Molecular characterisation of a serum-responsive, DAF-12-like nuclear hormone receptor of the fox-tapeworm Echinococcus multilocularis

As the primary mediators of lipophilic and steroid hormone signalling, the family of nuclear receptors (NRs) plays a central role in the regulation of metazoan development. Lipophilic hormones are also thought to be important players in the molecular interaction between larval cestodes and their hosts but no member of the NR family has yet been characterised in this group of parasites. In this work, we provide for the first time evidence for the presence of NRs in cestodes of the genus Echinococcus. By bioinformatic analyses, we identified a set of 17 NRs in the genomes of E. multilocularis and E. granulosus which broadly overlapped with the set of NRs that is expressed by schistosomes, but also contained several members that are unique to cestodes. One of these receptors, EmNHR1, displayed structural homologies to the DAF-12/HR-96 subfamily of NRs that regulates cholesterol homeostasis and longevity in metazoans. By RT-PCR analyses, we demonstrate that the EmNHR1 encoding gene is expressed in all Echinococcus larval stages that are involved in the infection of the intermediate host. By yeast two-hybrid analyses, we further demonstrate cross-communication between EmNHR1 and TGF-β signalling pathways in Echinococcus and that mammalian serum contains a ligand that induces homodimerisation of the EmNHR1 ligand-binding domain. EmNHR1 could thus play an important role in hormonal host-parasite cross-communication mechanisms during an infection. On the basis of our results, further investigations into the role of NR signalling in cestode development and host-parasite interaction will be greatly facilitated.     

A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling

Transforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. Smad2 and Smad3 are direct mediators of TGF-beta signaling, however little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-beta target genes, suggest that Smad2 and Smad3 have distinct roles in TGF-beta signaling. The observation that TGF-beta inhibits proliferation of Smad3-null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that Smad3 has a different role in epithelial cells and fibroblasts. Herein, the current understanding of Smad2 and Smad3-mediated TGF-beta signaling and their relative roles are discussed, in addition to potential mechanisms for the selective activation of Smad2 versus Smad3. Since alterations in the TGF-beta signaling pathway play an important role in promoting tumorigenesis and cancer progression, methods for therapeutic targeting of the TGF-beta signaling pathway are being pursued. Determining how Smad2 or Smad3 differentially regulate the TGF-beta response may translate into developing more effective strategies for cancer therapy.     

Dissection of inhibitory Smad proteins: both N- and C-terminal domains are necessary for full activities of Xenopus Smad6 and Smad7

Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-β family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-β related pathways, and causes spina bifida when misexpressed dorsally. We have performed structure-function analyses to identify domains of Anti-Smads that are responsible for their shared and unique activities. We find that the C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans.     

Expression of Smad2 and Smad4, transforming growth factor-beta signal transducers in rat endometrium during the estrous cycle, pre-, and peri-implantation

SMADs are intracellular signaling molecules that transmit signals elicited by members of transforming growth factor-β (TGF-β) superfamily. To decipher the mechanism of TGF-β signaling during the estrous cycle and implantation, we performed in situ hybridization to investigate the expression patterns of mRNAs for Smad2 and Smad4 in rat endometrium during the estrous cycle and on Days 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5 of pregnancy. Intense epithelial expression of Smad2 mRNA at diestrus and proestrus was reduced at estrus and metaestrus, while Smad4 maintained its constitutive expression during the estrous cycle. During pre-implantation, both Smads were accumulated in the luminal epithelium and the glandular epithelium. Contrary to the dramatic Smad4 expression, Smad2 was highly down-regulated on Day 2.5 and was increased on Day 3.5. During peri-implantation, both Smads were expressed in the luminal epithelium, subepithelial stroma, and the primary decidual zone. Smad4 was down-modulated on Day 5.5. These results suggest that (a) both Smads are involved in the tissue remodeling of cycling and pregnant rat uteri; (b) TGF-β signaling functions mainly in the epithelium during pre-implantation and Smad2 is involved in the endometrial switch from the neutral phase to the receptive phase; (c) TGF-β signaling is down-regulated at the time when trophoblast invasion begins and both Smads are involved in the formation of the primary decidual zone.     

Echinococcus multilocularis: identification and functional characterization of cathepsin B-like peptidases from metacestode

Cysteine peptidases have potent activities in the pathogenesis of various parasitic infections, and are considered as targets for chemotherapy and antigens for vaccine. In this study, two cathepsin B-like cysteine peptidases (EmCBP1 and EmCBP2) from Echinococcus multilocularis metacestodes were identified and characterized. Immunoblot analyses demonstrated that EmCBP1 and EmCBP2 were present in excretory/secretory products and extracts of E. multilocularis metacestodes. By immunohistochemistry, EmCBP1 and EmCBP2 were shown to localize to the germinal layer, the brood capsule and the protoscolex. Recombinant EmCBP1 and EmCBP2 expressed in Pichia pastoris, at optimum pH 5.5, exhibited substrate preferences for Z-Phe-Arg-MCA, Z-Val-Val-Arg-MCA, and Z-Leu-Arg-MCA, and low levels of hydrolysis of Z-Arg-Arg-MCA. Furthermore, recombinant enzymes degraded IgG, albumin, type I and IV collagens, and fibronectin. These results suggested that EmCBP1 and EmCBP2 may play key roles in protein digestion for parasites’ nutrition and in parasite–host interactions.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.     

TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.     

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.