Factor Transcriptional Activity Research Articles

Zic1 transcription factor in bone: neural developmental protein regulates mechanotransduction in osteocytes

A transcriptome analysis compared gene expression in human bone biopsy samples taken from lumbar spine and iliac crest, sites that experience high and low levels of mechanical stress, respectively. The analysis revealed that the zinc finger protein of cerebellum (Zic) family member transcription factor Zic1 was the most up-regulated gene in the lumbar spine (202-fold; P<10(-7)) in comparison with the iliac crest. Software analysis of differential gene expression in the biopsy samples identified the ciliary-related proteins PATCH1 and GLI-Kruppel family members Gli1 and Gli3 as part of a potential molecular network associated with Zic1. RT-PCR confirmed the expression of Zic1, Gli1, and Gli3 and other related key signaling mediators in osteoblastic cells and osteocytes in vitro. Zic1 was immunolocalized in the cytosol and nucleus of the murine osteocyte cell line MLO-Y4 and osteoblast-like cells MC3T3-E1 and in primary rat osteoblasts. MLO-Y4 cells subjected to prolonged oscillatory fluid flow showed increased localization of Zic1 in the nucleus with diminished levels in the cytosol, but no such changes were seen in MC3T3-E1 cells. A shear stress-induced increase in T-cell factor/lymphoid enhancer factor transcriptional activity was abolished by Zic1 gene silencing. These results suggest that Zic1, perhaps together with Gli1 and Gli3, may act as a link between mechanosensing and Wnt signaling. We conclude that Zic1, a neural developmental transcription factor, plays an important role in shear flow mechanotransduction in osteocytes.

Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-beta1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-1 have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3beta and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-beta1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3beta phosphorylation. GSK-3beta inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3beta mutant blocked BMP-4-, TGF-beta1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3beta phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-beta1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3beta-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-beta1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH.

Activation of NR2A Receptors Induces Ischemic Tolerance through CREB Signaling

Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

Wilms tumor cells with WT1 mutations have characteristic features of mesenchymal stem cells and express molecular markers of paraxial mesoderm

Wilms tumors (WTs) are genetically heterogeneous kidney tumors whose cells of origin are unknown. Tumors with WT1 mutations and concomitant loss of the wild-type allele represent a distinct subgroup, frequently associated with mutations in CTNNB1. Here, we describe the establishment and characterization of long-term cell cultures derived from five individual WTs with WT1 mutations. Three of these tumor cell lines also had CTNNB1 mutations and an activated canonical Wnt signaling pathway as measured by beta-catenin/T cell-specific transcription factor (TCF) transcriptional activity. Four of the five Wilms cell lines had a stable normal karyotype for at least 25 passages, and four lines showed loss of heterozygosity of chromosome 11p due to mitotic recombination in 11p11. Gene expression profiling revealed that the WT cell lines are highly similar to human mesenchymal stem cells (MSCs) and FACS analysis demonstrated the expression of MSC-specific surface proteins CD105, CD90 and CD73. The stem cell like nature of the WT cells is further supported by their adipogenic, chondrogenic, osteogenic and myogenic differentiation potentials. By generating multipotent mesenchymal precursors from paraxial mesoderm (PAM) in tissue culture using embryonal stem cells, gene expression profiles of PAM and MSCs were described. Using these published gene sets, we found coexpression of a large number of genes in WT cell lines, PAM and MSCs. Lineage plasticity is indicated by the simultaneous expression of genes from the mesendodermal and neuroectodermal lineages. We conclude that WTs with WT1 mutations have specific traits of PAM, which is the source of kidney stromal cells.

