Stat3 Activates the Receptor Tyrosine Kinase Like Orphan Receptor-1 Gene in Chronic Lymphocytic Leukemia Cells

Background

The receptor tyrosine kinase like orphan receptor (ROR)-1 gene is overexpressed in chronic lymphocytic leukemia (CLL). Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors γ-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1.

Methodology/Principal Findings

Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells.

Conclusion/Significance

Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.     

Thrombopoietin regulates Bcl-xL gene expression through Stat5 and phosphatidylinositol 3-kinase activation pathways.

Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor kappa B-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor kappa B, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.     

STAT3 but Not STAT1 Is Required for Astrocyte Differentiation

The JAK-STAT signaling pathway has been implicated in astrocyte differentiation. Both STAT1 and STAT3 are expressed in the central nervous system and are thought to be important for glial differentiation, as mainly demonstrated in vitro; however direct in vivo evidence is missing. We investigated whether STAT1 and STAT3 are essential for astrocyte development by testing the STAT responsiveness of astrocyte progenitors. STAT3 was absent in the ventricular zone where glial progenitors are born but begins to appear at the marginal zone at E16.5. At E18.5, both phospho-STAT1 and phospho-STAT3 were present in glial fibrillary acidic protein (GFAP)-expressing white matter astrocytes. Overexpression of STAT3 by electroporation of chicks in ovo induced increased numbers of astrocyte progenitors in the spinal cord. Likewise, elimination of STAT3 in Stat3 conditional knockout (cKO) mice resulted in depletion of white matter astrocytes. Interestingly, elimination of STAT1 in Stat1 null mice did not inhibit astrocyte differentiation and deletion of Stat1 failed to aggravate the glial defects in Stat3 cKO mice. Measuring the activity of STAT binding elements and the gfap promoter in the presence of various STAT mutants revealed that transactivation depended on the activity of STAT3 not STAT1. No synergistic interaction between STAT1 and STAT3 was observed. Cortical progenitors of Stat1 null; Stat3 cKO mice generated astrocytes when STAT3 or the splice variant Stat3β was supplied, but not when STAT1 was introduced. Together, our results suggest that STAT3 is necessary and sufficient for astrocyte differentiation whereas STAT1 is dispensable.     

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.     

G2/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6–10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC₅₀ 0.48 μM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G₂/M phase in a manner dependent on its ability to bind GlcNAc.     

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number (($\text{0.26±0.03) · 10}{}^6\text{sites/cell}$) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites of Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by $\text{N-}\text{acetylgalactosamine}$, the determinant of Dolichos lectin, as well as by Dolichos antiserum.     

A novel cis-acting element in Her2 promoter regulated by Stat3 in mammary cancer cells

Stat3 plays important roles in the development of breast malignancies and oncogenesis. In the present study, a palindromic cis-acting element displaying repression activity in breast cancer cells expressing low level of Her2 was found in Her2 promoter. Deletion analysis showed that the novel element was located within Pal2 region spanning nucleotides -529 to -505. The sequence analysis of Pal2 region revealed a DNA sequence (TTAAGATAA) homologous to the binding site of Stat3, starting from position -529 to -521bp. By reporter assay, Pal2 was found to be regulated by constitutive activated Stat3C. A stimulatory effect both on Her2 mRNA and protein expressions was observed in MCF-7 cells stably expressing Stat3C, suggesting that Stat3 regulated Her2 expression. Using ChIP assays the binding of Stat3 to Her2 promoter was confirmed. The data obtained in this study indicate constitutive activated Stat3 regulates Her2 expression. Further investigation of differential effects of Stat3 exerting on breast cancer cells expressing Her2 at different levels will provide more insights into the roles of Stat3 in Her2 expression as well as the regulation of diverse biological activities.     

Purification and Characterization of a Mucin Specific Mycelial Lectin from Aspergillus gorakhpurensis: Application for Mitogenic and Antimicrobial Activity

Background

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis.

Methods

Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay.

Results

Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5–9.5, while optimum temperature for lectin activity was 20–30°C. Lectin was stable within a pH range of 7.0–10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae.

Conclusion

This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin.     

Genome-wide survey identifies TNNI2 as a target of KLF7 that inhibits chicken adipogenesis via downregulating FABP4

Krüppel-like factor 7 (KLF7) negatively regulates adipocyte differentiation; however, the mechanism underlying its activity in mammals and birds remains poorly understood. To identify genome-wide KLF7-binding motifs in preadipocytes, we conducted a chromatin immunoprecipitation-sequencing analysis of immortalized chicken preadipocytes (ICP2), which revealed 11,063 specific binding sites. Intergenic binding site analysis showed that KLF7 regulates several novel factors whose functions in chicken and mammal adipogenesis are underexplored. We identified a novel regulator, troponin I2 (TNNI2), which is positively regulated by KLF7. TNNI2 is downregulated during preadipocyte differentiation and acts as an adipogenic repressor at least in part by repressing FABP4 promoter activity. In conclusion, we demonstrated that KLF7 functions through cis-regulation of TNNI2, which inhibits adipogenesis. Our findings not only provide the first genome-wide picture of KLF7 associations in preadipocytes but also identify a novel function of TNNI2.