P-selectin glycoprotein ligand 1 promotes T cell lymphoma development and dissemination.

P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.

Coenzyme Q production by metabolic engineered Escherichia coli strains in defined medium.

Coenzyme Q (CoQ) plays an important role as an electron transporter in the respiratory chain. It is formed from a benzoquinone ring and an isoprenoid chain of a specific length depending on the organism. We constructed an engineered Escherichia coli strain (menF) unable to produce demethylmenaquinone and menaquinone, compounds that compete for both chorismate, precursor of the benzoquinone ring, and the isoprenoid chain involved in CoQ biosynthesis. In addition, a mutant strain (entC) unable to produce enterobactin, high-affinity siderophore, synthesized from chorismate, and a double mutant (entC-menF) were constructed. The use of glucose or glycerol as carbon sources was also evaluated for the production of CoQ8 in these strains. The double mutant (entC-menF) showed 18% increase in CoQ8-specific content compared to the control strain; however, the single-mutant strains did not show statistically significant differences in CoQ8-specific content respect to the control, in glucose medium in bioreactor experiments. Glycerol was significantly superior compared to glucose for the production of CoQ8 in E. coli, where the CoQ8-specific content increased 126% and 53% in the control and double-mutant strain, respectively. The expression of genes related to CoQ8 biosynthesis is reported, where the entC-menF double-mutant strain showed a significant increase in the expression of CoQ8 biosynthesis-related genes when glycerol was used as sole carbon source. The control strain did not show gene expression difference between both carbon sources, indicating a possible regulation at a different level.

NF-κB-dependent RANKL expression in a mouse model of immature T-cell leukemia.

Activation of the receptor activator of nuclear factor-κB (RANK) by its ligand (RANKL) is involved in both solid and hematological malignancies, including multiple myeloma, acute myeloid leukemia and B-cell leukemia. Although RANKL expression has been described in normal T cells, a potential role in T-cell leukemia remains undefined. Here, we used a model of immature T-cell leukemia/lymphoma, the TEL-JAK2 transgenic mice, to assess RANKL expression in leukemic cells and its regulatory mechanisms. We found that Rankl mRNA was significantly overexpressed in leukemic T cells when compared to wild-type thymocytes, their nonmalignant counterparts. Moreover, Rankl mRNA and RANKL surface expression in leukemic cells was induced by T-cell receptor (TCR) signaling activation, dependently on the NF-κB signaling pathway. These results indicate that TCR-activated leukemic T cells express high levels of RANKL and are potential inducers of RANK signaling in microenvironmental cells.

FoxN1-dependent thymic epithelial cells promote T-cell leukemia development.

T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphomas (T-LBL) are aggressive malignancies of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL/LBL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition of Notch1 mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin (Krt) 5 and Krt8 expression, adjacently to epithelial-free areas negative for Krt expression. Both areas contained abundant laminin (extracellular matrix) and ER-TR7+ (fibroblasts) CD31+ (endothelial) and CD11c+ (dendritic) cells. Besides Krt5, Krt-positive areas harbored medullary thymic epithelial cells (TECs) labeled by Ulex europaeus agglutinin-1. By performing flow cytometry and RNA sequencing analyses of thymic lymphomas, we observed an enrichment in medullary TEC markers in detriment of cortical TEC markers. To assess whether TECs are important for T-ALL/LBL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factor nude null mutation (Foxn1+/nu). Strikingly, in TJ2-Tg;Foxn1+/nu compound mice, both emergence of malignant cells in preleukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays, leukemic cell expansion within the thymus of recipient Foxn1+/nu mice was reduced as compared with control littermates. Since thymopoesis is largely normal in Foxn1+/nu mice, these results indicate that FoxN1 haploinsufficiency in TECs has a more profound impact in thymic leukemogenesis.

Different responses in the expression of alginases, alginate polymerase and acetylation genes during alginate production by Azotobacter vinelandii under oxygen-controlled conditions.

Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate [Formula: see text] from 10.9 to 45.3 mmol g-1 h-1 by changes in the dilution rate (D) from 0.06 to 0.10 h-1, whereas under 1% DOT the [Formula: see text] was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.

CITED2 Cooperates with ISL1 and Promotes Cardiac Differentiation of Mouse Embryonic Stem Cells.

The transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC.

FBXL5 modulates HIF-1α transcriptional activity by degradation of CITED2.

CITED2 is a ubiquitously expressed nuclear protein exhibiting a high affinity for the cysteine-histidine-rich domain 1 (CH1) of the transcriptional co-activators CBP/p300. CITED2 is particularly efficient in the inhibition of the hypoxia-inducible factor-1α (HIF-1α) dependent transcription by competing with it for the interaction with the CH1 domain. Here we report a direct and specific interaction between CITED2 and the F-box and leucine rich repeat protein 5 (FBXL5), a substrate adaptor protein which is part of E3 ubiquitin ligase complexes mediating protein degradation by the proteasome. We demonstrated that depletion of FBXL5 by RNA interference led to an increase of CITED2 protein levels. Conversely, overexpression of FBXL5 caused the decrease of CITED2 protein levels in a proteasome-dependent manner, and impaired the interaction between CITED2 and the CH1 domain of p300 in living cells. In undifferentiated mouse embryonic stem cells, the overexpression of FBXL5 also reduced Cited2 protein levels. Finally, we evidenced that FBXL5 overexpression and the consequent degradation of CITED2 enabled the transcriptional activity of the N-terminal transactivation domain of HIF-1α. Collectively, our results highlighted a novel molecular interaction between CITED2 and FBXL5, which might regulate the steady state CITED2 protein levels and contribute to the modulation of gene expression by HIF-1α.

StemChecker: a web-based tool to discover and explore stemness signatures in gene sets.

Stem cells present unique regenerative abilities, offering great potential for treatment of prevalent pathologies such as diabetes, neurodegenerative and heart diseases. Various research groups dedicated significant effort to identify sets of genes-so-called stemness signatures-considered essential to define stem cells. However, their usage has been hindered by the lack of comprehensive resources and easy-to-use tools. For this we developed StemChecker, a novel stemness analysis tool, based on the curation of nearly fifty published stemness signatures defined by gene expression, RNAi screens, Transcription Factor (TF) binding sites, literature reviews and computational approaches. StemChecker allows researchers to explore the presence of stemness signatures in user-defined gene sets, without carrying-out lengthy literature curation or data processing. To assist in exploring underlying regulatory mechanisms, we collected over 80 target gene sets of TFs associated with pluri- or multipotency. StemChecker presents an intuitive graphical display, as well as detailed statistical results in table format, which helps revealing transcriptionally regulatory programs, indicating the putative involvement of stemness-associated processes in diseases like cancer. Overall, StemChecker substantially expands the available repertoire of online tools, designed to assist the stem cell biology, developmental biology, regenerative medicine and human disease research community. StemChecker is freely accessible at http://stemchecker.sysbiolab.eu.

Acute loss of Cited2 impairs Nanog expression and decreases self-renewal of mouse embryonic stem cells.

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.