The regulatory proteins DSCR6 and Ezh2 oppositely regulate Stat3 transcriptional activity in mesoderm patterning during Xenopus development.

Embryonic cell fate specification and axis patterning requires integration of several signaling pathways that orchestrate region-specific gene expression. The transcription factor signal transducer and activator of transcription 3 (Stat3) plays important roles during early development, but it is unclear how Stat3 is activated. Here, using Xenopus as a model, we analyzed the post-translational regulation and functional consequences of Stat3 activation in dorsoventral axis patterning. We show that Stat3 phosphorylation, lysine methylation, and transcriptional activity increase before gastrulation and induce ventral mesoderm formation. Down syndrome critical region gene 6 (DSCR6), a RIPPLY family member that induces dorsal mesoderm by releasing repressive polycomb group proteins from chromatin, bound to the Stat3 C-terminal region and antagonized its transcriptional and ventralizing activities by interfering with its lysine methylation. Enhancer of zeste 2 polycomb-repressive complex 2 subunit (Ezh2) also bound to this region; however, its methyltransferase activity was required for Stat3 methylation and activation. Loss of Ezh2 resulted in dorsalization of ventral mesoderm and formation of a secondary axis. Furthermore, interference with Ezh2 phosphorylation also prevented Stat3 lysine methylation and transcriptional activity. Thus, inhibition of either Ezh2 phosphorylation or Stat3 lysine methylation compensated for the absence of DSCR6 function. These results reveal that DSCR6 and Ezh2 critically and post-translationally regulate Stat3 transcriptional activity. Ezh2 promotes Stat3 activation in ventral mesoderm formation independently of epigenetic regulation, whereas DSCR6 specifies dorsal fate by counteracting this ventralizing activity. This antagonism helps pattern the mesoderm along the dorsoventral axis, representing a critical facet of cell identity regulation during development.

Uraemia-induced immune senescence and clinical outcomes in chronic kidney disease patients.

BACKGROUND: Patients with chronic kidney disease (CKD) are more prone to develop premature age-related diseases. Data on immune senescence are scarce in CKD populations, except in end-stage renal disease and dialysis. We designed a longitudinal prospective study to evaluate immune senescence at different CKD stages and its influence on CKD patient outcomes. METHODS: Clinical and biological data collections were performed on 222 patients at different CKD stages [1-2 (n = 85), 4 (n = 53) and 5 (n = 84)]. Immune senescence biomarkers were measured by cytometry on T cells (CD28, CD57, CD45RA, CD31, γH2A.X) or by quantitative polymerase chain reaction [relative telomere length (RTL)] on peripheral blood mononuclear cells and analysed according to CKD stages and outcomes. RESULTS: CKD was associated with an increase in immune senescence and inflammation biomarkers, as follows: low thymic output (197 ± 25 versus 88 ± 13 versus 73 ± 21 CD4+CD45RA+CD31+ T cells/mm3), an increased proportion of terminally differentiated T cells (CD8+CD28-CD57+) (24 ± 18 versus 32 ± 17 versus 35 ± 19%) restricted to cytomegalovirus-positive patients, telomere shortening (1.11 ± 0.36 versus 0.78 ± 0.24 versus 0.97 ± 0.21 telomere:single copy ratio) and an increase in C-reactive protein levels [median 2.9 (range 1.8-4.9) versus 5.1 (27-9.6) versus 6.2 (3.4-10.5) mg/L]. In multivariate analysis, shorter RTL was associated with death {hazard ratio [HR] 4.12 [95% confidence interval (CI) 1.44-11.75]}. Low thymic output was associated with infections [HR 1.79 (95% CI (1.34-9.58)] and terminally differentiated CD8+ T-cell expansion with a risk of cardiovascular events [CEs; HR 4.86 (95% CI 1.72-13.72)]. CONCLUSION: CKD was associated with premature immune ageing. Each of these alterations increased the risk of specific age-related diseases, such as RTL and death, thymic function and infections and terminally differentiated CD8+ T-cell expansion and CEs.

Pretransplant thymic function predicts acute rejection in antithymocyte globulin-treated renal transplant recipients.