The low-density lipoprotein receptor-related protein 10 is a negative regulator of the canonical Wnt/β-catenin signaling pathway

Wnt signaling pathways play fundamental roles in the differentiation, proliferation and functions of many cells as well as developmental, growth, and homeostatic processes in animals. Low-density lipoprotein receptor (LDLR)-related protein (LRP) 5 and LRP6 serve as coreceptors of Wnt proteins together with Frizzled receptors, triggering activation of canonical Wnt/β-catenin signaling. Here, we found that LRP10, a new member of the LDLR gene family, inhibits the canonical Wnt/β-catenin signaling pathway. The β-catenin/T cell factor (TCF) transcriptional activity in HEK293 cells was activated by transfection with Wnt3a or LRP6, which was then inhibited by co-transfection with LRP10. Deletion of the extracellular domain of LRP10 negated its inhibitory effect. The inhibitory effect of LRP10 was consistently conserved in HEK293 cells even when GSK3β phosphorylation was inhibited by incubation with lithium chloride and co-transfection with constitutively active S33Y-mutated β-catenin. Nuclear β-catenin accumulation was unaffected by LRP10. The present studies suggest that LRP10 may interfere with the formation of the β-catenin/TCF complex and/or its binding to target DNA in the nucleus, and that the extracellular domain of LRP10 is critical for inhibition of the canonical Wnt/β-catenin signaling pathway.

Functional Identification of a C-repeat Binding Factor Transcriptional Activator from Blueberry Associated with Cold Acclimation and Freezing Tolerance

Highbush blueberry (Vaccinium corymbosum) is susceptible to winter freezing injury and frost damage in the spring. As part of an ongoing project to understand the process of cold acclimation, we isolated a C-repeat binding factor (CBF) transcriptional activator gene-coding region from the highbush blueberry cultivar Bluecrop. Expression of the highbush blueberry CBF gene was compared in floral buds of the cold-tolerant northern highbush cultivar Bluecrop and the more cold-sensitive southern rabbiteye (V. virgatum) blueberry cultivar Tifblue. Relative gene expression was higher in ‘Bluecrop’ than in ‘Tifblue’. Expression in both cultivars was highest at the earliest time point in the fall (coincident with the first stage of cold acclimation), declined during the later fall and winter, and, in ‘Bluecrop’, increased again as buds deacclimated, when temperatures tend to fluctuate. To confirm the putative identity of the gene as a member of the CBF gene family, and to determine if expression in a heterologous system could enhance freezing tolerance, the blueberry gene coding sequence was overexpressed in transgenic Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Transgenic plants expressing the putative blueberry CBF gene exhibited induced expression of the A. thaliana cold-regulated (COR) genes COR78 and COR6.6, under non-inducing conditions (i.e., 23 °C); however, expression of two other COR genes was unaffected. Transgenic plants also exhibited enhanced freezing tolerance under non-acclimated conditions, but not to the level of acclimated control plants. Thus, the expression pattern in floral buds and the ability of the isolated gene to turn on a subset of COR genes and increase freezing tolerance in a heterologous system suggest it is a functional member of the CBF gene family in blueberry.

Lack of conservation of bacterial type promoters in plastids of Streptophyta

We demonstrate the scarcity of conserved bacterial-type promoters in plastids of Streptophyta and report widely conserved promoters only for genes psaA, psbA, psbB, psbE, rbcL. Among the reasonable explanations are: evolutionary changes of sigma subunit paralogs and phage-type RNA polymerases possibly entailing the loss of corresponding nuclear genes, de novo emergence of the promoters, their loss together with plastome genes; functional substitution of the promoter boxes by transcription activation factor binding sites.ReviewersThis article was reviewed by Dr. Arcady Mushegian, and by Dr. Alexander Bolshoy and Dr. Yuri Wolf (both nominated by Dr. Purificación López-García).

DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA contacts. We use this effect to establish the separate contributions of transcription factor binding and DNA dynamics to transcriptional activity. Our results argue against a purely ‘transcription factor-centric’ view of transcription initiation, suggesting that both DNA dynamics and transcription factor binding are necessary conditions for transcription initiation.

