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1.
Mol Cancer Res ; 7(8): 1342-53, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19671686

ABSTRACT

Smad2 and Smad3 are intracellular mediators of transforming growth factor beta (TGFbeta) signaling that share various biochemical properties, but data emerging from functional analyses in several cell types indicate that these two Smad proteins may convey distinct cellular responses. Therefore, we have investigated the individual roles of Smad2 and Smad3 in mediating the cytostatic and proapoptotic effects of TGFbeta as well as their function in epithelial-to-mesenchymal transition. For this purpose, we transiently depleted mouse mammary epithelial cells (Nme) of Smad2 and/or Smad3 mainly by a strategy relying on RNaseH-induced degradation of mRNA. The effect of such depletion on hallmark events of TGFbeta-driven epithelial-to-mesenchymal transition was analyzed, including dissolution of epithelial junctions, formation of stress fibers and focal adhesions, activation of metalloproteinases, and transcriptional regulation of acknowledged target genes. Furthermore, we investigated the effect of Smad2 and Smad3 knockdown on the TGFbeta-regulated transcriptome by microarray analysis. Our results identify Smad3 as a key factor to trigger TGFbeta-regulated events and ascribe tumor suppressor as well as oncogenic activities to this protein.


Subject(s)
Epithelial Cells/metabolism , Mammary Glands, Animal/cytology , Signal Transduction , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Animals , Apoptosis/drug effects , Cell Shape/drug effects , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Enzyme Induction/drug effects , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Female , Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Humans , Intercellular Junctions/drug effects , Intercellular Junctions/metabolism , Matrix Metalloproteinases/biosynthesis , Mice , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Smad2 Protein/genetics , Smad2 Protein/metabolism , Smad3 Protein/genetics
2.
Proc Natl Acad Sci U S A ; 105(42): 16183-8, 2008 Oct 21.
Article in English | MEDLINE | ID: mdl-18852455

ABSTRACT

Insulin/insulin-like growth factor (IGF) signaling constitutes an evolutionarily conserved pathway that controls growth, energy homeostasis, and longevity. In Drosophila melanogaster, key components of this pathway are the insulin-like peptides (Dilps). The major source of Dilps is a cluster of large neurons in the brain, the insulin-producing cells (IPCs). The genetic control of IPC development and function is poorly understood. Here, we demonstrate that the Pax6 homolog Eyeless is required in the IPCs to control their differentiation and function. Loss of eyeless results in phenotypes associated with loss of insulin signaling, including decreased animal size and increased carbohydrate levels in larval hemolymph. We show that mutations in eyeless lead to defective differentiation and morphologically abnormal IPCs. We also demonstrate that Eyeless controls IPC function by the direct transcriptional control of one of the major Dilps, dilp5. We propose that Eyeless has an evolutionarily conserved role in IPCs with remarkable similarities to the role of vertebrate Pax6 in beta cells of the pancreas.


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Eye Proteins , Homeodomain Proteins , Insulin/biosynthesis , Insulins/metabolism , Neurons/cytology , Neurons/metabolism , Paired Box Transcription Factors , Repressor Proteins , Animals , Base Sequence , Cell Differentiation , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Female , Gene Expression Regulation, Developmental , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Insulins/genetics , Male , Molecular Sequence Data , Mutation/genetics , PAX6 Transcription Factor , Signal Transduction , Transcription, Genetic/genetics
3.
Hum Mol Genet ; 15(22): 3369-77, 2006 Nov 15.
Article in English | MEDLINE | ID: mdl-17038485

ABSTRACT

Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.


Subject(s)
Body Patterning/genetics , Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins/metabolism , Signal Transduction/genetics , Transforming Growth Factor beta/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Animals , Animals, Genetically Modified , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Mutation/genetics , Nodal Protein , Protein Binding , Zebrafish/metabolism , Zebrafish Proteins/deficiency , Zebrafish Proteins/genetics
4.
Genes Cells ; 10(9): 897-906, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16115198

ABSTRACT

Ligand-bound receptors of the Transforming Growth Factor-beta (TGF-beta) family promote the formation of complexes between Smad proteins that subsequently accumulate in the nucleus and interact there with other transcriptional regulators, leading to modulation of target gene expression. We identified a novel nuclear protein, Smicl, which binds to Smad proteins. Smicl and Smads cooperate and enhance TGF-beta mediated activation of a Smad-responsive reporter gene. A domain with five CCCH-type zinc fingers in Smicl is structurally and functionally, at least in vitro, similar to a domain in CPSF-30, the 30 kDa subunit of Cleavage and Polyadenylation Specificity Factor (CPSF). Like CPSF-30, Smicl can associate with some other CPSF subunits characterized previously. Its effect on the induction of a reporter gene for TGF-beta requires the cleavage/polyadenylation signal downstream of the coding sequence of that gene. Thus, Smicl is a novel protein that displays CPSF-30-like activities, interacts in the nucleus with activated Smads, and potentiates in TGF-beta stimulated cells Smad-dependent transcriptional responses, possibly in conjunction with the activity of CPSF complexes.


Subject(s)
Carrier Proteins/metabolism , Cleavage And Polyadenylation Specificity Factor/metabolism , Nuclear Proteins/metabolism , Activin Receptors, Type I/metabolism , Animals , Base Sequence , CHO Cells , COS Cells , Carrier Proteins/chemistry , Cells, Cultured , Chlorocebus aethiops , Cleavage And Polyadenylation Specificity Factor/chemistry , Cloning, Molecular , Cricetinae , Humans , Mice , Models, Biological , Molecular Sequence Data , Nuclear Proteins/chemistry , RNA Precursors/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Time Factors , Transcription, Genetic , Transfection , Transforming Growth Factor beta/metabolism , Two-Hybrid System Techniques
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