Lack of clear identification of patients at high risk of acute rejection hampers the ability to individualize immunosuppressive therapy. Here we studied whether thymic function may predict acute rejection in antithymocyte globulin (ATG)-treated renal transplant recipients in 482 patients prospectively studied during the first year post-transplant of which 86 patients experienced acute rejection. Only CD45RA(+)CD31(+)CD4(+) T cell (recent thymic emigrant [RTE]) frequency (RTE%) was marginally associated with acute rejection in the whole population. This T-cell subset accounts for 26% of CD4(+) T cells. Pretransplant RTE% was significantly associated with acute rejection in ATG-treated patients (hazard ratio, 1.04; 95% confidence interval, 1.01-1.08) for each increased percent in RTE/CD4(+) T cells), but not in anti-CD25 monoclonal (αCD25 mAb)-treated patients. Acute rejection was significantly more frequent in ATG-treated patients with high pretransplant RTE% (31.2% vs. 16.4%) or absolute number of RTE/mm(3) (31.7 vs. 16.1). This difference was not found in αCD25 monclonal antibody-treated patients. Highest values of both RTE% (>31%, hazard ratio, 2.50; 95% confidence interval, 1.09-5.74) and RTE/mm(3) (>200/mm(3), hazard ratio, 3.71; 95% confidence interval, 1.59-8.70) were predictive of acute rejection in ATG-treated patients but not in patients having received αCD25 monoclonal antibody). Results were confirmed in a retrospective cohort using T-cell receptor excision circle levels as a marker of thymic function. Thus, pretransplant thymic function predicts acute rejection in ATG-treated patients.

The Xenopus homologue of Down syndrome critical region protein 6 drives dorsoanterior gene expression and embryonic axis formation by antagonising polycomb group proteins.

Mesoderm and embryonic axis formation in vertebrates is mediated by maternal and zygotic factors that activate the expression of target genes. Transcriptional derepression plays an important role in the regulation of expression in different contexts; however, its involvement and possible mechanism in mesoderm and embryonic axis formation are largely unknown. Here we demonstrate that XDSCR6, a Xenopus homologue of human Down syndrome critical region protein 6 (DSCR6, or RIPPLY3), regulates mesoderm and embryonic axis formation through derepression of polycomb group (PcG) proteins. Xdscr6 maternal mRNA is enriched in the endoderm of the early gastrula and potently triggers the formation of dorsal mesoderm and neural tissues in ectoderm explants; it also dorsalises ventral mesoderm during gastrulation and induces a secondary embryonic axis. A WRPW motif, which is present in all DSCR6 homologues, is necessary and sufficient for the dorsal mesoderm- and axis-inducing activity. Knockdown of Xdscr6 inhibits dorsal mesoderm gene expression and results in head deficiency. We further show that XDSCR6 physically interacts with PcG proteins through the WRPW motif, preventing the formation of PcG bodies and antagonising their repressor activity in embryonic axis formation. By chromatin immunoprecipitation, we demonstrate that XDSCR6 releases PcG proteins from chromatin and allows dorsal mesoderm gene transcription. Our studies suggest that XDSCR6 might function to sequester PcG proteins and identify a novel derepression mechanism implicated in embryonic induction and axis formation.

New Insights on End-Stage Renal Disease and Healthy Individual Gut Bacterial Translocation: Different Carbon Composition of Lipopolysaccharides and Different Impact on Monocyte Inflammatory Response.

Chronic kidney disease induces disruption of the intestinal epithelial barrier, leading to gut bacterial translocation. Here, we appreciated bacterial translocation by analyzing circulating lipopolysaccharides (LPS) using two methods, one measuring only active free LPS, and the other quantifying total LPS as well as LPS lipid A carbon chain length. This was done in end-stage renal disease (ESRD) patients and healthy volunteers (HV). We observed both higher LPS concentration in healthy volunteers and significant differences in composition of translocated LPS based on lipid A carbon chain length. Lower LPS activity to mass ratio and higher concentration of high-density lipoproteins were found in HV, suggesting a better plasma capacity to neutralize LPS activity. Higher serum concentrations of soluble CD14 and pro-inflammatory cytokines in ESRD patients confirmed this hypothesis. To further explore whether chronic inflammation in ESRD patients could be more related to LPS composition rather than its quantity, we tested the effect of HV and patient sera on cytokine secretion in monocyte cultures. Sera with predominance of 14-carbon chain lipid A-LPS induced higher secretion of pro-inflammatory cytokines than those with predominance of 18-carbon chain lipid A-LPS. TLR4 or LPS antagonists decreased LPS-induced cytokine production by monocytes, demonstrating an LPS-specific effect. Thereby, septic inflammation observed in ESRD patients may be not related to higher bacterial translocation, but to reduced LPS neutralization capacity and differences in translocated LPS subtypes.