Short hairpin RNA targeting β-catenin suppresses cell proliferation and induces apoptosis in human gastric carcinoma cells

Objective. Aberrant activation of Wnt/β-catenin signaling is involved in various cancers, including human gastric cancer. Here we investigate the role of Wnt/β-catenin signaling in regulating gastric cancer cell apoptosis. Material and methods. Expression of β-catenin was investigated after transfection with β-catenin short hairpin RNA (shRNA) in gastric cancer cells by Western blotting and immunofluorescence analysis. β-catenin/T-cell factor transcriptional activity was also investigated by using a luciferase reporter assay. Next, the effects of β-catenin shRNA on cell proliferation and apoptosis were evaluated by the 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide assay and flow cytometric analysis. To investigate the precise mechanism of these effects, a comprehensive analysis was performed using a cDNA microarray. Results. shRNA targeting β-catenin resulted in a significant decrease in β-catenin expression, and its nuclear localization and cell proliferation. Meanwhile, increased cell apoptosis was confirmed. The comprehensive analysis showed that shRNA targeting β-catenin upregulated 26 apoptosis-related genes (including PERP, TRAF3, PDCD2, TNFRSF25, AKT2 and YWHAZ) and downregulated 48 apoptosis-related genes (including MALT1, IRAK1, TNFAIP3, PPP1R13L, TRIP and YWHAB) in gastric cancer cells. Pathway analysis suggested that the nuclear factor-kappaB pathway was involved in β-catenin knockdown-induced apoptosis. Conclusions. Attenuation of β-catenin by shRNA resulted in suppressed cell proliferation and apparent apoptosis, suggesting that β-catenin may be a target for therapy of gastric cancer.

AV-65, a Novel Inhibitor of the Wnt/β-Catenin Signaling Pathway, Inhibits the Proliferation of Myeloma Cells.

Abstract 2866Poster Board II-842Although new molecular targeting agents against multiple myeloma (MM) have been developed, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. β-catenin is the downstream effector of Wnt signaling and it regulates genes implicated in malignant progression. We have demonstrated that blockade of Wnt/β-catenin signaling pathway inhibits the progression of MM by using RNA interference methods with an in vivo mouse model (Ashihara E, et al. Clin Cancer Res 15:2731, 2009.). In this study, we investigated the effects of AV-65, a novel inhibitor of the Wnt/β-catenin signaling pathway, on MM cells.The system to identify a series of small molecule compounds using a biomarker driven approach has been established. A gene expression biomarker signature reporting on the inhibition of Wnt/β-catenin signaling was generated upon treatment of a colon cancer cell line with β-catenin siRNA. This gene expression signatiure was used to screen a small molecule compound library to identify compounds which mimic knockdown of β-catenin and thus potentially inhibit the Wnt/β-catenin signaling pathway. One compound series, LC-363, was discovered from this screen and validated as novel Wnt/β-catenin signaling inhibitors (Strovel JW, et al. ASH meeting, 2007.). We investigated the inhibitory effects of AV-65, one of LC-363 compounds, on MM cell proliferation. AV-65 inhibited the proliferation of MM cells in a time- and a dose-dependent manner and the values of IC50 at 72 hrs were ranging from 11.7 to 82.1 nM. AV-65 also showed an inhibitory effect on the proliferation of RPMI8226/LR-5 melphalan-resistant MM cells (provided from Dr. William S. Dalton). In flow cytometric analysis, apoptotic cells were increased by AV-65 treatment in a time- and a dose-dependent manner. Western blotting analysis showed that β-catenin was ubiquitinated and that the expression of nuclear β-catenin diminished (Figure 1). Moreover, AV-65 suppressed T-cell factor transcriptional activities, resulting in the decrease of c-myc expression. Taken together, AV-65 promotes the degradation of β-catenin, resulting in the induction of apoptosis of MM cells.We next investigated the in vivo effects of AV-65 using an orthotopic MM-bearing mouse model. AV-65 inhibits the growth of MM cells and significantly prolongs the survival rates (Figure 2). In conclusion, AV-65 inhibited the proliferation of MM cells via inhibition of the Wnt/β-catenin signaling pathway. AV-65 is a promising therapeutic agent for treatment of MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr Virus Latent Membrane Protein 1 is not Associated with Vessel Density nor with Hypoxia Inducible Factor 1 Alpha Expression in Nasopharyngeal Carcinoma Tissue

