammary epithelial cells. However, such actions were blocked in the absence of Smad4. These findings indicate that TGFbeta/Smad4 signals play a role in cell fate maintenance during mammary gland development and neoplasia.     

Integrin-linked kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition

The role of integrin-linked kinase (ILK) in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFbeta-mediated E-cadherin delocalization. TGFbeta-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFbeta stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFbeta was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFbeta-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K-ILK-Akt pathway that is independent of the TGFbeta-induced Smad pathway is required for TGFbeta-mediated epithelial to mesenchymal transition.     

Smad3-ATF3 signaling mediates TGF-beta suppression of genes encoding Phase II detoxifying proteins

This study provides evidence that in mammary epithelial cells the pluripotent cytokine TGF-beta1 repressed expression of multiple genes involved in Phase II detoxification. GCLC, the gene that encodes the catalytic subunit of the enzyme glutamate cysteine ligase, the rate-limiting enzyme in the biosynthesis of glutathione, was used as a molecular surrogate for investigating the mechanisms by which TGF-beta suppressed Phase II gene expression. TGF-beta was found to suppress luciferase reporter activity mediated by the human GCLC proximal promoter, as well as reporter activity mediated by the GCLC antioxidant response element, ARE4. TGF-beta downregulated expression of endogenous GCLC mRNA and GCLC protein. TGF-beta suppression of the Phase II genes correlated with a decrease in cellular glutathione and an increase in cellular reactive oxygen species. Ectopic expression of constitutively active Smad3E was sufficient to inhibit both reporters in the absence of TGF-beta, whereas dominant negative Smad3A blocked TGF-beta suppression. Smad3E suppressed Nrf2-mediated activation of the GCLC reporter. We demonstrate that TGF-beta increased ATF3 protein levels, as did transient overexpression of Smad3E. Ectopic expression of ATF3 was sufficient to suppress the GCLC reporter activity, as well as endogenous GCLC expression. These results demonstrate that Smad3-ATF3 signaling mediates TGF-beta repression of ARE-dependent Phase II gene expression and potentially provide critical insight into mechanisms underlying TGF-beta1 function in carcinogenesis, tissue repair, and fibrosis.     

Activation and roles of ALK4/ALK7-mediated maternal TGFbeta signals in zebrafish embryo

Activin, Nodal, and Vg1, members of the transforming growth factor beta (TGFbeta) superfamily, transduce signal through type I receptors ALK4 or ALK7 and play important roles in mesoderm induction and patterning during vertebrate embryogenesis. However, the timing and magnitude of the ALK4/ALK7-mediated maternal TGFbeta signals are not clear. SB-431542 is identified as an inhibitor of the ALK4/ALK5/ALK7-mediated TGFbeta signals and its specificity in vertebrate embryos has not been reported. We demonstrate that SB-431542 is able to specifically and reproducibly block the Smad2/3-mediated TGFbeta signals in zebrafish embryo. Embryos exposed to SB-431542 exhibit various defects phenocopying Nodal-deficient mutants. SB-431542 treatments starting at different cell cycles before the midblastula transition lead to different degrees of developmental defects in mesoderm induction and patterning, suggesting that maternal TGFbeta signals are activated right after fertilization and required for mesoderm formation and patterning.     

Inhibition of TGFbeta1-mediated PAI-1 induction by oltipraz through selective interruption of Smad 3 activation

Transforming growth factor-beta1 (TGFbeta1) induces plasminogen activator inhibitor-1 (PAI-1) as a major target protein. PAI-1 is associated with fibrosis, thrombosis, and metabolic disorders. TGFbeta1 induces PAI-1 via phosphorylation and nuclear translocation of Smads. Oltipraz inhibits TGFbeta1 expression and also regenerates cirrhotic liver. Nevertheless, whether oltipraz modulates TGFbeta1-mediated cell signaling is unclear. First, this study examined the effect of oltipraz on PAI-1 expression in cirrhotic rat liver. The cells immunochemically stained with anti-PAI-1 antibody accumulated around and within fibrous nodules in cirrhotic liver, which was notably decreased by oltipraz treatment. Next, whether oltipraz inhibits TGFbeta1-mediated Smads activation or Smad-mediated PAI-1 induction was determined in L929 fibroblasts. Oltipraz inhibited the ability of TGFbeta1 to induce PAI-1, as indicated by repression of TGFbeta1-mediated luciferase induction from the plasmid comprising the human PAI-1 promoter and of TGFbeta1-induced Smad-DNA-binding activity. TGFbeta1 induced nuclear transport of receptor-regulated Smad 2 and Smad 3, of which oltipraz selectively inhibited the transport and phosphorylation of Smad 3, thereby reducing formation of Smad 3/4 complex in the nucleus. In summary, oltipraz inhibits PAI-1 induction via a decrease in the formation of Smad 3/4 complex due to selective interruption of Smad 3 activation, indicating that oltipraz regulates the cellular responses downstream of ligand-activated TGFbeta1 receptor.