End-Stage Renal Disease-Associated Gut Bacterial Translocation: Evolution and Impact on Chronic Inflammation and Acute Rejection After Renal Transplantation.

Chronic inflammation in end-stage renal disease (ESRD) is partly attributed to gut bacterial translocation (GBT) due to loss of intestinal epithelium integrity. Increased levels of circulating lipopolysaccharide (LPS) -a surrogate marker of GBT- contribute to maintain a chronic inflammatory state. However, circulating LPS can be neutralized by lipoproteins and transported to the liver for elimination. While ESRD-associated GBT has been widely described, less is known about its changes and impact on clinical outcome after kidney transplantation (KT). One hundred and forty-six renal transplant recipients with serum samples obtained immediately before and 1 year after transplantation (1-Year post KT) were included. Intestinal epithelium integrity (iFABP), total LPS (by measuring 3-hydroxymyristate), LPS activity (biologically active LPS measured by the LAL assay), inflammatory biomarkers (sCD14 and cytokines), lipoproteins and LPS-binding proteins (LBP and phospholipid transfer protein [PLTP] activity) were simultaneously measured. At 1-Year post KT, iFABP decreased but remained higher than in normal volunteers. Total LPS concentration remained stable while LPS activity decreased. Inflammation biomarkers decreased 1-Year post KT. We concomitantly observed an increase in lipoproteins. Higher sCD14 levels before transplantation was associated with lower incidence of acute rejection. Although GBT remained stable after KT, the contemporary increase in lipoproteins could bind circulating LPS and contribute concomitantly to neutralization of LPS activity, as well as improvement in ESRD-associated chronic inflammation. Chronic exposure to LPS in ESRD could promote endotoxin tolerance and explain why patients with higher pre-transplant sCD14 are less prompt to develop acute rejection after transplantation.

Late Persistent Positive EBV Viral Load and Risk of Solid Cancer in Kidney Transplant Patients.

BACKGROUND: Recent studies reported that posttransplant Epstein-Barr virus (EBV) replication is frequent and indicates overimmunosuppression. We hypothesized that long-term EBV replication may identify overimmunosuppressed patients at higher risk of cancer. METHODS: We analyzed a prospective cohort of renal transplant recipients having routine EBV PCR surveillance. All cancers (except EBV-related neoplasia) were recorded. RESULTS: Mean follow up was 94 + 23 months. Samples (8412) were available in 669 patients. Three hundred eighty-eight of the 669 patients (58%) had at least 1 positive viremia during follow-up.Epstein-Barr virus D+/R- patients (P = 0.046) as well as those having received antithymocyte globulin (P < 0.001) were more likely to develop persistent EBV viremia. Eighty-six patients (12.9%) developed a cancer during follow-up. The cumulated incidence of cancer was higher in patients with persistent high EBV replication (22.4% vs 10.2%, P = 0.005). The effect of persistent EBV infection remained significant even after adjustment for all confounding factors (hazard ratio, 1.69; 95% confidence interval, 1.10-2.61; P = 0.018). Age, history of antithymocyte globulin use, smoking, and history of cancer were also associated with cancer occurrence. CONCLUSIONS: Persistent high EBV viral load is associated with the occurrence of solid cancer. In this setting, more intensive screening and/or minimization of immunosuppressive treatment are probably required.

Antagonistic interaction between IGF and Wnt/JNK signaling in convergent extension in Xenopus embryo.

The homeobox gene Otx2 is expressed during gastrulation in the anterior domain of the vertebrate embryo and is involved in neural and head induction during Xenopus early development. It also prevents convergent extension movements in trunk and posterior mesoderm. Insulin-like growth factors (IGFs) were shown to have similar function. However, whether they interact and the mechanism by which they affect convergent extension remain unclear. We show that IGF pathway specifically induces the expression of Otx2 in the early gastrula and blocks convergent extension of neuroectoderm and mesoderm through the transcriptional activation of Otx2 gene. Otx2 represses the expression of Xbra and Xwnt-11, and the effects of IGF on gastrulation movements can be partially rescued by antisense Otx2 morpholino oligonucleotide. These indicate that IGF pathway interacts with Otx2 to restrict Xbra and Xwnt-11 expression in the trunk and posterior regions. Consistent with this, we show that inhibition of IGF signaling or Otx2 function induces Xbra and Xwnt11 expression and convergent extension in ectodermal cells. Furthermore, the blockade of convergent extension by IGF-I and Otx2 can be rescued by coexpression of Xwnt-11 or a constitutively active Jun N-terminal kinase (JNK). Because Xbra and Xwnt-11 are required for convergent extension movements and Xwnt-11 activates the non-canonical Wnt-11/JNK pathway, our results reveal a mutually exclusive function between IGF and Wnt-11/JNK pathways in regulating cell behaviours during vertebrate head and trunk development.