Hypoxia-inducible factor-1alpha (HIF-1alpha) and the neo-angiogenic factors induced as a result of hypoxia-inducible factor transcriptional activation may contribute to tumorigenesis by inducing vessel formation that in turn provides oxygen and nutrients promoting tumor expansion. In vitro studies of nasopharyngeal carcinoma (NPC), an aggressive malignancy that is nearly always infected by Epstein-Barr virus, show HIF-1alpha is upregulated by viral latent membrane protein 1 (LMP1). The current study used immunohistochemistry to examine the extent to which HIF-1alpha and LMP1 are co-expressed in naturally infected NPC tissues. Analytic procedures were optimized for sensitive localization of HIF-1alpha and LMP1 in fixed tissue sections using immunohistochemistry with sensitive fluorescent and signal amplification technologies. Vessel density was quantified by CD31 immunohistochemistry. LMP1 was expressed focally in all 18 NPCs examined, including 7/8 in situ lesions. There was no consistent co-localization with HIF-1alpha which was usually only weakly expressed in a subset of neoplastic cells. Neither LMP1 nor HIF-1alpha expression correlated with vessel density, and degree of vascularization varied widely among cases. Advanced immunohistochemical technologies reveal that LMP1 is expressed more commonly than previously reported in NPC. There is no consistent relationship between LMP1 and either HIF-1alpha expression or degree of microvasculature. The biologic basis for the wide variation in vessel density deserves further investigation.

Repression of classical nuclear export by S-nitrosylation of CRM1

The karyopherin chromosomal region maintenance 1 (CRM1) is the major receptor for classical nuclear protein export. However, little is known about the regulation of CRM1 itself. Here, we report that cellular CRM1 became S-nitrosylated after extensive exposure to endogenous or exogenous nitric oxide (NO). This abrogated the interaction of CRM1 with nuclear export signals (NESs) and repressed classical protein export. Analysis by mass spectrometry and involving the use of S-nitrosylation mimetic mutations indicated that modification at either of two specific cysteines of CRM1 was sufficient to abolish the CRM1-NES association. Moreover, ectopic overexpression of the corresponding S-nitrosylation-resistant CRM1 mutants rescued NO-induced repression of nuclear export. We also found that inactivation of CRM1 by NO facilitated the nuclear accumulation of the antioxidant response transcription factor Nrf2 and transcriptional activation of Nrf2-controlled genes. Together, these data demonstrate that CRM1 is negatively regulated by S-nitrosylation under nitrosative stress. We speculate that this is important for promoting a cytoprotective transcriptional response to nitrosative stress.

The transcriptional activity of Pygopus is enhanced by its interaction with cAMP-response-element-binding protein (CREB)-binding protein

Pygopus is a core component of the beta-catenin/TCF (T-cell factor) transcriptional activation complex required for the expression of canonical Wnt target genes. Recent evidence suggests that Pygopus could interpret histone methylation associated with target genes and it was shown to be required for histone acetylation. The involvement of a specific acetyltransferase, however, was not determined. In this report, we demonstrate that Pygopus can interact with the HAT (histone acetyltransferase) CBP [CREB (cAMP-responsive-element-binding protein)-binding protein]. The interaction is via the NHD (N-terminal homology domain) of Pygopus, which binds to two regions in the vicinity of the HAT domain of CBP. Transfected and endogenous hPygo2 (human Pygopus2) and CBP proteins co-immunoprecipitate in HEK-293 (human embryonic kidney 293) cells and both proteins co-localize in SW480 colorectal cancer cells. The interaction with CBP also enhances both DNA-tethered and TCF/LEF1 (lymphoid enhancing factor 1)-dependent transcriptional activity of Pygopus. Furthermore, immunoprecipitated Pygopus protein complexes displayed CBP-dependent histone acetyltransferase activity. Our data support a model in which the NHD region of Pygopus is required to augment TCF/beta-catenin-mediated transcriptional activation by a mechanism that includes both transcriptional activation and histone acetylation resulting from the recruitment of the CBP histone acetyltransferase.

