Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin-null mouse model

6 pages, 3 figures, 2 tables.-- Presented in part in abstract form at the 26th Annual Meeting of the European Thyroid Association, Milan, Italy, August 28 to September 1, 1999.-- This is part of the Ph.D. thesis of J.A.P., University of Porto, Porto, Portugal.Transthyretin (TTR) is the major T4-binding protein in rodents. Using a TTR-null mouse model we asked the following questions. 1) Do other T4 binding moieties replace TTR in the cerebrospinal fluid (CSF)? 2) Are the low whole brain total T4 levels found in this mouse model associated with hypothyroidism, e.g. increased 5'-deiodinase type 2 (D2) activity and RC3-neurogranin messenger RNA levels? 3) Which brain regions account for the decreased total whole brain T4 levels? 4) Are there changes in T3 levels in the brain? Our results show the following. 1) No other T4-binding protein replaces TTR in the CSF of the TTR-null mice. 2) D2 activity is normal in the cortex, cerebellum, and hippocampus, and total brain RC3-neurogranin messenger RNA levels are not altered. 3) T4 levels measured in the cortex, cerebellum, and hippocampus are normal. However T4 and T3 levels in the choroid plexus are only 14% and 48% of the normal values, respectively. 4) T3 levels are normal in the brain parenchyma. The data presented here suggest that TTR influences thyroid hormone levels in the choroid plexus, but not in the brain. Interference with the blood-choroid-plexus-CSF-TTR-mediated route of T4 entry into the brain caused by the absence of TTR does not produce measurable features of hypothyroidism. It thus appears that TTR is not required for T4 entry or for maintenance of the euthyroid state in the mouse brain.This work was supported by Grants PRAXIS (Portugal) SAU/2/96 and BIA/459/94.Peer reviewe

Directed Neural Differentiation of Mouse Embryonic Stem Cells Is a Sensitive System for the Identification of Novel Hox Gene Effectors

The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes

Graded Smad2/3 Activation Is Converted Directly into Levels of Target Gene Expression in Embryonic Stem Cells

The Transforming Growth Factor (TGF) β signalling family includes morphogens, such as Nodal and Activin, with important functions in vertebrate development. The concentration of the morphogen is critical for fate decisions in the responding cells. Smad2 and Smad3 are effectors of the Nodal/Activin branch of TGFβ signalling: they are activated by receptors, enter the nucleus and directly transcribe target genes. However, there have been no studies correlating levels of Smad2/3 activation with expression patterns of endogenous target genes in a developmental context over time. We used mouse Embryonic Stem (ES) cells to create a system whereby levels of activated Smad2/3 can be manipulated by an inducible constitutively active receptor (Alk4*) and an inhibitor (SB-431542) that blocks specifically Smad2/3 activation. The transcriptional responses were analysed by microarrays at different time points during activation and repression. We identified several genes that follow faithfully and reproducibly the Smad2/3 activation profile. Twenty-seven of these were novel and expressed in the early embryo downstream of Smad2/3 signalling. As they responded to Smad2/3 activation in the absence of protein synthesis, they were considered direct. These immediate responsive genes included negative intracellular feedback factors, like SnoN and I-Smad7, which inhibit the transcriptional activity of Smad2/3. However, their activation did not lead to subsequent repression of target genes over time, suggesting that this type of feedback is inefficient in ES cells or it is counteracted by mechanisms such as ubiquitin-mediated degradation by Arkadia. Here we present an ES cell system along with a database containing the expression profile of thousands of genes downstream of Smad2/3 activation patterns, in the presence or absence of protein synthesis. Furthermore, we identify primary target genes that follow proportionately and with high sensitivity changes in Smad2/3 levels over 15–30 hours. The above system and resource provide tools to study morphogen function in development

Arkadia Activates Smad3/Smad4-Dependent Transcription by Triggering Signal-Induced SnoN Degradation▿ †