Specification of anteroposterior axis by combinatorial signaling during Xenopus development.

The specification of anteroposterior (AP) axis is a fundamental and complex patterning process that sets up the embryonic polarity and shapes a multicellular organism. This process involves the integration of distinct signaling pathways to coordinate temporal-spatial gene expression and morphogenetic movements. In the frog Xenopus, extensive embryological and molecular studies have provided major advance in understanding the mechanism implicated in AP patterning. Following fertilization, cortical rotation leads to the transport of maternal determinants to the dorsal region and creates the primary dorsoventral (DV) asymmetry. The activation of maternal Wnt/ß-catenin signaling and a high Nodal signal induces the formation of the Nieuwkoop center in the dorsal-vegetal cells, which then triggers the formation of the Spemann organizer in the overlying dorsal marginal zone. It is now well established that the Spemann organizer plays a central role in building the vertebrate body axes because it provides patterning information for both DV and AP polarities. The antagonistic interactions between signals secreted in the Spemann organizer and the opposite ventral region pattern the mesoderm along the DV axis, and this DV information is translated into AP positional values during gastrulation. The formation of anterior neural tissue requires simultaneous inhibition of zygotic Wnt and bone morphogenetic protein (BMP) signals, while an endogenous gradient of Wnt, fibroblast growth factors (FGFs), retinoic acid (RA) signaling, and collinearly expressed Hox genes patterns the trunk and posterior regions. Collectively, DV asymmetry is mostly coupled to AP polarity, and cell-cell interactions mediated essentially by the same regulatory networks operate in DV and AP patterning. For further resources related to this article, please visit the WIREs website.

The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development.

RNA-binding proteins play an important role to post-transcriptionally regulate gene expression. During early development they exhibit temporally and spatially regulated expression pattern. The expression of Xenopus laevis Seb4 gene, also known as RBM24 in other vertebrates, is restricted to the lateral and ventral mesoderm during gastrulation and then localized to the somitic mesoderm, in a similar pattern as XMyoD gene. Using a hormone-inducible form of MyoD to identify potential direct MyoD target genes, we find that Seb4 expression is directly regulated by MyoD at the gastrula stage. We further show that a 0.65kb X. tropicalis RBM24 regulatory region contains multiple E boxes (CANNTG), which are potential binding sites for MyoD and other bHLH proteins. By injecting a RBM24 reporter construct into the animal pole of X. laevis embryos, we find that this reporter gene is indeed specifically activated by MyoD and repressed by a dominant negative MyoD mutant. Knockdown of Seb4 produces similar effects as those obtained by the dominant negative MyoD mutant, indicating that it is required for the expression of myogenic genes and myogenesis in the embryo. In cultured ectodermal explants, although overexpression of Seb4 has no obvious effect on myogenesis, knockdown of Seb4 inhibits the expression of myogenic genes and myogenesis induced by MyoD. These results reveal that Seb4 is a target of MyoD during myogenesis and is required for myogenic gene expression.

Dynamic expression pattern of distinct genes in the presomitic and somitic mesoderm during Xenopus development.

Whole-mount and sectioned in situ hybridization was used to identify genes with restricted expression pattern in the presomitic and somitic mesoderm during Xenopus early development. Here we report the dynamic expression pattern of six distinct genes differentially expressed in these regions. These include Xenopus homologues of purine nucleoside phosphorylase, acetylcholine receptor, aspartate aminotransferase, glycine amidinotransferase and brain and muscle isoform creatine kinases. Purine nucleoside phosphorylase was initially expressed in the marginal zone at gastrula stage and then localized to the tail bud. Although the other genes showed no significantly localized expression at gastrula or neurula stages, they are progressively restricted in the somitic mesoderm as development proceeds. A common feature for these genes is that their deficiency in humans leads to an impairment of several tissue or cell functions. An analysis of their expression pattern could provide information regarding their implication in early development.

Down syndrome critical region protein 5 regulates membrane localization of Wnt receptors, Dishevelled stability and convergent extension in vertebrate embryos.