A-form DNA structure is a determinant of transcript levels from the Xenopus gata2 promoter in embryos

We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.

Glucose-dependent Insulinotropic Polypeptide and Glucagon-like Peptide-1 Modulate β-Cell Chromatin Structure

Chromatin can exert a regulatory effect on gene transcription by modulating the access of transcription factors to target genes. In the present study, we examined whether nuclear actions of the incretin hormones, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1, involve modulation of beta-cell chromatin structure. Stimulation of INS-1(832/13) beta-cells or dispersed mouse islets with glucose-dependent insulinotropic polypeptide or glucagon-like peptide-1 resulted in the post-translational modification of core H3 histones, through acetylation and phosphorylation. Both increased histone H3 acetyltransferase and reduced histone deacetylase activities contributed. Subsequent studies demonstrated that incretin-mediated histone H3 modifications involved activation of protein kinase A, p42/44 mitogen-activated protein kinase (MAPK), and p38 MAPK signaling modules, resulting in the activation of mitogen- and stress-activated kinase-1. Additionally, modification of histone H3 increased its association with the transcription factor, phosphorylated cAMP-response element-binding protein (phospho-CREB) and with cAMP-responsive CREB coactivator 2. Incretin-activated CREB-related Bcl-2 transcription was greatly reduced by a histone acetyltransferase inhibitor, demonstrating the functional importance of histone H3 modification. This appears to be the first demonstration of beta-cell chromatin modification in response to the incretins and the studies indicate that their regulatory effects involve coordinated nuclear interactions between specific signaling modules, chromatin-modifying enzymes and transcription factors.

Activator protein 2α suppresses intestinal tumorigenesis in the Apcmin mouse

Activator protein 2α (AP-2α) is a putative tumor suppressor, and various reports have described the loss or reduction of AP-2α expression in cutaneous malignant melanomas, as well as in cancers of the prostate, breast and colon. Previously, AP-2α was shown to attenuate β-catenin/T-cell factor-4 (TCF-4) nuclear interactions and β-catenin/TCF-4-dependent transcriptional activity in human colorectal cancer cells [Q. Li, R.H. Dashwood, Activator protein 2alpha associates with adenomatous polyposis coli/beta-catenin and Inhibits beta-catenin/T-cell factor transcriptional activity in colorectal cancer cells, J. Biol. Chem. 279 (2004) 45669–45675]. Here, we show that in vivo gene delivery of AP-2α suppressed intestinal polyp formation in the Apc min mouse, and protected against the development of anemia and splenomegaly. Immunoblot analyses and immunohistochemistry following gene delivery revealed an increase in AP-2α expression in the mouse intestinal mucosa and liver. Co-immunoprecipitation experiments provided evidence for interactions between AP-2α, β-catenin, and adenomatous polyposis coli (APC) proteins in mouse intestinal mucosa, as well as in a primary human colorectal cancer. Collectively, these studies support a tumor suppressor role for AP-2α in the gastrointestinal tract, and suggest that AP-2α represents a novel target for therapeutic intervention in human cancers characterized by dysregulated Wnt signaling.

Wnt inhibitory factor (WIF)-1 inhibits osteoblastic differentiation in mouse embryonic mesenchymal cells

Wnt inhibitory factor (WIF)-1 belongs to the members of secreted modulators of Wnt proteins. Secreted frizzled-related proteins (sFRPs), another member of Wnt modulators, have been shown to play differential roles in Wnt signaling depending on the subtypes and cell models. This study was undertaken to investigate the functional role of WIF-1 in osteoblastic differentiation of mouse mesenchymal C3H10T1/2 cells. C3H10T1/2 cells express endogenous WIF-1 and its expression level decreases during osteoblastogenesis. Treatment of C3H10T1/2 cells with WIF-1 significantly reduced alkaline phosphatase (ALP) activities induced by either osteogenic medium (OM, ascorbic acid and β-glycerophosphate) or Wnt-3a conditioned medium (CM) in a dose-dependent manner. In contrast, the expression level of endogenous WIF-1 increased during adipogenesis and WIF-1 treatment resulted in increased adipogenesis. C3H10T1/2 cells transduced with WIF-1 retrovirus also exhibited reduced ALP activity and decreased mRNA expression of Runx2, collagen type 1, ALP and osteocalcin during osteoblastic differentiation compared to empty virus-transduced cells. Moreover, treatment with WIF-1 dose-dependently attenuates β-catenin/T-cell factor (TCF) transcriptional activity in this cell line. Finally, knockdown of WIF-1 in C3H10T1/2 cells by RNA interference leads to increase in ALP activities. Collectively, these results indicate that WIF-1 plays as a negative regulator of osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells in vitro.