E3 ubiquitin ligases play important roles in regulating transforming growth factor β (TGF-β)/Smad signaling. Screening of an E3 ubiquitin ligase small interfering RNA library, using TGF-β induction of a Smad3/Smad4-dependent luciferase reporter as a readout, revealed that Arkadia is an E3 ubiquitin ligase that is absolutely required for this TGF-β response. Knockdown of Arkadia or overexpression of a dominant-negative mutant completely abolishes transcription from Smad3/Smad4-dependent reporters, but not from Smad1/Smad4-dependent reporters or from reporters driven by Smad2/Smad4/FoxH1 complexes. We show that Arkadia specifically activates transcription via Smad3/Smad4 binding sites by inducing degradation of the transcriptional repressor SnoN. Arkadia is essential for TGF-β-induced SnoN degradation, but it has little effect on SnoN levels in the absence of signal. Arkadia interacts with SnoN and induces its ubiquitination irrespective of TGF-β/Activin signaling, but SnoN is efficiently degraded only when it forms a complex with both Arkadia and phosphorylated Smad2 or Smad3. Finally, we describe an esophageal cancer cell line (SEG-1) that we show has lost Arkadia expression and is deficient for SnoN degradation. Reintroduction of wild-type Arkadia restores TGF-β-induced Smad3/Smad4-dependent transcription and SnoN degradation in these cells, raising the possibility that loss of Arkadia function may be relevant in cancer

E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase

Arkadia (RNF111) is a positive regulator of the TGF-β signaling that mediates the proteasome-dependent degradation of negative factors of the pathway. It is classified as an E3 ubiquitin ligase and a SUMO-targeted ubiquitin ligase (STUBL), implicated in various pathological conditions including cancer and fibrosis. The enzymatic (ligase) activity of Arkadia is located at its C-terminus and involves the RING domain. Notably, E3 ligases require E2 enzymes to perform ubiquitylation. However, little is known about the cooperation of Arkadia with various E2 enzymes and the type of ubiquitylation that they mediate. In the present work, we study the interaction of Arkadia with the E2 partners UbcH5B and UbcH13, as well as UbcH7. Through NMR spectroscopy, we found that the E2–Arkadia interaction surface is similar in all pairs examined. Nonetheless, the requirements and factors that determine an enzymatically active E2–Arkadia complex differ in each case. Furthermore, we revealed that the cooperation of Arkadia with different E2s results in either monoubiquitylation or polyubiquitin chain formation via K63, K48, or K11 linkages, which can determine the fate of the substrate and lead to distinct biological outcomes

Unveiling the Essential Role of Arkadia’s Non-RING Elements in the Ubiquitination Process

Arkadia is a positive regulator of the TGFβ-SMAD2/3 pathway, acting through its C-terminal RING-H2 domain and targeting for degradation of its negative regulators. Here we explore the role of regions outside the RING domain (non-RING elements) of Arkadia on the E2-E3 interaction. The contribution of the non-RING elements was addressed using Arkadia RING 68 aa and Arkadia 119 aa polypeptides. The highly conserved NRGA (asparagine-arginine-glycine-alanine) and TIER (threonine-isoleucine-glutamine-arginine) motifs within the 119 aa Arkadia polypeptide, have been shown to be required for pSMAD2/3 substrate recognition and ubiquitination in vivo. However, the role of the NRGA and TIER motifs in the enzymatic activity of Arkadia has not been addressed. Here, nuclear magnetic resonance interaction studies with the E2 enzyme, UBCH5B, C85S UBCH5B-Ub oxyester hydrolysis, and auto-ubiquitination assays were used to address the role of the non-RING elements in E2-E3 interaction and in the enzymatic activity of the RING. The results support that the non-RING elements including the NRGA and TIER motifs are required for E2-E3 recognition and interaction and for efficient auto-ubiquitination. Furthermore, while Arkadia isoform-2 and its close homologue Arkadia 2C are known to interact with free ubiquitin, the results here showed that Arkadia isoform-1 does not interact with free ubiquitin