The Glypican family of heparan sulfate proteoglycans regulates Wnt signaling and convergent extension (CE) in vertebrate embryos. They are predicted to be glycosylphosphatidylinositol (GPI)-tethered membrane-bound proteins, but there is no functional evidence of their regulation by the GPI synthesis complex. Down syndrome critical region protein 5 (Dscr5, also known as Pigp) is a component of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex, and is associated with specific features of Down syndrome. Here we report that Dscr5 regulates CE movements through the non-canonical Wnt pathway. Both dscr5 overexpression and knockdown impaired convergence and extension movements. Dscr5 functionally interacted with Knypek/Glypican 4 and was required for its localization at the cell surface. Knockdown of dscr5 disrupted Knypek membrane localization and caused an enhanced Frizzled 7 receptor endocytosis in a Caveolin-dependent manner. Furthermore, dscr5 knockdown promoted specific Dishevelled degradation by the ubiquitin-proteosome pathway. These results reveal a functional link between Knypek/Glypican 4 and the GPI synthesis complex in the non-canonical Wnt pathway, and provide the new mechanistic insight that Dscr5 regulates CE in vertebrate embryos by anchoring different Wnt receptors at the cell surface and maintaining Dishevelled stability.

FGF8, Wnt8 and Myf5 are target genes of Tbx6 during anteroposterior specification in Xenopus embryo.

The T-box transcription factor Tbx6 is required for somite formation and loss-of-function or reduced activity of Tbx6 result in absence of posterior paraxial mesoderm or disorganized somites, but how it is involved in a regulatory hierarchy during Xenopus early development is not clear. We show here that Tbx6 is expressed in the lateral and ventral mesoderm of early gastrula, and it is necessary and sufficient to directly and indirectly regulate the expression of a subset of early mesodermal and endodermal genes. Ectopic expression of Tbx6 inhibits early neuroectodermal gene expression by strongly inducing the expression of posterior mesodermal genes, and expands the mesoderm territory at the expense of neuroectoderm. Conversely, overexpression of a dominant negative Tbx6 mutant in the ventral mesoderm inhibits the expression of several mesodermal genes and results in neural induction in a dose-dependent manner. Using a hormone-inducible form of Tbx6, we have identified FGF8, Xwnt8 and XMyf5 as immediate early responsive genes of Tbx6, and the induction of these genes by Tbx6 is independent of Xbra and VegT. These target genes act downstream and mediate the function of Tbx6 in anteroposterior specification. Our results therefore identify a regulatory cascade governed by Tbx6 in the specification of posterior mesoderm during Xenopus early development.

v-SRC specifically regulates the nucleo-cytoplasmic delocalization of the major isoform of TEL (ETV6).

TEL is a frequent target of chromosomal translocations in human cancer and an alleged tumor suppressor gene. TEL encodes two isoforms: a major TEL-M1 isoform as well as TEL-M43, which lacks the first 42 amino acid residues of TEL-M1. Both isoforms are potent transcriptional repressors that can inhibit RAS-induced transformation. Here we show that the v-SRC protein-tyrosine kinase relieves the repressive activity of TEL-M1, an activity that is associated with the v-SRC-induced delocalization of TEL-M1 from the nucleus to the cytoplasm. TEL-M1 delocalization requires the kinase activity of v-SRC and is not induced by oncogenic RAS or AKT. Cytoplasmic delocalization of TEL-M1 in response to v-SRC critically depends upon its unique amino-terminal domain (SRCD domain) because (i). v-SRC did not inhibit the repressive properties of TEL-M43, nor affected TEL-M43 nuclear localization; (ii). fusion of the first 52 amino acid residues of TEL-M1 to FLI-1, an ETS protein insensitive to v-SRC-induced delocalization, is sufficient to confer v-SRC-induced delocalization to this TEL/FLI-1 chimeric protein. The v-SRC-induced nucleo-cytoplasmic delocalization of TEL-M1 does not involve phosphorylation of the SRCD and does not require TEL self-association and repressive domains. Finally, enforced expression of the v-SRC-insensitive TEL-M43, but not of TEL-M1, inhibits v-SRC-induced transformation of NIH3T3 fibroblasts. These results identify a regulatory domain in TEL that specifically impinges on the subcellular localization of its major TEL-M1 isoform. They, furthermore, indicate that inhibition of TEL-M1 nuclear function is required for v-SRC to induce cellular transformation.

Frizzled receptor dimerization is sufficient to activate the Wnt/beta-catenin pathway.

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activate beta-catenin-dependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/beta-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/beta-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/beta-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/beta-catenin signal.