PGF2α stimulates FP prostanoid receptor mediated crosstalk between Ras/Raf signaling and Tcf transcriptional activation

Prostaglandin-F2α (PGF2α) is a product of the cyclooxygenase pathway and is a local signaling molecule that activates a G-protein coupled prostanoid receptor named FP. FP receptors can stimulate T-cell factor (Tcf) transcriptional activation by stabilization of β-catenin and can upregulate the expression of mRNA encoding cysteine-rich protein 61 (Cyr61), a secreted extracellular matrix protein that stimulates angiogenesis. We now show in both HEK cells and human microglial cells that the induction of Cyr61 protein expression by the human FP receptor utilizes a novel mechanism involving the activation of Ras and Raf followed by a MEK/ERK independent activation of Tcf signaling. The upregulation of Cyr61 in microglial cells may contribute to glioma tumorigenesis and could be a potential therapeutic target.

Induction Of Functional Hypoxia-inducible Factor-1α And Angiogenesis By Derivatives Of 8-hydroxyquinoline

Hypoxia-inducible factor-1 (HIF-1) as a complex of α and β subunits mediates a ubiquitous pathway by which mammalian cells sense and respond to hypoxia. In mammalian cells, the levels and activity of HIF-1α are regulated by its post-translational hydroxylation as catalyzed HIF hydroxylases, whose inhibition is thus attractive from the perspective of developing pharmaceuticals that activate the HIF pathway and induce a pro-angiogenic response. We found that 8-hydroxyquinoline and its derivatives inhibit hydroxylation of proline and asparagine of HIF-1α with varying degrees. In addition, they completely block ubiquitination of HIF-1α, which leads to its accumulation and activation of HIF-1-mediated vascular endothelial growth factor transcription and reporter gene activity. Furthermore, in vivo organ models based on the chick chorioallantoic membrane assay demonstrate promotion of new blood vessel formation. Therefore, our results indicate that CQ analogs possess a pro-angiogenesis potential and thus might have the therapeutic utility in the treatment of ischemic diseases.

The selenocysteine tRNA STAF-binding region is essential for adequate selenocysteine tRNA status, selenoprotein expression and early age survival of mice

STAF [Sec (selenocysteine) tRNA gene transcription activating factor] is a transcription activating factor for a number of RNA Pol III- and RNA Pol II-dependent genes including the Trsp [Sec tRNA gene], which in turn controls the expression of all selenoproteins. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated transgenic mice expressing the Trsp transgene lacking the STAF-binding site and made these mice dependent on the transgene for survival by removing the wild-type Trsp. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced approximately 60% in liver and kidney, approximately 70% in lung and spleen and approximately 80% in brain and muscle compared with the corresponding organs in control mice. Moreover, the ratio of the two isoforms of Sec tRNA that differ by methylation at position 34 (Um34) was altered significantly, and the Um34-containing form was substantially reduced in all tissues examined. Selenoprotein expression in these animals was most affected in tissues in which the Sec tRNA levels were most severely reduced. Importantly, mice had a neurological phenotype strikingly similar to that of mice in which the selenoprotein P gene had been removed and their life span was substantially reduced. The results indicate that STAF influences selenoprotein expression by enhancing Trsp synthesis in an organ-specific manner and by controlling Sec tRNA modification in each tissue examined.